Silk fibroin (SF) is the major component of the Bombyx mori cocoons (70% ca) and readily available in nature. Its peculiar structural, mechanical and biological properties make it a very attractive biopolymer (Omenetto and Kaplan, 2010; Ling et al., 2018) which offers unlimited opportunities for functionalization, processing and biological modifications (Rockwood et al., 2011; Reddy, 2020). Due to its biocompatibility and biodegradability, SF is an excellent biomaterial for a wide range of biomedical applications, for example drug delivery, tissue engineering, including cartilage and bone, skin tissue, ligaments, cornea, tympanic membrane, and implantable devices such as artificial kidney, and vascular grafts wound dressings. (Lawrence et al., 2008; Kundu et al., 2013; Koh et al., 2015; Melke et al., 2016). It can be processed from aqueous solutions and converted into a variety of versatile systems (Qi et al., 2017; Huang et al., 2018; Guo et al., 2020), such as hydrogels sponges, foams, solid matrices, ultra-resistant, optically transparent and flexible films among others. Moreover, doping with various organic and inorganic dopants opens up the way to a wide variety of applications ranging from supported catalysts (Rizzo et al., 2020a) to environmentally sustainable devices for photonics, electronics, and optoelectronics (Tao et al., 2012; Zhu et al., 2016). In fact, SF exhibits a moderate fluorescence (it can be excited at 277 nm and emits light at 345 nm) (Georgakoudi et al., 2007) that has been ascribed to the aromatic moieties of Tryptophan and Tyrosine, commonly used, together with Phenylalanine, as fluorescent probes to study the structure of proteins. SF fluorescence can be enhanced by the incorporation of organic molecules, ions and nanoparticles within the fibers, and the possibility of doping with lanthanide (Ln) ions opens new possibilities for photonic systems (Lee et al., 2020).

The use of Ln-doped fibers in photonics is well-known, and several studies have reported that the presence of lanthanides can confer photochromic, luminescent and fluorescent properties as well as up-conversion ability to fibers. Examples could be Eu⁺³ or Dy⁺³ strontium aluminate luminescent fibers produced by a special spinning technology, with the substrate of polyester, nylon, or polypropylene resin (Guo et al., 2011), fluorescent Pr⁺³ and Dy⁺³-doped Chalcogenide fibers (Chahal et al., 2016), Er⁺³/Yb⁺³/Tm⁺³ tri-doped tellurite glass microsphere coupled by tapered fibers (Liu et al., 2020), fluorescent silks obtained from silkworms fed with rare-earth upconverting phosphors (Zheng et al., 2018). Moreover, Ln-doped fibers have been employed in the production of lasers (amplifiers rare-earth-doped silica and fluorozirconate fibers, particularly erbium-doped fibers) (Digonnet, 2001) and smart Dy⁺³ and Eu⁺³ doped stimuli-responsive textiles (Shen et al., 2021). In addition, Tryptophan and Tyrosine can act as sensitizers for lanthanide ions enhancing their emission (da Rocha et al., 2020). Therefore, combining the mechanical and optical properties of SF with those of lanthanides ions could be an interesting approach for the development of new photonic systems, particularly appealing due to the high biocompatibility and biodegradability of SF.

Few examples of SF films and powders doped with lanthanides have been reported so far and are limited to Eu⁺³ and Tb⁺³ (Pugina et al., 2019a,b; da Rocha et al., 2020). In a previous work (Rizzo et al., 2020b) we have described a simple method for doping fibrous SF with Ce⁺³. In particular, in the framework of a systematic study on the effects of changing the chaotropic salt in Ajisawa’s solvent (CaCl₂ in H₂O/EtOH in a molar ratio 1:8:2), used as standard protocol to solubilize SF (Ajisawa, 1998), we found that when replacing CaCl₂ with the hydrated lanthanide salt CeCl₃⋅7H₂O while maintaining all the other conditions unchanged, not only SF was completely dissolved, but it reprecipitated in a fibrous form very quickly and spontaneously during the dialysis purification. The structural characterization of the resulting material demonstrated that the fibers obtained preserved most of the morphological and structural properties of the original degummed SF (Marsh et al., 1955; Guo et al., 2018), with a highly ordered molecular organization, and that they were doped with Ce⁺³ ions, in a concentration of about 0.3 wt%, distributed both within its amorphous regions and the β-crystalline domains.

