Hybrid Conjugates Formed between Gold Nanoparticles and an Amyloidogenic Diphenylalanine-Cysteine Peptide

We investigated the effect of gold nanoparticles (AuNPs) on the aggregation of a CFF (C = cysteine; F = phenylalanine) tripep- tide (derived from Ab peptide) in aqueous medium. Special attention was dedicated to the role of AuNPs as inducers and inhibitors during nucleation kinetics and the structure of the resulting scaffolds was carefully investigated. At millimolar concentrations, the tripeptide was found to form b-sheet structures organized into long filaments. Spectral signatures and topography of the filaments were studied by Raman spectroscopy and atomic force microscopy (AFM), revealing that conjugation to AuNPs not only stabilizes the system, but also inhibit or enhance amyloid-like features depending on the synthesis route used in the preparation of AuNPs. Sodium borohydride (NaBH4) mediated synthesis of AuNPs gave rise to a strong absorption peak close to 520nm, indicating that AuNPs were dispersed, independently of the peptide concen- tration added in the reaction. However, when the peptide/gold salt mixture was heated at 608C, AuNPs and AuNP-decorated filaments were both formed in solution and the fractions of which population were found to be dependent on the [HAuCl4]/[CFF] ratio, as illustrated by TEM images. In addition, the insertion of AuNPs at the surface of CFF nanostructures can promote electron transfer from the metallic nanoparticles to the CFF surface, creating an n-type semiconductor, and causing a peak shift of the phenylalanine absorption band.


INTRODUCTION
The formation of metal nanoparticles decorated by short peptides is based on the reducing capability of metal ions and the capping ability of the peptides. Peptide sequence-activity relationships can be used to achieve shape and size control of the metal nanoparticles by varying the ratio of the metal ion to the peptide concentration. [1][2][3][4] Achieving control of such interactions is important for developing diagnostic and therapeutic strategies where protein and peptide aggregation is associated with disease. [5][6][7][8][9][10][11][12] Recent studies have shown that nanoparticles can have an impact on the aggregation properties of peptide or protein structures, where the cooperative binding of biomolecules to nanoparticles may be either beneficial or deleterious to the formation of amyloid structures. [13][14][15][16][17][18] Interaction with nanoparticles may either affect the structure of amyloid-prone peptides, inducing the formation of -structures that serve as nucleation centers for fibrillization, or bind intermediate units, and even temporary reverse the growth of fibrils or aggregate structures. [13,[19][20][21] Identification of possible targets in the formation of amyloid structures is crucial for future application of nanotechnology to enhance specificity and efficacy of prevention and control methods of neurodegenerative diseases.
At the same time, amyloid-like peptide nanostructures (ALPNs) were shown to be able to control aggregation of metal nanoparticles into 1D nanostructures. [22,23] ALPNs can be specifically designed to have chemically active groups offering metal binding sites on their external surface. Previously, Schebeil et al. [24] applied layer by layer electrodeposition of Au and Ag on ALPNs, obtaining metal-peptide conjugates with conductive properties comparable to that of a solid metal wire. [24] In this connection, Chen and Rosi [25] reported that peptides can be used as scaffolds to control the nucleation and growth of inorganic materials. [25] 4 Diphenylalanine (FF) peptide, the core recognition motif of Alzheimerʼs -amyloid polypeptide, is one of the most studied for the formation of self-organized structures. [26,27] The polymorphism of FF-based nanostructures can be controlled by the experimental incubation conditions such as solvent, peptide concentration, pH and temperature. [27][28][29][30][31][32] It has been shown that triphenylalanine peptides (FFF) self-assemble into nanospheres or nanorods, which are different from the nanovesicles and nanotubes formed by simply tuning the FF peptide concentration. [33][34][35][36] In a pioneering study by Reches and Gazit, [26] it was found that FF self-assemblies serve as excellent sacrificial templates for fabrication of Ag nanowires through a reductive process. [26] Furthermore, a morphological transition was observed when phenylglycine (Phg) was used as the dipeptide motif. [37] Phg offers constrained rotational freedom around the C-C bond, which helps in the formation of very stable Phg-Phg nano vesicles. A similar morphological switch was also observed when FF dipeptide was conjugated to a cysteine residue (CFF). [38,39] This tripeptide self-assembled into soft nanospheres suggesting that the disulfide bridge formation results in the closure of nanotubes. However, a fine control of the CFF aggregation process has not yet been elucidated, making difficult to control the stability, morphology and polydispersity of the formed nanostructures.
