Adducts between a nanocellulose and a pentapeptide and its halogenated derivatives

Abstract

The present invention generally relates to the field of nanocellulose-based nanocomposites. In particular, the present invention relates to adducts between a nanocellulose, in particular a nanofibrillated cellulose, and a peptide of sequence DFNKF, wherein one or both of the phenylalanine residues can optionally be halogenated on the aromatic ring. The present invention further relates to a process for the preparation of said adducts, films based on said adducts and the process for obtaining them, and uses of said adducts and films.

Description

[0001] TITLE

[0002] ADDUCTS BETWEEN A NANOCELLULOSE AND A PENTAPEPTIDE AND ITS HALOGENATED DERIVATIVES

[0003] * * * * * * * * * * * * *

[0004] FIELD OF THE INVENTION

[0005] The present invention relates generally to the field of nanocellulose-based nanocomposites.

[0006] In particular, the present invention relates to adducts between a nanocellulose, in particular a nanofibrillated cellulose, and a peptide of sequence SEQ ID NO: 1 DF*NKF*, wherein one or both of the phenylalanine residues can optionally be halogenated on the aromatic ring. The present invention further relates to a process for the preparation of said adducts, films based on said adducts and the process for obtaining them, and uses of said adducts and films.

[0007] PRIOR ART

[0008] Cellulose is the most abundant renewable and sustainable material on our planet today. The interest for this material does not only lie in its abundance and affordability, but it is also due to its characteristics such as biodegradability, reusability, ease of chemical functionalization, ability to take on different morphologies, as well as its excellent mechanical characteristics, high strength, high modulus and low density that make it a versatile material suitable for a wide range of applications.

[0009] Mechanical and / or chemical treatment of cellulose allows to obtain nanocellulose, a nano-structured cellulose in the form of either cellulose nanocrystal (CNC) or cellulose nanofibers also called nanofibrillated cellulose (CNF), which is of particular technological interest as a renewable and biodegradable biomass for the design of nanoscale functional systems and composite materials. However, the high concentration of hydroxyl groups at the surface of nanocellulose leads to the formation of a large number of hydrogen bonds strongly affecting its reactivity, for example leading to the formation of a dense tangle of fibrils in nanofibrillated cellulose, thus limiting or even preventing its interaction with other matrices / materials. To favour interaction with other materials, research efforts have been focused on the development of covalently functionalized nanocelluloses, through processes such as oxidation, phosphorylation, sulphonation, carbomethylation and silylation. Among the possible derivatives obtainable, TEMPO-oxidized nanocellulose, in which the hydroxyl groups are functionalized with negatively charged carboxylate groups, is the most studied and most widely used to date for a wide range of applications such as in packaging solutions, drug delivery systems, optical materials and energy storage devices. The use of TEMPO-oxidized nanocellulose in combination with other substances, for example of a plastic / metallic nature or of a protein nature, to obtain materials with improved properties is well known in the art.

[0010] CN1 1289226 and CN113019140 disclose protein nanocellulose composite separation membranes, in which TEMPO-oxidized nanocellulose is coupled with an amyloid oligomer, specifically lysozyme. Nanocomposites based on TEMPO-oxidized cellulose and an amyloid protein, the Curli-specific gene A (CsgA) protein, are also disclosed in Khatri, V. et al, Biomacromolecules 2023, 24 (11 ), 5290-5302.

[0011] These systems, however, suffer from numerous drawbacks.

[0012] First of all, the TEMPO-functionalization of nanocellulose is a multi-step, long and costly process requiring the use of solvents and reagents with a strong environmental impact, which can even lead to the deterioration of the nanocellulose by, for example, lowering its polymerization degree.

[0013] Moreover, in addition to the chemical functionalization with TEMPO, these systems make use of large proteins which require further processing before being combined with the nanocellulose, thus further lengthening and increasing the difficulty and cost of the process. Indeed, the use of proteins or high-weight protein derivatives poses limits in terms of versatility and customization, in addition to the difficulties that these large peptide entities present regarding their purification and characterization.

[0014] Nanocellulose has also been widely studied as a base material for films in various applications like food packaging and electronic displays. Nonetheless, these films exhibit a detrimental response when exposed to water or highly humid environments due to the nanocellulose strong hygroscopic nature. Many of the strategies proposed, to date, to tailor nanocellulose surface properties, rely on chemical hydrophobization involving the use of potentially hazardous chemicals and solvents, leading to high processing costs and environmental concerns. As an example, a traditional way to increase nanocellulose barrier properties is to surface coat per- and polyfluorinated substances (PFASs), especially for food packaging purposes. However, the use of PFASs in consumer products has raised significant environmental and health concerns due to their persistence, bioaccumulation, and potential toxicity.