This protocol represents the first method to obtain SF in a fibrous form after solubilization (the fibroin is normally re-obtained as aqueous solutions or gels). In addition, the SF obtained is doped with Ce⁺³ ions. In principle, this simple process could be extended to other lanthanide ions, possibly resulting in a more generally applicable protocol to produce new silk-based doped materials.

Here we report the results of a systematic investigation of the behavior of degummed SF solution in the presence of all lanthanide ions in the Periodic Table, from Lanthanum to Lutetium, in the form of chloride salts, with the only exclusion of Promethium, due to its radioactivity. The main goals of our paper are: (i) disclosing a new protocol to obtain doped fibroin in fibrous form after solubilization in the presence of Ln⁺³ ions and (ii) characterizing structural and morphological properties of the fibrous materials obtained.

All hydrated lanthanide chlorides were purchased from NOVA ELEMENTS SAS (Palermo, Italy). All other reagents and solvents were purchased from Sigma Aldrich and were used without any further purification.

According to Ajisawa’s standard method (Ajisawa, 1998), hydrated LnCl₃ in H₂O/EtOH (in a molar ratio 1:8:2), were used to dissolve degummed SF. The mixtures were heated at 60°C for 4 h. The resulting SF solutions were dialyzed against bidistilled water to remove the residual salt and ethanol. Regenerated fibers were rinsed several times with water and let air-dry. Solutions and hydrogel were stored at 4°C.

All lanthanides chlorides in H₂O/EtOH were able to dissolve degummed SF (Figure 1). As already found for CeCl₃⋅7H₂O, when using hydrated LaCl₃, PrCl₃, ErCl₃, TmCl₃, YbCl₃, and LuCl₃ as chaotropic salts, a fibrous material reprecipitated within the dialysis cassette (Table 1). In all these cases the recovery of the fiber was almost quantitative. With DyCl₃ the recovery of the fiber was only partial (48–51%) whilst with HoCl₃ a hydrogel was obtained. Intermediate lanthanides of the series (NdCl₃, SmCl₃, EuCl₃, GdCl₃, and TbCl₃) did not induce precipitation of a regenerated fibrous material leading to, at the end of the purification process, clear and stable solutions that could be used to obtain films and/or other scaffolds.

The structural and morphological properties of the solid fibers recovered after treatment with La⁺³, Pr⁺³, Er⁺³, Tm⁺³, Yb⁺³, and Lu⁺³ salts (hereafter indicated in the text as Ln⁺³/SF) in H₂O/EtOH were analyzed. Further, drop-casted films from SF solutions obtained by Nd⁺³, Sm⁺³, Eu⁺³, Gd⁺³, and Te⁺³ solution treatment were characterized by ATR-FTIR. All these experiments showed that the material recovered is, in fact, SF.

The screening analysis of the behavior of SF solubilized by substituting the chaotropic salt CaCl₂ with hydrated lanthanides chlorides, LnCl₃⋅nH₂O, in the standard Ajisawa’s method led to some very interesting results. First of all, all LnCl₃⋅nH₂O are able to solubilize SF when used as chaotropic salts, but lanthanides at the beginning and at the end of the series of the Periodic Table (La⁺³, Ce⁺³, Pr⁺³, Dy⁺³, Er⁺³, Tm⁺³, Yb⁺³, and Lu⁺³) are able to induce the regeneration of the fiber during purification by dialysis. When using intermediate lanthanides (Nd⁺³, Pr⁺³, Sm⁺³, Eu⁺³, Gd⁺³, and Tb⁺³) aqueous solutions of SF are obtained, as in the standard Ajisawa’s method. Dy⁺³ and Ho⁺³ can be considered borderline cases: using Dy⁺³ only 45% of the fiber is recovered during dialysis and the remaining part is in the aqueous solution, Ho⁺³ induces the formation of a SF hydrogel.

The fibers reprecipitated from Pr⁺³, Er⁺³, Tm⁺³, Yb⁺³, and Lu⁺³ solutions in H₂O/EtOH keep a morphology very similar to that of degummed SF and Ce⁺³/SF: their integrity is almost recovered, although with a reduced smoothness and straightness. The typical hemicylindrical shape of the cross-section of the fibers is also retained, with an increase in the average diameter, probably due to swelling effects. Fibers obtained after treatment with La⁺³ and Dy⁺³ are constituted by small and disordered fragments with very coarse surfaces and irregular cross sections.