The aim of the present work is to gain insights into the self-assembly of CFF in aqueous solution and verify the influence of peptide conformational changes at the interface of AuNPs. We explored the effects of nanoparticles on the peptide fibrillization process from both the perspectives of inducers and inhibitors of the CFF nucleation kinetics. We show that the latter dependent on the nanoparticle synthesis route, i.e., by using sodium borohydride (NaBH4) as a reducing agent, or by using the peptide-catalyzed 5 redox technique for the in situ synthesis and stabilization of AuNPs under heating at 60ºC, as schematized in Figure 1.

Spectroscopy and Morphological Studies of CFF
To evaluate the phenomena of self-organization and study the aggregation mechanism of CFF in aqueous media, seven CFF solutions in a range of 0.3-2.0 mmol•L -1 peptide concentrations were analyzed by steady-state fluorescence spectroscopy. The critical aggregation concentration (cac) was assessed by the normalized intensity of the phenylalanine fluorescence emission at λ= 289 nm. [40][41][42][43] In this way, the CFF cac (~0.8 mmol•L -1 at 25°C) was estimated from the crossover between linear fits corresponding to different emission regimes (Figure 2A). In aqueous solutions, CFF forms spontaneously a -sheet conformation mainly stabilized by hydrogen bonding interaction (see Figure 2C).
In addition, both electrostatic forces between the charged residues, and the hydrophobic 6 effect on the FF group contribute to the aggregation process, which is identified by the inflection point in the fluorescence intensity vs. peptide concentration curve. The latter indicates the transition from a monomer-like phase to the formation of an aggregate structure, characterized by a higher fluorescence due to water exclusion that cannot exert its quenching activity on the F fluorophore. This is confirmed by the slight blue shift of the F emission, due to the lower polarity of the water-excluded environment in the aggregate.
Moreover, we also observed that the CFF self-assembly can be influenced upon changing the pH of the solution. Although the CD spectra of Figure 2C are typical of an antiparallel β-sheet arrangement at all the pHs investigated (pH= 3.0, 7.0 and 10), their relative rotational strengths are not the same, suggesting that a different amount of βsheet conformers populate the solutions at different pHs. As the pH decreases from 10 to 3.0, the ionization degree of CFF decreases, as can be seen in the titration curve ( Figure   2B). Therefore, pH changes affect the interchain interactions of the aromatic peptide amphiphiles, [44] promoting CFF aggregation as A-like structures.
CD spectra of CFF pre-assemblies ( Figure 2C) in aqueous solution showed a negative peak at 190 nm, and two positive peaks at 200 nm and 217 nm corresponding to the π-π * transition and n-π * transition, respectively. [45] The shape of the CD curves was similar to that of β-turn peptides and -stacked phenylalanine residues, which was characterized by a negative peak at 180-190 nm and two positive peaks at 200-205 nm and 220-230 nm. [41,46] It is inferred that at this concentration CFF achieves a higher level of organization that could be associated to the beginning of fibrillization in solution. This finding strongly suggests the possibility that this simple tripeptide can form amyloid-like fibers. 7 To investigate the morphology of the CFF aggregates, AFM images ( Figure 2D) were acquired on dried samples obtained from a solution of peptide above the cac. The images show a network of entangled fibers at neutral pH with diameters ranging from 100 to 150 nm and lengths of up to a few micrometers, in coexistence with globular particles with a few tens of nanometers in diameter (see Figure 2D). The fibers are formed by smaller elliptical particles, which may be related to an Ostwald-like ripening process of coherent β-precipitates during the internal rearrangement of structures formed in a kinetically driven self-assembly process. [47] Spherical or granular aggregates were in some cases considered as precursors or nuclei for fibrillization as in the case of amyloid -protein. [48]  Recently, Gazit and co-workers [47] studied the morphological transition of Boc-FF from a globular to a tubular phase, indicating that the transition from spheres to tubes is exothermic and that the concentration of monomers in equilibrium with the tubular phase is lower than that found in the case of monomers-spheres equilibrium. This finding suggests that the thermodynamic stability of the tubular phase is higher than that of the globular phase, and hence that the transition is governed by Ostwald´s rule of stages. [47]

Manipulating the assembly of cysteine-diphenylalanine in solution using AuNPs
AuNPs were synthesized via the NaBH4 reduction of HAuCl4 in the presence of the which is characteristic of spherical nanoparticles. [49] The observed blue-shift in plasmon resonance absorption band of the hybrids with respect to the CFF concentration is due to the reduction of the nanoparticle diameter from 55 to 20 nm. As it can be seen in Figure   3A, the intensity of the band decreases with the increase of the CFF concentration.