[0015] Therefore, there remains the need for nanocellulose-based systems and materials with a lower environmental impact obtainable through more cost and time efficient processes, maintaining or even improving the rheological performances of the materials obtained.

[0016] SUMMARY OF THE INVENTION

[0017] The Applicant addressed the need for a nanocellulose-based system overcoming, at least in part, the drawbacks described above.

[0018] In particular, the Applicant has tackled the problem of providing nanocellulose based materials endowed with optimized and tunable rheological properties, at the same time reducing the environmental impact and optimizing the efficiency of the processes to obtain these materials.

[0019] The Applicant has long been studying the DFNKF sequence pentapeptide and its halogenated derivatives (SEQ ID NO: 1 ; see e.g. Bertolani, A. et al., Nature communications 2015, 6, 1 -9). The DFNKF pentapeptide (SEQ ID NO: 1 ) is a fragment of the human calcitonin protein, the mutation of which may lead to the formation of insoluble fibril aggregates typical of several diseases. In addition to the implications in the human field, the amyloidogenic power of the DFNKF peptide (SEQ ID NO: 1 ) has been investigated with a view to its use in the field of nanomaterials; the effects of its halogenation have also been investigated and led for example to super-gelling peptides, resulting in the formation of stronger and more resistant hydrogels compared to the wild-type peptide. After extensive experimentation, the Applicant surprisingly found that it is possible to obtain stable non-covalent adducts between a nanocellulose, in particular a nanofibrillated cellulose, and a DFNKF peptide (SEQ ID NO: 1 ), forming strong and stable hydrogels with excellent rheological properties, without the need for prior chemical functionalization of the nanocellulose. The Applicant further surprisingly found that it is possible to modulate the intrinsic characteristics of the nanocellulose-peptide adducts according to the present invention, and consequently their rheological properties, by introducing halogen atoms in key positions of said peptides.

[0020] Therefore, in a first aspect the present invention relates to an adduct between a nanocellulose, in particular a nanofibrillated cellulose, and a peptide of sequence SEQ ID NO: 1 : DF*NKF*, wherein each of the phenylalanine (F*) residues, independently from one another, can be optionally substituted on the aromatic ring with from 1 to 5 halogen atoms selected from fluorine, iodine, chlorine, bromine, and mixtures thereof.

[0021] In a second aspect, the present invention relates to a process for the preparation of an adduct between a nanocellulose and a DF*NKF* peptide (SEQ ID NO: 1 ) as defined above, comprising the following steps: a) providing an aqueous suspension of a nanocellulose; b) providing an aqueous solution of at least one peptide of sequence SEQ ID NO: 1 DF*NKF* as defined above; c) adding the aqueous solution obtained in step b) to the aqueous suspension obtained in step a); and d) agitating and then sonicating the mixture obtained in step c) until formation of an adduct.

[0022] In a third and in a fourth aspect, the present invention relates to a film based on an adduct between a nanocellulose and a peptide of sequence SEQ ID NO: 1 DF*NKF* as defined above, and to a process to obtain said film.

[0023] Finally, in a fifth aspect the present invention relates to the use of the adducts, or the films, as defined above as a coating, as a non-woven textile material, as a packaging material, or as a separation and / or filtration membrane. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The description is disclosed herein with reference to the accompanying drawings, provided solely by way of a non-limiting example of the invention.

[0025] Figure 1 shows Transmission Electron Microscopy (TEM) images of a 0.8wt% CNF hydrogel (panel A) and of a hydrogel of an adduct according to the present invention (CNF-DF(l)NKF(0.05wt%); panel B).

[0026] Figure 2 shows the IR spectra of adducts according to the present invention (CNF-DFNKF(0.05wt%) panel a, CNF-DF(l)NKF(0.05wt%) panel b, CNF- DF(F5)NKF(0.05wt%) panel c), in comparison with that of pure nanofibrillated cellulose (CNF) and the reference peptide alone.

