Fibronectin-derived protein forms a protein corona on gold nanoparticles: synthesis, Raman and optical properties of a new plasmonic nanocarrier

Synthetic ﬁbronectin III-derived protein scaffolds represent a new generation of proteins that can overcome some clinical limitations of therapeutic monoclonal antibodies. However, one major disadvantage of smaller protein scaffolds is their rapid renal clearance and correspondingly short circulating serum half-lives. A complex formation of these protein scaffolds with nanoparticles can be a valuable route to overcome the short half-life in human serum. Here we present the synthesis and characterization of a ﬁrst example of ﬁbronectin III, 14th domain-derived scaffold, called Pronectin ™ , with gold nanoparticle of around 30-nm diameter to form a protein corona. The obtained functionalized nanoparticles were characterized by Raman spectroscopy and electron microscopy. Their plasmonic properties, due to the gold core, and the luminescence, attributed to the protein, were measured in two cases (nanoparticles with albumin or Pronectin ™ ), and conﬁrmed that the formation of a protein corona induces some form of denaturation of the proteins themselves.


Introduction
Gold nanoparticles are surprising materials, in particular for their plasmonic properties which occur when they interact with light [1][2][3][4]. These properties are related to their nanoscale structure, depending on their shape, size, coating agent and assembly [5][6][7][8][9][10]. This implies that through an appropriate synthetic strategy, it is possible to obtain nanoparticles whose plasmonic properties adapt to the fields of application, which include, in biomedical, imaging, drug delivery, hyper-thermal and photothermal therapies [11][12][13]. While much remains to be understood about how nanoparticles interact with individual biological systems, it is generally believed that when nanoparticles come into contact with a biological medium, such as serum, the biomolecules within, mainly simple proteins and lipids, will adsorb on their surface.
Serum proteins to a large extent include albumin, immunoglobulin G (IgG), fibrinogen and apolipoproteins [14][15][16][17]. The proteins that adsorb on the surface of the metal, based on their affinity with that surface, are divided into two classes: the ''hard'' corona proteins, that is tightly bound proteins, and the ''soft'' corona proteins, that is low-binding proteins. Among the various hypotheses formulated in the literature, the ''hard'' corona proteins interact directly with the metal surface, while the ''soft'' ones interact with the hard corona, and exploit the protein-protein interactions [18,19]. However, the chemical surface of the nanoparticles plays a key role in the formation of the protein corona; a stabilizing nanoparticle coating such as thiolate polyethylene glycol covalently bonded to the gold surface affects the formation of the protein corona. On the other hand, a citrate coating, which uses a negative electrostatic interaction to bind the metal surface, is easily replaceable and allows for spontaneous adsorption of proteins [20][21][22][23][24].
Adsorption of proteins can lead to changes in the size, shape and surface charge of the nanoparticles, giving them a kind of biological identity [25]. Specific biological reactions such as fibrillation, cell uptake, circulation time, bioavailability and even toxicity can therefore be attributed to a specific nanoparticleprotein complex.
Pronectins™ represents a new class of single domain, non-immunoglobulin, cysteine-free proteins with exquisite binding affinity and specificity similar to those of monoclonal antibodies (Mabs) [26,27]. Their protein structure is derived from the 14th domain of fibronectin III (14Fn3) with molecular weight of approximately 10,000 KDa (94-100 amino acids). Pronectins™ are similar in structure to other unglycosylated single domain, cysteine-free proteins derived from the 10th human fibronectin III domain (10Fn3), like monobodies [28], Adnectins [29] and Centryins [30] [31]and share great thermostability in plasma, and high tissue penetration properties [32]; nevertheless, their small size can be a drawback in maintaining a steady, clinically important and concentration in the blood stream for an effective acute therapy. The serum half-life of these proteins is ca. 1-1.5 [33]hours in comparison to the more stable Mabs that can circulate in blood for weeks. Several methods have been developed to extend their half-life, including the covalent link to PEG [30], a moiety of human serum albumin [33] and their recombinant expression as part of larger fusion protein (Fc-, dimer, trimer, etc.) [32,34]. A bi-specific Pronectin™ linked to a single chain anti-CD3 has been developed to study its therapeutic effect on epithelial ovary cancer [35] as T-cell engager.
A complex formation of Pronectin(s) with nanoparticles can be a valuable route to overcome the short half-life in human serum. In addition, the Pronectins' affinity and specificity may be of great use in delivering nanoparticles to specific cells (i.e., tumor specific receptors), tissues and organs for both diagnostics and therapeutic applications [34,36,37].
In this work we report the synthesis, morphology and spectroscopic characterization of spherical gold nanoparticles (AuNS) coated with Pronectin™ AXL-54-Cys (AXL), specially synthesized to be directed specifically against the tumor cell receptor AXL [38], and suitably functionalized with a Cys termination (at its carboxy-terminus) to effectively bind a gold nanoparticle. To optimize the synthetic method and conveniently choose the coating agent of the AuNSs, they were coated with a covalently bonded agent, O-(2-mercaptoethyl)-O′-methylpolyethylene glycol, PEG-SH (AuNS@PEG-SH), or with an electrostatically bonded agent, cetyltrimethylammonium chloride, CTAC (AuNS@CTAC). These two types of AuNSs were coated with the well-studied human serum albumin, HSA (AuNS@PEG-SH@HSA and AuNS@HSA, respectively). After a comparative study of these two protein-nanoparticle complexes, it was decided to use the CTAC-coated nanoparticles (AuNS@CTAC) to form a protein corona with AXL. The obtained nanoparticles, AuNS@AXL were characterized by Raman spectroscopy, which has been used with considerable success in the study of many different systems, [39][40][41][42][43][44] and their morphology and optical properties were studied by TEM and by UV-Vis absorption and emission spectroscopy.