Furthermore, the presence of the peptide in the AuNPs solution promotes the stabilization of the system (see Figure 3B).    On the other hand, there are various approaches to accelerate the spontaneous fibrillization of proteins, including agitation of the solution by shaking or stirring. [50] In this work, we found that increasing the time incubation of the CFF and HAuCl4 reaction mixture at 60°C, but without the use of external reducing agents, induced fibrillization, depending on the ratio of the Au salt and capping agent ( Figure 5). Reduction of the 11 chloroaurate ion by a cysteine moiety implies oxidation of the amino acid and selfreduction of the gold salt precursor. [HAuCl4]/[CFF] molar ratios are spherical and monodisperse with a particle size which may be varied within the range comprised between 10 and 15 nm (see Figure 5A).
Increasing the amount of CFF in the system affected the stability of AuNPs in solution, promoting the formation of different polymorphs, including a mixture of small spheres, some amorphous aggregates and fibrils ( Figure 5B). Some of the aggregation properties of the CFF peptides were thus transferred to the Au nanoparticles, and this may provide a strategy for the control of the AuNPs assembly. 12 At lower [HAuCl4]/[CFF] ratios, i.e. with increasing the CFF concentration above the cac, a remarkably large fraction of peptide aggregates was present in the solution instead than on the nanoparticle surface. Thus, peptide aggregation as A-like fibers predominantly occurs in solution as seen in AFM images ( Figure 2D), instead than on the nanoparticle surface.
CFF forms fibrils the peptide layers of which could serve as a template for the formation of geometrically confined Au seeds ( Figure 5C). Some smaller fibers had diameters of approximately 50 nm with lengths in the micrometer range. The observed morphology change proves that the addition of peptide-AuNPs facilitates the subsequent assembly of peptides into stable fibril aggregates by increasing the local concentration of those peptides. Nanoparticles may act like conventional catalysts by reducing the energy barrier to fibrillization increasing the population of prefibrillar aggregates. [19] A possible model illustrating both the formation of particle and fiber aggregates are schematized in  Table 1.
For the hybrids, in all cases, colloidal solutions containing CFF-coated nanoparticles were stable for months with little evidence of precipitation. The scattering profiles can be divided in three distinct parts: at low q region (q < 0.3 nm -1 ) the observed behavior is typical of the Guinier's region, which is used for the determination of the overall size of the particles. At q ~1.0 nm -1 , a subtle oscillation in the curve can be related to the overall size of AuNPs and its polydispersity. At high q region (q > 1.1 nm -1 ) the curve has little information because this region, in our data, is dominated by the background and flexible parts signals.