[0027] Figure 3 shows the N-H stretching region in the FTIR spectra of adducts according to the present invention (CNF-DFNKF(0.05wt%) panel a, CNF- DF(l)NKF(0.05wt%) panel b, CNF-DF(F5)NKF (0.05wt%) panel c), after subtracting the contribute of neat nanofibrillated cellulose, in comparison with the spectra of the respective peptides alone.

[0028] Figure 4 shows scanning electron microscopy (SEM) images of nanofibrillated cellulose (panel A) and of an adduct according to the present invention (CNF-DF(l)NKF(0.05wt%), panel B). Also shown are the energy dispersive X-ray spectroscopy (EDS) maps of the corresponding SEM images of exemplary adducts according to the invention (CNF-DF(l)NKF(0.05wt%) panel C, CNF-DF(F5)NKF(0.05wt%) panel D).

[0029] Figure 5 shows Scanning Electron Microscopy (SEM) images of a nanofibrillated cellulose based film (panel a), and of a CNF- DF(Fs)NKF based film according to the invention (panel b) and its corresponding fluorine elemental map (panel c).

[0030] Figure 6 shows the water uptake values after 24 hours immersion (panel A) and the water vapor transmission rates (WVTR) values (panel B) for a nanofibrillated cellulose-based film (label: CNF) and of a CNF-DF(Fs)NK based film according to the present invention (label: CNF-Fs).

[0031] Figure 7 shows the WTR value of a CNF-DF(Fs)NK based film according to the present invention compared to the WVTR values of films based on neat nanofibrillated cellulose and other industrially relevant cellulose derivatives (Labels: CA / OLA = Cellulose acetate-oleic acid; CAP= Cellulose acetate propionate; CE = Cellulose esters; CNF-F5 = CNF-DF(Fs)NK adduct according to the present invention; W+C / AC = Wax + Cellulose-aleuritic acid; CA / Ac = Cellulose acetate / Acetone; CA = Cellulose acetate; C / CA = Cellulose-aleuritic acid; CA / TFA = Cellulose acetate / TFA; CNF = nanofibrillated cellulose).

[0032] DETAILED DESCRIPTION OF THE INVENTION

[0033] Definitions

[0034] According to the present invention, the term “adduct” is understood to mean a stable composite obtained by combining two or more components; more specifically, the term adduct indicates composites whose components, bound by means of intermolecular interactions, retain their individuality in some ways. In particular according to the present invention, the term “adduct(s)” refer to an adduct obtained from the interaction, via noncovalent bonds, between a nanocellulose and a peptide of sequence SEQ ID NO: 1 DF*NKF*.

[0035] According to the present invention, the term “nanocellulose” is intended to mean a nanostructured material obtained from cellulose, having at least one of the three-dimensional sizes in the nano-region (1 -100 nm).

[0036] According to the present invention, the term “nanofibrillated cellulose” or “CNF” is intended to mean a type of nanocellulose comprising a bundle of cellulose nanofibers, in which the nanofibers are entangled with each other but flexible. More specifically, nanofibrillated cellulose comprises long, soft nanosized cellulose fibrils with a high aspect ratio (length to width ratio), typically with a diameter (or width) of 5-20 nanometers and a wide range of lengths, typically in the micrometer-scale (few micrometers).

[0037] According to the present invention, the term “hydrogel(s)” is intended to mean a three-dimensional hydrophilic network formed by the assembly of small molecules or macromolecular polymers, capable of absorbing large amounts of water despite being insoluble. In particular, in the context of the present specification and the appended claims, the term “hydrogel(s)” refers to a gelled material consisting essentially of water and at least one of the adducts according to the present invention.

[0038] Detailed description In a first aspect the present invention relates to an adduct between a nanocellulose and a peptide of sequence SEQ ID NO: 1 DF*NKF*, wherein each of the phenylalanine (F*) residues, independently from one another, can be optionally substituted on the aromatic ring with from 1 to 5 halogen atoms selected from fluorine, iodine, bromine, chlorine atoms, and mixtures thereof. Preferably, said one or more halogen atoms are selected from fluorine and iodine.

[0039] Preferably, said nanocellulose is a nanofibrillated cellulose (CNF).

[0040] Nanofibrillated cellulose (CNF) is a material composed of nanosized cellulose fibrils with a high aspect ratio (length to width ratio). Typical fibril widths are 5-20 nanometers with a wide range of lengths, typically several micrometers. It is pseudo-plastic and exhibits thixotropy, the property of certain gels or fluids that are thick (viscous) under normal conditions but become less viscous when shaken or agitated. When the shearing forces are removed the gel regains much of its original state. The fibrils can be isolated from any cellulose containing source including wood-based fibers (pulp fibers) through high-pressure, high temperature and high velocity impact homogenization, grinding or microfluidization.