Instruments
A Perkin Elmer Lambda 900 spectrophotometer was employed to obtain the extinction spectra [45][46][47]. Excitation and emission spectra were recorded on a Horiba Jobin Yvon Fluorolog 3 FL3-211 spectrofluorometer with a 450-W xenon lamp, double grating monochromators and a TBX-04 photomultiplier. The size and morphology of the gold nanoparticles were measured using a transmission electron microscope (Jeol JEM-1400 Plus 120 kV). The samples for transmission electron microscopy (TEM) were prepared by depositing a drop of a diluted solution on 300 mesh copper grids. After evaporation of the solvent in air at room temperature, the particles were observed at an operating voltage of 80 kV. A Jobin Yvon micro-Raman LABRAM supplied by a CCD (25691024 pixels) detector cooled at −70°C with the 633 nm line emitted by a He:Ne laser was used. The exciting power of the sample was modulated by the application of an optical density filter (D03). A 509 Olympus objective was used to visually explore the surface of the samples and collect the spectra. The spectral resolution was 1 cm −1 .
The particle size distribution was determined by dynamic light scattering (DLS), using a Zetasizer (Nano-ZS, Malvern Instrument, UK). Particle size measurements were carried out inserting the sample in a disposable cuvette with a detection angle of 173°. ζ-potential was assessed on the samples inserted in a capillary cell. Three measurements were taken for each sample and the results are expressed as the mean and standard deviation.

AXL-54-Cys Pronectin
AXL-54-Cys (AXL) is a 94 amino acid protein isolated from a library of 26 billion analogs produced by Protelica, Inc. in Howard, California. Its sequence is reported in SI. The protein was identified by a combination of phage panning and yeast display and selected by an in vitro set of assays for its high affinity and specificity toward AXL cell receptor. AXL-54-Cys was expressed in E. Coli and purified by size exclusion gel chromatography. Its M.W. is 11,307 Da. The purified protein was stored in a pH= 4.5 buffer (50 mM sodium acetate/150 mM NaCl).

