Multivalent Clustering of Adhesion Ligands in Nanofiber-Nanoparticle Composites

Because the positioning and clustering of biomolecules within the extracellular matrix dictates cell behaviors, the engineering of biomaterials incorporating bioactive epitopes with spatial organization tunable at the nanoscale is of primary importance. Here we used a highly modular composite approach combining peptide amphiphile (PA) nanofibers and silica nanoparticles, which are both easily functionalized with one or several bioactive signals. We show that the surface of silica nanoparticles allows the clustering of RGDS bioactive signals leading to improved adhesion and spreading of fibroblast cells on composite hydrogels at an epitope concentration much lower than in PA-only based matrices. Most importantly, by combining the two integrin-binding sequences RGDS and PHSRN on nanoparticle surfaces, we improved cell adhesion on the PA nanofiber/particle composite hydrogels, which is attributed to synergistic interactions known to be effective only for peptide intermolecular distance of ca. 5 nm. Such composites with soft and hard nanostructures offer a strategy for the design of advanced scaffolds to display multiple signals and control cell behavior.

improved adhesion and spreading of fibroblast cells on composite hydrogels at an epitope concentration much lower than in PA-only based matrices. Most importantly, by combining the two integrin-binding sequences RGDS and PHSRN on nanoparticle surfaces, we improved cell adhesion on the PA nanofiber/particle composite hydrogels, which is attributed to synergistic interactions known to be effective only for peptide intermolecular distance of ca. 5 nm. Such composites with soft and hard nanostructures offer a strategy for the design of advanced scaffolds to display multiple signals and control cell behavior.

INTRODUCTION
The natural extracellular matrix (ECM) surrounding cells plays a critical role in directing cell function by providing essential structural and biochemical cues. One mechanism by which ECMs regulate cell signaling is clustering of biological ligands with variable densities and separation. [1][2][3] For example, focal adhesions are triggered by the formation of an effective integrin cluster with a specific lateral spacing. This has been experimentally demonstrated by controlling the density and interspacing of arginine-glycine-aspartate (RGD) ligands in synthetic materials, showing that the peptide spacing (ca. 70 nm) within a local cluster is more essential than its bulk density to trigger cell adhesion. [4][5][6][7] In addition to RGD clustering, integrin-binding proteins contain domains that operate synergistically with RGD to elicit cell response. For instance, the PHSRN sequence within fibronectin synergizes with RGD in a distance-dependent manner. 8-9 Different approaches have been developed to control ligand positioning and inter-ligand distances, including their conjugation onto amphiphilic [10][11][12][13] and PEGylated constructs, 14,15 oligopeptide backbones, [16][17][18] DNA constructs [19][20][21] or the functionalization of titanium surfaces to display distinct bioactive motifs in a chemically-controlled fashion. 22 The use of self-assembling peptide amphiphiles (PAs), which consist of a short peptide sequence linked to a hydrophobic alkyl tail, has been particularly promising in engineering bioactive artificial scaffolds for cells. 23,24 The facile incorporation of multiple bioactive signals at controlled concentrations, together with their structural similarity to extracellular matrix fibres makes PA assemblies useful as bioactive artificial extracellular matrix components for cell signalling. 25,26 Interestingly, peptide amphiphile supramolecular systems were shown to have both fully dynamic and kinetically inactive areas in the aggregate, which can be used to generate useful cluster morphologies. 27,28 Over the last few years, the nanocomposite approach has emerged as an efficient alternative to generate biofunctional scaffolds. 29 Bionanocomposites based on the association between bio-based polymers and inorganic colloids combine the chemical diversity, hierarchical structure and biocompatibility of biomacromolecules with the robustness and functionality of the inorganic phase. 30 Depending on the chemical nature of the nanoparticles (NPs), different properties can be imparted to the resulting composite to design conductive, optical and magnetic devices, and also to tune the mechanical properties and the bioactivity of hydrogels. 31,32 Silver NPs have often been incorporated within matrices of biological or synthetic origin for their antimicrobial properties, [33][34][35][36] as well as to design plasmonic sensors 37 . Gold, [38][39][40] cobalt, 41 nickel 42 and copper 43,44 metal NPs together with iron oxides NPs [45][46][47][48] have also been encapsulated to design composites, many of which finding applications in drug delivery. In parallel, the incorporation of silica nanoparticles (SiNPs) has been shown to enhance the mechanical properties of hydrogels, 49-53 enhances biological activity of biomaterials 54 and has been widely studied in the field of drug delivery. [55][56][57][58][59][60] SiNPs are particularly interesting candidates due to their low cost, limited cytotoxicity, ease of synthesis, and the versatility of sol-gel chemistry that offers various routes to conjugate biomolecules at the NP surface, while preserving their molecular recognition properties. 61,62 Here we combine self-assembled PA matrices with SiNPs to design novel SiNP-PA composite biomaterials (Figure 1). The ability to independently modify the chemistries of both PA and NP substrates to link distinct bioactive motifs on which cells would grow allows us to cluster signals in variable patterns positioned through the composite material to impart biological functionality.
We show that clustering of the fibronectin derived RGDS peptide on the surface of Stöber SiNPs (ca. 200 nm in diameter, Figure 1A) triggers cell adhesion onto SiNP-PA scaffolds. In addition, the multiple display of RGDS and PHSRN bioactive epitopes can be achieved within SiNP-PA composites ( Figure 1B) to trigger synergistic effects on cell behavior. This strategy offers a unique modularity by the ability to introduce functionality through both the nanofiber scaffold and the incorporated modified NPs, making composites highly promising biomaterials to display bioactive sequences with synergistic effects. Simultaneous display of two bioactive signals in PA nanofibers (left) or after the clustering of two signals at the surface of SiNPs embedded in a PA matrix (right).