WAXS experiments, confirmed by ATR-FTIR and birefringence, demonstrated that in all cases the samples belonging to the SF-fiber regenerating lanthanides group retain an ordered crystalline structure, but they show two different behaviors:

(i) the fibers obtained with Pr⁺³ (at the beginning of the lanthanide series, as Ce⁺³) and Tm⁺³, Yb⁺³, Lu⁺³ (at the end of the series) exhibit 2D WAXS patterns similar to degummed SF and Ce⁺³/SF, with a typical cross β-diffraction pattern, thus indicating that, during reprecipitation, the fibers partially recover a highly oriented structure, rich of stacked β-sheet nanocrystals;

(ii) the fibers obtained with La⁺³, Dy⁺³, and Er⁺³ show 2D WAXS patterns similar to natural silk cocoon, i.e., without any preferential orientation.

Anyway, WAXS experiments show no sign of inclusion of lanthanide ions within the crystalline domains of the fibers, contrary to what was found in the case of Ce⁺³/SF.

XPS characterization indicates that the fibers with La⁺³, Tm⁺³, and Lu⁺³ do not show any relevant contamination of lanthanide ions, whilst in all other fibers the Ln⁺³ concentration is between 0.2 and 0.4% at, but, except for Ce⁺³, Ln⁺³ ions are not coordinated to SF. This evidence could suggest that lanthanide ions could be present as impurities, likely on the fiber surface.

In summary, only when using CeCl₃⋅7H₂O as chaotropic salt in the Ajisawa’s method it is possible to obtain SF doped by Ce⁺³ in a significant amount. In all other cases, SF is regenerated, but it is not doped with Ln⁺³ ions and, in some cases, it has different structural properties. Regarding intermediate lanthanide ions, it is interesting to point out that only SF aqueous solutions can be obtained at the end of the Ajisawa’s process and the features of films prepared by solvent evaporation are very similar to the films obtained when using CaCl₂.

As already stressed when discussing the results obtained in our previous work on Ce⁺³/SF (Rizzo et al., 2020b), this behavior is difficult to rationalize, firstly because silk solubilization and recovery are complex processes, driven by chemical and physical interactions that are not completely understood to date. It is known that SF solubilization is possible only in the presence of chaotropic salts that seemingly disrupt the hydrogen bonding network between the hydration shell of the protein and the bulk, so weakening the hydrophobic effects. However, the roles of different chaotropic salts and especially of solvent molecules have not been clarified yet.

In this context, it is necessary to consider that when using lanthanides as solubilizing agents, the situation is much more complicated due to the particular properties of lanthanides. Lanthanides series consist of 15 elements, from lanthanum (Z = 57) to lutetium (Z = 71), with high coordination numbers and characteristic oxidation state +3 (Zheng, 2012; Peters et al., 2020). It is well-known that the interactions between Ln⁺³ cations and ligands are mainly of electrostatic origin, since the formation of covalent bonds with donor atoms of ligand is virtually impossible. Due also to the absence of directionality of bonding electrons, the coordination number and the stereochemistry of Ln⁺³-complexes are dictated by the steric requirements of the ligands and not by electronic effects. In addition, the so-called “lanthanide contraction” (Peters et al., 2020), which is a unique characteristic of lanthanide metals, must be taken into account: Ln⁺³ ionic radium decreases with increasing atomic number, that is with increasing charge density. Lanthanide contraction further affects the capability of lanthanide ions to form stable complexes. Ln coordination numbers in the range 3–12 have been observed and with 7, 8, and 9 as the most common ones. Lanthanides ions with larger ionic radii can, in principle, have larger coordination numbers. On the other hand, the decrease of ionic radium with the consequent increase of charge density increases the tendency to form more stable complexes. In any case, due to their electrostatic nature, Ln⁺³-complexes are quite labile and the coordination sphere is not well-defined, since the coordination number is primarily affected by the size of the ligands, the number of donors of each ligand that can pack around the ion and, in the case of bulky ligands, by intermolecular interactions. Because of the small radius and the high nuclear charge, the Ln⁺³ ions are strong Lewis acids that coordinate with hard bases such as carboxylates and highly electronegative donors such as N or O (Zheng, 2012), that is to say that Ln⁺³ ions are good complexing agents for amino acids and peptide bonds in proteins.

In our previous paper (Rizzo et al., 2020b), comparing the behavior of SF in the presence of Ce⁺³ ions to the results reported in the literature on the influence of other metal ions on SF, it seemed reasonable to infer that an important role is played by Ce⁺³ charge and coordination number. The results obtained for the complete series of lanthanides are consistent with this hypothesis.