AuNS@CTAC
Seed solution: A total of 25 µL of 5.0 E-2 M HAuCl 4 water solution were added to 4.7 mL of 0.1 M CTAC water solution and 300 µL of a freshly prepared 1.0 E-2 M NaBH 4 water solution was then injected with vigorous stirring. Excess borohydride was consumed by keeping the seed solution at room temperature for 30 min before use. Gold nanospheres of 5-7 nm in diameter have been obtained and used as seeds [48].
Growth solution: Aqueous solutions of CTAC (0.2 M, 20 mL), ascorbic acid (0.1 M, 15 mL) and the seed solution (1 mL) were shacked, by hand, in a 20-mL glass vial. Then, an aqueous solution of HAuCl 4 (0.5 E-3 M, 20 mL) has been added in a oneshot injection. Gold spheres of 30 nm in diameter have been obtained [49] (see Fig. S1 in SI). A ζ-potential of 22 mV at pH=7 has been recorded.

AuNS@PEG-SH
CTAC capping agent of AuNS@CTAC was replaced with PEG-SH by dissolving 30 mg of polymer in 1 mL of deionized water, then adding this solution to 25 mL of 5.0 E-4 M water-dispersed AuNS@CTAC [4]. The sample was left under stirring overnight, and then purified by unlinked PEG-SH (three centrifuge cycles, 600 rpm). The solid residue was dissolved in water.

Photophysical characterization
The absorption spectrum of HSA in water solution shows a principal band at 278 nm and a shoulder at 284 nm, while the emission spectrum presents a band at 344 nm and a prominent shoulder at 309 nm ( Fig. 1): the principal peak is due to the tryptophan residue de-excitation, the shoulder corresponds to the tyrosine residue emission [50], while in physiological condition it results quenched due to an energy-   transfer process toward tryptophan. The absorption spectrum of AXL in water solution shows a principal band at 275 nm and a shoulder at 284 nm, while the emission spectrum presents two bands at 317 and 341 nm, (Fig. 2) attributed to tryptophan and tyrosine emission, respectively. The extinction spectra of AuNS@CTAC and AuN-S@PEG-SH are reported in Fig. 3. The plasmonic band falls around 555 nm for both samples, as expected cause the solvent and size are identical, while the different monolayered coating doesn't interfere with plasmon resonance [51,52]. Figures 4 and 5 report the absorption and emission spectra of AuNS@HSA and AuNS@PEG-SH@HSA, while in SI (Figures S2 and S3) TEM images are showed, that confirms the same morphology and size of the obtained nanoparticles (30 nm in diameter).
Absorption spectra (Fig. 4) show the plasmon band at 565 nm, redshifted respect to nanoparticles without proteins: this is attributed to the protein-corona formation which acts as a dielectric coating [18,20]. Absorption spectra show also a wide unstructured band in the UV region, due to the scattered light by protein-coated nanoparticles, which submerge the absorption bands of proteins (Fig. 2). The coated proteins presence is confirmed by emission spectra (Fig. 5), which clearly show the luminescence bands of HSA. By comparing the emission spectra of AuNS@HSA with AuNS@PEG-SH@HSA (Fig. 5), it is possible to observe that the luminescence intensity of the first sample is greater than that of the last, although the reaction conditions, in particular the stoichiometric ratio between nanoparticles and proteins, were kept identical. To explain this, it is useful to consider the different binding nature of CTAC and PEG-SH with respect to the gold surface: CTAC interacts with the gold surface through weak electrostatic interactions: for this reason, it is easy for albumin to replace CTAC. Conversely, since PEG-SH covalently binds to the metal surface, albumin does not replace it on this surface and both contribute to the coverage of the nanoparticles. Consequently, despite the same amount of protein administered, the AuNS@CTACs were coated with a greater amount of HSA than AuNS@PEG-SH. Figure 6 reports the absorption and emission spectra of AuNS@AXL, while Figure S4 in SI reports TEM image of the sample, showing 30 nm diameter nanoparticles. In the absorption spectrum the plasmon band at 565 nm is well sharped, proving a  higher monodispersity of this sample than that coated with HSA; moreover, in the UV regions the bands attributed to the AXL protein are clearly visible. By comparing the emission spectrum of Pronectin-coated nanoparticles with that of the free protein (Fig. 2), it is possible to note the presence of series of features, in particular at 341 and at about 317 nm, already present in the spectrum of the observed protein, and at 300 nm, due to the phenylalanine residue de-excitation [50], and not observed in the previous spectrum. This behavior can be due to the protein denaturation caused by corona formation [53][54][55]; in fact, in native proteins the emission of phenylalanine (and of tyrosine) is often quenched, due to its interaction with the peptide chain or energy transfer to tryptophan. Denaturation of proteins frequently involves a conformational change which prevents proper energy transfer.
The occurrence of protein denaturation due to the corona formation can be confirmed by lifetime measurement of AuNS@AXL which give a value of 0.38 ns (see Fig. S5 in SI), that, compared with the value of the free protein (τ=3.0 ns), results reduced [50].
The several difference in the zeta potential value between AuNS@CTAC (22 mV) and AuNS@AXL (-17 mV) and DLS measurement (see Fig. S6 in SI), further demonstrate the corona formation.