EXPERIMENTAL SECTION
PA and Peptide synthesis. PAs and peptides were synthesized using a standard fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS) on Rink Amide MBHA resin as described previously. 63 Amino acid couplings were performed either manually or on a CEM Liberty microwave-assisted peptide synthesizer. Rink Amide MBHA resin, Fmoc-protected amino acids and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Novabiochem; Fmoc-NH-PEG4-CH2COOH was purchased from ChemPep Inc.; palmitic acid was purchased from Acros Organics; Fmoc-(4-amino)benzoic acid and Fmoc-(4-aminomethyl)benzoic acid were purchased from VWR and Chem-Impex International Inc., respectively. All other reagents and solvents were purchased from Sigma Aldrich and used as received. Fmoc deprotection was performed using 30% piperidine in N,Ndimethylformamide (DMF) and amino acid and palmitic acid couplings were performed with 4 molar equivalent (eq.) protected amino acid or palmitic acid, 3.95 eq. HBTU, and 6 eq. of N,Ndiisopropylethylamine (DIEA) in DMF alone or in a solvent mixture of 1:1:1 DMF/dichloromethane (DCM)/N-methyl-2-pyrrolidone (NMP). The coupling reaction for the PEGylated amino acid was performed similarly to other standard Fmoc-protected amino acids, using Fmoc-PEGylated amino acid (3 eq.), HBTU (2.95 eq.), and DIEA (4.5 eq.) in DMF. For the coupling of Fmoc-(4-amino)benzoic acid and Fmoc-(4-aminomethyl)benzoic acid, both were converted into acid chloride first (procedure described below) to increase the coupling yields. The coupling reaction was performed by using 4 eq. of Fmoc-(4-amino)-benzoyl chloride or Fmoc-(4aminomethyl)benzoyl chloride and 6 eq. of DIEA in NMP. Synthesized PA and peptide molecules were cleaved from the resin using a mixture of 95% trifluoroacetic acid (TFA), 2.5% water, and 2.5% triisopropylsilane (TIPS). After removing TFA by rotary evaporation, the product was precipitated with cold diethyl ether, dried, and purified using preparative scale reverse phase high performance liquid chromatography on a Varian Prostar Model 210 system equipped with a Phenomenex Jupiter Proteo column (C12 stationary phase, 10 mm, 4 µm particle size and 90 Å pore size, 150 × 30 mm). A linear gradient of acetonitrile (2 to 100%) and water with 0.1% ammonium hydroxide (added to aid PA solubility) was used as the mobile phase for purification.
Electrospray ionization mass spectrometry (Agilent 6510 Q-TOF LC/MS) was used to identify the pure fractions ( Figure S1,S2), which were then combined together and lyophilized after removing excess acetonitrile by rotary evaporation.

Synthesis of peptide-conjugated SiNPs.
The synthesis of peptide-conjugated SiNPs proceeded in three steps, whose success was checked by zeta potential measurements ( Figure S4).
Subsequently, the reaction mixture was heated to 80°C and the total volume was reduced to approximately two-third by distillation of ethanol and ammonia at ambient pressure. The mixture was left to cool down to RT and was subsequently washed three times with ethanol (by centrifugation at 12 000 rpm for 15 min) before drying under vacuum. Successful surface modification was ascertained by the increase of the  potential value of the recovered nanoparticles at all pH values compared to initial SiNPs. Quantification of surface functionalization using Cy3-Azide. SiNP-DBCO or nude SiNPs were redispersed in water before addition of 1.2 µmol of Cy3-azide (Cy3-N3, 90%, Aldrich) in DMSO (4 mmol.g -1 silica). The mixture was stirred for 12 h at RT and subsequently washed as many times as necessary (at least 5 times) with water (by centrifugation at 12 000 rpm for 15 min). Absorbance and fluorescence of the samples were then measured to quantify the conjugation rate at the surface of SiNPs, providing a density of 0.2 Cy3 per nm 2 of silica surface ( Figure S5).