[0041] In embodiments of the present invention, said peptide is a peptide of sequence SEQ ID NO: 2 DF*NKF, wherein the phenylalanine (F*) residue in position 2 can be optionally substituted on the aromatic ring with from 1 to 5 halogen atoms selected from fluorine, iodine, bromine, chlorine atoms, and mixtures thereof. Preferably, said one or more halogen atoms are selected from fluorine and iodine.

[0042] In embodiments of the present invention, said phenylalanine (F*) residue in position 2 can be optionally substituted on the aromatic ring with one halogen atom selected from an iodine, a chlorine, a bromine and a fluorine atom. Preferably, said halogen atom is a fluorine, a bromine or an iodine atom. Preferably, said halogen atom is in the para-position.

[0043] In embodiments, said phenylalanine (F*) residue in position 2 can be optionally substituted on the aromatic ring with two halogen atoms selected from iodine, chlorine, bromine, fluorine atoms, and mixtures thereof. Preferably, said halogen atoms are selected from fluorine and bromine atoms, and mixtures thereof. Even more preferably, said halogen atoms are both fluorine atoms or both bromine atoms. Preferably, said halogen atoms are in positions 3 and 5 of the aromatic ring.

[0044] In embodiments, said phenylalanine (F*) residue in position 2 can be optionally substituted on the aromatic ring with three, four or five halogen atoms selected from iodine, chlorine, bromine, fluorine atoms, and mixtures thereof. Preferably, said halogen atoms are selected from fluorine and iodine atoms, and mixtures thereof. Even more preferably, said halogen atoms are all fluorine atoms.

[0045] In particularly preferred embodiments of the present invention, said peptide is selected from:

[0046] • a peptide of sequence DFNKF (SEQ ID NO: 1 ),

[0047] • a peptide of sequence DF*NKF (SEQ ID NO: 2) wherein the phenylalanine (F*) residue in position 2 is substituted on the aromatic ring in the para-position with an iodine atom,

[0048] • a peptide of sequence DF*NKF (SEQ ID NO: 2) wherein the phenylalanine (F*) residue in position 2 is substituted on the aromatic ring in the para-position with a fluorine atom,

[0049] • a peptide of sequence DF*NKF (SEQ ID NO: 2) wherein the phenylalanine (F*) residue in position 2 is substituted on the aromatic ring in the para-position with a bromine atom,

[0050] • a peptide of sequence DF*NKF (SEQ ID NO: 2) wherein the phenylalanine (F*) residue in position 2 is substituted on the aromatic ring with two fluorine atoms in positions 3 and 5,

[0051] • a peptide of sequence DF*NKF (SEQ ID NO: 2) wherein the phenylalanine (F*) residue in position 2 is substituted on the aromatic ring with two bromine atoms in positions 3 and 5, and

[0052] • a peptide of sequence DF*NKF (SEQ ID NO: 2) wherein the phenylalanine (F*) residue in position 2 is substituted on the aromatic ring with five fluorine atoms.

[0053] Even more preferably, said peptide is selected from: • a peptide of sequence DFNKF (SEQ ID NO: 1 ),

[0054] • a peptide of sequence DF*NKF (SEQ ID NO: 2) wherein the phenylalanine (F*) residue in position 2 is substituted on the aromatic ring in the para-position with an iodine atom, and

[0055] • a peptide of sequence DF*NKF (SEQ ID NO: 2) wherein the phenylalanine (F*) residue in position 2 is substituted on the aromatic ring with five fluorine atoms.

[0056] Typically, in the adducts according to the present invention, the weight ratio between said nanocellulose and said peptide is of from 2:1 to 100:1 , preferably of from 8:1 to 80:1. In embodiments, the weight ratio between said nanocellulose and said peptide can for example be of about 4:1 , 8:1 , 10:1 , 10.7:1 , 16:1 , 24:1 , 32:1 , 40:1 , 64:1 , 80:1 , 90:1 , etc..

[0057] Typically, the adducts according to the present invention are in the form of a hydrogel. Typically, the hydrogels essentially consist of water and at least one of the adducts according to the present invention, meaning that they do not comprise any other solvent, but for traces amounts. Preferably, the water is ultrapure water, such as milliQ water.