Micro-Raman spectroscopy
The representative Raman spectra of the AXL protein water solution are shown in Fig. 7.
The Raman spectra show vibrational features attributable to the presence of different amino acids. The Raman band at 369 cm −1 is assigned to the bending out of plane of the S-H bond in L-Cysteine (L-Cys). The presence of the S-S bridges between cysteine residues can be confirmed by the bands located at 452 cm −1 and 531 cm −1 [56]. These bands describe the stretching mode of the S-S bond of the C α C β S-S'C β C α ' moiety. In particular, the gauchegauche-gauche conformer (GGG) of the S-S bond is confirmed by the band at 452 cm −1 , while the conformer gauche-gauche-trans (GGT) produced the band at 531 cm −1 . The S-S bridges between oxidized cysteines stabilize the secondary structures of the protein, which is, in this sample, α-helical as confirmed by the Raman feature at 938 cm −1 .
In the spectrum of Fig. 7b there are not Raman features detectable. It is quite amazing that the usual modes of protein due to the presence of the peptide bond denominated Amide I, Amide II and Amide III fall in the ranges at 1640−1678, 1520−1570 and 1230− 1270 cm −1 , respectively, are not detected [60].
The free S-H stretching mode of the cysteine amino acid appears in the region between 2400 and 2700 cm −1 as broadband as shown in Fig. 7c [61].
The stretching of the O-H bond in the water occurs as broadband in the region between 3200 and 3600 cm −1 and is shown in the Raman spectrum of Fig. 7d.
The representative Raman spectrum of AuNS@C-TAC in water solution is shown in Fig. 8, while in Fig.  S7 SI, is reported the Raman spectrum of CTAC in water.
Because, in our knowledge, there are no previous works on the Raman characterization of the vibrational modes of the CTAC deposited on gold nanosphere, a tentative attribution of the Raman modes to the AuNS@CTAC water solution was done according to the previous literature [65] ascribed to the similar quaternary ammonium surfactant, cetyltrimethylammonium bromide (CTAB).
As shown in Fig. 8 (spectrum a) the bands at 379 and 455 cm −1 are assigned to the deformation of the C 4 N ? group. The stretching of the bond C-N ? is confirmed by the bands at 761 and 965 cm −1 . The peaks at 1033, 1062, 1124 and 1143 cm −1 correspond to the stretching of the C-C bonds and the peak at 1231 cm −1 , together with the peak at 1298 cm −1 are attributed to the CH 2 wagging modes. In Fig. 8  (spectrum b), the scissoring mode of the CH 2 groups is found at 1445 cm −1 . The peaks at 1500, 1538, 1559, 1580 and 1636 cm −1 are ascribed to the CH 3 deformations modes.
In the spectrum c of Fig. 8, the broadband which falls in the region between 2800 and 3000 cm −1 is due to the combination of the CH 2 symmetric stretching modes and the terminal-CH 3 asymmetric stretching modes. As shown in Fig. 8.d the bands around 3233 and 3440 cm −1 are ascribed to O-H stretching modes of the water.
The representative Raman spectrum of AuN-S@AXL in water solution is shown in Fig. 9. As shown in Fig. 9a the peak at 366 cm −1 is attributed to the out-of-plane S−H bending mode of the L-Cys residue. The S-S stretching mode of the gauchegauche-trans conformer is still present at 534 cm −1 , while the peak ascribed to the S-S stretching mode of the GGG conformer is absent. This result is quite interesting because as already confirmed, the variation of the S−S bond seems to indicate that the adsorption of protein on the surface of gold nanoparticles has been obtained.
The band at 706 cm −1 is due to the C−S stretching of the trans conformer. In this spectrum, some of the main Raman features ascribed to the aromatic amino acid residues of the AXL protein are observed. The bands at 1001, and 1029 cm −1 are attributed to the Phe amino acid as above. The others bands at 595, 758, 863, 880, 1017, 1232 and 1316 cm −1 , are assigned to the L-Trp and the bands at 706, 844, 863 and 1176 cm −1 , are due to the presence of Tyr. The bands that fall at 403 and 496 cm −1 are assigned to glycine while the bands at 477 and 676 cm −1 are due to L-Glutamine and L-Glutamic acid, respectively.
The spectrum of Fig. 9b shows the following Raman features: 1516, 1531, 1552, 1560, 1576 and 1618 cm −1 . Such bands are ascribed to the modes Amide I and Amide II, which are related to the peptide bond that, most probably, become detectable, because surface-enhanced Raman spectroscopy (SERS) effect: the gold surface enhances such modes. The band at 1531 cm −1 correspond to the bending of O-H asym stretch [59] Nt and Ct refer to the atoms belonging to the terminal groups W=water, ω=out-of-plane bending, τ=torsional internal coordinate the N-H bond in Cys [66]. In addition, such modes are ascribed to the tryptophan and tyrosine amino acid units of the protein [57]. Very interesting is the intensity decreasing of the bands in the range between 2500 and 2700 cm −1 due to the presence of S−H groups (Fig. 9c). This behavior indicates that the AXL molecules are bonded by the sulfur atoms to the gold atoms surfaces in a bond: AXL−S−Au. The cleavage of the S−S bond in the GGG conformer confirmed the presence of bonds AXL−S−Au [62].
The bands associated with the O-H stretching modes of water at 3233 and 3440 cm −1 are shown in Fig. 9d.