As in the case of CFF in water, we used the model of aggregated polydisperse spheres with flexible parts to describe the SAXS data for the synthesis of AuNPs using NaBH4 at room temperature ( Figure 6B As shown in Table 1 For the AuNPs obtained by synthesis under heating ( Figure 6C), oscillations in the mid q region (0.3 < q < 1.0 nm -1 ) were observed; this finding is associated with the presence of spherical particles with well-defined sizes, as imaged in the TEM experiments ( Figure 5). As shown in Table 1, for the ratios 2.7 and 1.3 there is an enhanced formation of nanoparticles characterized by a very narrow distribution and without the presence of larger aggregates. For the ratio 0.88 there is a much weaker scattering intensity which indicates a lower concentration of particles. In this case the size of spherical particles decreases and larger aggregates can be readily observed. Above the cac (ratio 0.88) somewhat similar SAXS results were obtained, indicating that concentration induced aggregates largely predominate. Table 1. Summary of the model parameters obtained from the fitting procedure of SAXS data. The insertion of AuNPs at the surface of CFF nanostructures can promote the formation of negative charges (-31 mV, as obtained by Zeta potential measurements) that can be excited using a 532 nm laser radiation. This can promote electron transfer from the AuNPs to the CFF surface, creating an n-type semiconductor, and causing a peak shift of the F absorption band. Comparing the methodologies for the nucleation of AuNPs, the intensity of the band is higher for the NaBH4 reduction method than for heating at 60°C sample; this can be indicative of the stronger n-doping of CFF applying the former procedure. [54,55] In order to monitor the excimer formation process, [56] the samples were excited at 330 nm to reveal the characteristic emission band of the formed aggregates (λem=423 nm).
The emission band was measured after a 24 hours preincubation of CFFH2O stock solution in the presence and absence of gold nanoparticles under different reaction conditions. As shown in Figure 8, the tripeptide shows two bands at 370 and 420 nm, which can be associated with the formation of globules (micellar aggregates) and fibers, respectively. [56] The control (CFFH2O) presents a very high intensity emission typical of micellar aggregates, indicating that in this system spherical aggregates predominate. For the sample under heating, both aggregate bands are present, but the intensity of the excimer band is definitely higher, signalling the formation of more ordered, crystalline structures, favoring the fibrillization of the peptide material. The hybrid sample obtained by NaBH4 reduction presents almost the same contribution for both the aggregate types, suggesting a slower kinetics for the formation of crystalline aggregates, making the fibrillization process more difficult, in fair agreement with the TEM and SAXS results. Therefore, we provided a single-step process that allows for the simultaneous formation of highly ordered nanostructures, where the size of peptide-gold nanoparticle hybrid systems could be controlled by using different [HAuCl4]/[CFF] ratios, reducing agents, and temperature-dependent reaction conditions. This method, which combines peptide-based supramolecular structures with desirable functional inorganic materials, may provide a novel approach for "bottom-up" fabrication of hierarchical structures with unique physical, chemical, and biological properties.

CONCLUSIONS
In summary, we have demonstrated the fabrication of hybrid nanostructures containing CFF and gold nanoparticles in water. Furthermore, the size of AuNPs in the selfassembly of an amyloid-like peptide can be tuned by adjusting the [HAuCl4]/ [CFF] proportion, temperature of the reaction, and reducing agent. The fibrillization process was faster for the CFF-AuNP conjugates under heating, promoting the formation of crystalline aggregates. However, amorphous structures were obtained in aqueous solution at room temperature by using NaBH4 as the reducing agent. Another parameter to be 19 considered is the peptide concentration. Below the cac, the system gave rise to the formation of polydisperse globular nanoparticles; while above the cac, the formation of fibrillar compounds with diameters in the order of ~50 nm takes place. In both cases, the nanoparticles are distributed inside and on the surface of the peptide nanostructures, indicating that the simultaneous self-assembly of the peptides and nucleation of the gold salt via specific interaction, propagate the molecular-scale ordering to the nanoscale morphology. For both synthetic methods, it was verified that the systems self-organize as polydisperse globules, with the presence of flexible parts. The insertion of AuNPs at the surface of CFF nanostructures can promote electron transfer from the metallic nanoparticles to the CFF surface, creating an n-type semiconductor. The method developed here may be useful to produce various multifunctional hybrid nanostructures that may find potential applications in nanotechnology and biomedical science.

SUPPORTING INFORMATION SUMMARY
The experimental procedures and additional characterization data are provided in supporting information.