Dibenzocyclooctyne-N-hydroxysuccinimidyl ester grafting on
Peptide Amphiphile (PA) and SiNP/PA Composite Gel Preparation. The desired amount of PA powder was weighed out in an Eppendorf tube in order to make 100 μL of a 1 wt% PA stock solution in H2O. The PA solution was subsequently annealed at 80°C in a PCR machine for 30 min and slowly cooled down to room temperature (RT) over 90 min. The self-assembled structures resulting from the different PA mixtures were characterized using TEM ( Figure S6). SiNP/PA composite gels were prepared following the same protocol except that a water suspension of SiNP was added to the PA solution at different ratios and the mixture was sonicated before annealing. supplemented with SiNPs at various ratios was added onto the surface and the layer was gelled with a 10 mM CaCl2 aqueous solution. These layers were characterized by SEM ( Figure S7). SiNP-Cy3/PA layers were also prepared. The PA were stained by DAPI and images of the sample were obtained using an inverted confocal laser scanning microscope (Nikon A1R).   First, amine-modified SiNPs were mixed with a 1 wt% (10 mg.mL -1 ) PA solution before gel formation. In this case, the SiNP concentration (from 3 to 25 mg.mL -1 ) was selected so as to target a peptide epitope concentration of 0.2 to 2.6 mol%, assuming that all surface amines are modified with peptide epitopes. The different co-assemblies all formed a gel, incorporating SiNPs within the nanofiber network, as observed by TEM and SEM for 1.3 and 2.6 mol% SiNPs (Figure 3A,B   and D,E). This indicates that the presence of SiNPs does not disturb the PA self-assembly. The localization of SiNPs within the PA matrix could further be visualized by the conjugation of azidecyanine 3 dye to SiNPs and of DAPI to the PA. Observations by confocal microscopy confirmed the good dispersion of SiNPs within the 3D gel (Figure 3C,F).

Preparation and characterization of SiNP
Next, the rheological properties of the PA and SiNP-PA (1.3 and 2.6 mol%) scaffolds were assessed, Figure 3G. The SiNP PA scaffolds remained in similar range of mechanical stability with storage moduli in the range of 100-500 Pa.  (Figure 4Aa). In contrast, improved cell adhesion and spreading was clearly observed for PA-RGDS matrices when reaching 2.6 mol% RGDS, and the cells showed formation of focal adhesions. The lower concentrations (0.6 and 1.3 mol%) of PA-RGDS were not sufficient to promote cell adhesion and spreading (Figure 4Ab-c).
Interestingly, the incorporation of SiNP-RGDS showed a positive effect on cell spreading at a concentration as low as 0.6 mol% (Figure 4Af). Quantitative assessments obtained by image analysis confirmed that PA-RGDS at 2.6 mol% and SiNP-RGDS PA at 0.6 mol% were equally efficient in promoting cell spreading. .Incorporation of SiNPs bearing the mutated peptide RGES (Figure 4Ai,j) or non-functionalized SiNPs ( Figure S8) did not result in cell spreading. While it is difficult to completely rule out a local mechanobiology effect, the observed decrease in RGDS concentration required to improve cell spreading between the PA-RGDS and PA-NP composite systems suggests that differences in epitope display within the scaffold and the local high concentration of SiNP RGDS PA are playing a role.
These results indicate that the clustering of the RGDS bioactive epitopes on the SiNP surface offers an efficient strategy to improve fibroblast cell adhesion on PA matrices.  promoted the most spreading. This strongly suggests that the peptide epitopes grafted on the silica nanoparticles are in optimal clusters as well as inter-epitope distance to allow for their synergistic effect on cells.

DISCUSSION AND CONCLUSION
In this work we integrated silica nanoparticles into peptide amphiphile fibrous networks to yield composite hydrogels. The ability to modify the surface of the particles with bioactive ligands independently from the peptide networks allows for a modular approach to introduce biological cues and control their local density. We demonstrated that surface modification of SiNPs with a diameter of 200 nm ensures the formation of effective peptide clusters with a statistical inter-ligand spacing that can effectively promote cell adhesion and spreading. [4][5][6][7] Furthermore, chemically-engineering the particles to simultaneously display two synergistic bioactive peptides enabled enhanced cell adhesion and spreading. Varying the concentration of surface ligands allowed the control of their density. which was calculated ( Figure   S5) to be 0.2 molecules per nm 2 SiNP, i.e 1 peptide every 5 nm 2 . This corresponds to a distance of ca. 5 nm between the two peptides RGDS and PHSRN, mimicking their separation distance in native fibronectin. It is important to point out that inter-ligand distances can also be controlled by the selected particle size, the synthesis conditions including solvent, surfactant and silica precursor species to vary silanol surface density. 65 Further control can be achieved by localizing the distribution of peptide epitopes into functional domains or patches. 66 Major progress in the field of regenerative medicine can be expected from the design of artificial scaffolds that mimic the various features of the ECM. While significant advances have already been achieved in reproducing the structural and mechanical features of the ECM, fine control over the incorporation and positioning of multiple biological cues that play a key role on regulating cell behavior remains highly challenging. This work shows that combining organic and inorganic building blocks with easy, versatile and orthogonal bioconjugation chemistries provides a highly modular approach to engineer 3D scaffolds displaying multiple epitopes. Silica nanoparticles allow for single epitope clustering and multivalent clustering. The possibility to tune their diameters and surface chemistry make them versatile platforms that may be engineered to display multiple epitopes. Integrating bioactive silica nanoparticles with the powerful peptide-

Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.