Conclusions
Pronectins represent a next generation of protein therapeutics that can overcome some clinical limitations of therapeutic MAbs, due to their complex structure (that implies intensive and costly preparation process and thermal instability), and their large size (that implies a limited ability to penetrate and accumulate in tissues). However, one major disadvantage of smaller protein scaffolds is their rapid renal clearance and correspondingly short circulating serum half-lives: typically, proteins smaller than 50-60 kDa have serum half-lives of less than one hour. To overcome these drawbacks, we used a special Pronectin with a Cys termination (AXL) to coat a gold nanoparticle in order to effectively form a protein corona around it. Since the coating of a gold nanoparticle is known to influence the formation of the protein corona, we first coated two gold nanosphere samples, of identical size (30 nm), with the well-known protein human serum albumin, one coated with CTAC, i.e., a soft coating agent, the other with PEG-SH, i.e., a coating agent covalently and firmly bonded to the surface of the nanoparticle. The spectroscopic results confirmed that CTAC-coated nanoparticles form a protein corona more effectively than those coated with PEG-SH; therefore, AXL was reacted with the CTAC-coated nanoparticles. The samples obtained were characterized by Raman spectroscopy and electron microscopy, and the plasmonic properties, due to the gold core, and the luminescence, attributed to the protein, were measured, showing how the formation of the protein corona induces some form of denaturation of the proteins themselves, observed for both albumin and Pronectin.
This new plasmonic nanocarrier is interesting from a double point of view: a) it allows to increase the half-life of the Pronectin, thanks to the increased size of the protein/nanoparticle system; b) by using the properties of Pronectin as an agent toward specific cellular targets, it is possible to convey into the cells nanoplatforms with phototherapeutic properties, such as photodynamic and photothermal.

Funding
Open access funding provided by Università della Calabria within the CRUI-CARE Agreement. The authors are grateful to NLHT-Nanoscience Laboratory for Human Technologies POR Calabria FESR-FSE 14/20, and to "Prog etto STAR 2-PIR01_00008"-Ministero dell'Università e Ricerca/Italian Ministry of University and Research.

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