The behavior of SF when Ln⁺³ ions in the middle of the lanthanide series (Nd⁺³, Pr⁺³, Sm⁺³, Eu⁺³, Gd⁺³, and Tb⁺³) are used as chaotropic salts is similar to the behavior observed in the presence of Ca⁺² ions (standard Ajisawa’s method). This can be explained as an effect of the ionic radius. In fact, as it can be seen in Table 1, the ionic radii of Ln⁺³ from Nd⁺³ to Tb⁺³, are within 1.08 and 1.01 Å, comparable to the ionic radius of Ca⁺² (1.03 Å).

For lanthanides at the beginning and at the end of the series, the Ln⁺³ coordination number seems to play a key role, besides the ionic radius. During the purification step after solubilization, the removal of Ln⁺³ ions, as already found for Ce⁺³, seems to promote the self-assembly of the fiber in the transition from solution to solid state, with the fibers undergoing a structural transition from random coil to a highly ordered structure.

Ce⁺³ ion has ionic radius slightly larger than that of Ca⁺², but also a larger charge and coordination number. Pr⁺³ has an ionic radius slightly lower than Ce⁺³, and presumably a lower coordination number. Hence, the fibers obtained at the end of the purification process are very similar to Ce⁺³/SF, although not doped by Pr⁺³ ions, that are more easily removed during the dialysis step, due to its lower coordination number.

Ln⁺³ ions at the end of the series, from Er⁺³ to Lu⁺³, have ionic radii that are significantly lower than Ce⁺³. Even if their density charges are larger than Ce⁺³, their coordination number is smaller, considering that the steric hindrance of the environment is the same. This could imply that these ions can easily induce the solubilization of SF with a mechanism very similar to the one hypothesized for Ce⁺³ ions, but can also been removed more easily than Ce⁺³ ions without remaining trapped within the fiber. The SF reprecipitated after dialysis is highly ordered, and richer of stacked β-sheet nano-crystals as the ionic radius decreases.

We have reported the results of a systematic investigation on the behavior of degummed SF when solubilized by replacing CaCl₂ with LnCl₃⋅nH₂O in the standard Ajisawa’s reagent. This study builds on previous work using CeCl₃⋅7H₂O (Rizzo et al., 2020b) and extends to all the other lanthanides in the Periodic Table, given the potential that lanthanide doping of SF holds for the manufacturing of devices for photonics and electronics.

It would seem that Cerium constitutes a unique case for obtaining lanthanide doped SF. Indeed, using CeCl₃ as chaotropic salt in water and ethanol, not only it is possible to regenerate SF in a fibrous form, but the fiber obtained is doped with Ce⁺³ ions and preserves the morphological and molecular structure of the pristine SF. Ce⁺³ ions should be coordinated to SF preferentially by interactions with the oxygen atoms of protein backbone and should be distributed within the highly ordered hydrophobic β-sheet regions of SF, as well as amorphous regions (Rizzo et al., 2020b). For all the other re-precipitated fibers (with the exception of La⁺³/SF, Tm⁺³/SF, and Lu⁺³/SF), a surface concentration of Ln⁺³ between 0.2 and 0.4% at was measured, comparable to that measured for Ce⁺³/SF. However, contrary to what was found in the case of Ce⁺³/SF, there is no evidence that the ions are coordinated with the fiber fibroin, nor is the presence of Ln⁺³ ions detectable within the ordered domains. This could suggest that Ln⁺³ ions could be distributed on the fiber surface, although it is not possible to exclude their inclusion within the amorphous regions.

However, the results obtained for the other lanthanides can be considered interesting for other reasons. Indeed, our work shows that all lanthanides in H₂O/EtOH (8:1) are effective chaotropic salts for SF and that they constitute a valid alternative to CaCl₂ in the Ajisawa’s method. Using hydrated LnCl₃ we have obtained a new protocol which enables to regenerate SF in the form of fibrous material by direct reprecipitation during dialysis.

A further appealing feature of the protocol described is its environmental sustainability, given both the possibility of near-full recovery of the lanthanide salts from water and the absence of toxic solvents or hazardous reagents throughout the whole process (Rizzo et al., 2020b).

Further studies are in progress to investigate the optical properties and applications of the fibers regenerated in the presence of lanthanide solutions and also to optimize possible protocols for a controlled inclusion of lanthanide ions other than Cerium into the fibers.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.