[0058] Preferably, the concentration of the nanocellulose in said hydrogels is of from 0.2 wt% to 2 wt%, preferably of from 0.4 wt% to 1 wt%, even more preferably is of about 0.8 wt%.

[0059] Preferably, the concentration of the peptide in said hydrogels is of from 0.002 wt% to 1 wt%, preferably of from 0.008 wt% to 0.4 wt%, even more preferably of from 0.01 wt% to 0.1 wt%. In embodiments, the concentration of the peptide in said hydrogels is for example of about 0.008 wt%, 0.01 wt%, 0.025 wt%, 0.05 wt%, 0.075 wt%, 0.1 wt%, 0.2 wt%, 0.4 wt%, 0.8 wt%, etc..

[0060] In a second aspect, the present invention relates to a process for the preparation of an adduct between a nanocellulose and a DF*NKF* peptide (SEQ ID NO: 1 ) as defined above, comprising the following steps: a) providing an aqueous suspension of a nanocellulose; b) providing an aqueous solution of at least one peptide of sequence DF*NKF* (SEQ ID NO: 1 ) as defined above; c) adding the aqueous solution obtained in step b) to the aqueous suspension obtained in step a); and d) agitating and then sonicating the mixture obtained in step c) until formation of an adduct.

[0061] Typically, step b) of the process according to the present invention is carried out under heating, for example at 70-100°C, and under sonication, for example using an ultrasound bath, until complete dissolution of the peptide.

[0062] Advantageously, in step d) the mixture can be agitated using any conventional instrument, such as for example a benchtop vortex.

[0063] Typically, the sonication in step d) of the process according to the present invention can be carried out using any sonicator instrument known in the art, preferably a tip sonicator is used.

[0064] Preferably, the sonication in step d) of the process according to the present invention is carried out at 100-400 W, thus providing the system with a total energy of about 5-30 kJ. Preferably, step d) is carried out for a time from 0.5 to 2 minutes.

[0065] Without being bound to any theory, it is believed that the lysine residue of the peptides of sequence DF*NKF* (SEQ ID NO: 1 ) defined above can promote the formation of supramolecular, non-covalent interactions with the nanocellulose, leading to the formation of the nanocellulose-peptide adducts according to the present invention. Moreover, the different variants of the peptide of sequence DF*NKF* (SEQ ID NO: 1 ) disclosed above possess different hydrophobicity depending on the halogenated moieties introduced on the phenylalanine residues, thus adducts according to the present invention provide a straightforward and sustainable way to enhance and tune hydrophobicity of nanocellulose. Finally, by avoiding covalent functionalization of nanocellulose with hydrophobic groups, the inherent properties of cellulose are mostly preserved, including its biodegradability.

[0066] In a third aspect, the present invention relates to a film based on an adduct between a nanocellulose and a peptide of sequence SEQ ID NO: 1 DF*NKF* as defined above.

[0067] In a fourth aspect, the present invention relates to a process for the preparation of a film based on an adduct between a nanocellulose and a peptide of sequence SEQ ID NO: 1 DF*NKF* as defined above, said process comprising the steps of: i. providing a hydrogel comprising an adduct between a nanocellulose and a peptide of sequence SEQ ID NO: 1 DF*NKF* as defined above; and ii. filtering under vacuum said hydrogel.

[0068] Typically, said step ii. is carried out at a pressure of about 20 mbar for about 30-60 minutes.

[0069] Finally, in a fifth aspect the present invention relates to the use of the adducts, or the films, according to the present invention, as a coating, as a non-woven textile material, as a packaging material, or as a separation and / or filtration membrane.

[0070] The adducts according to the present invention are endowed with excellent rheological properties, e.g. a more than tenfold increase in both dynamic moduli with respect to pristine nanocellulose, thus indicating high mechanical strength and stability. Moreover, the films obtained from the adducts according to the present invention exhibit increased hydrophobicity and affinity to oils with respect to those based on pristine nanocellulose, as well as enhanced hydrodynamic properties, i.e. reduced water uptake capacity and excellent water-vapor transmission rate (WVTR) values, thus being endowed with high resistance to moisture while at the same time showing good oxygen permeation.

[0071] These features render the adducts and the films according to the present invention particularly suitable for use as coatings, for example being applied on surfaces such as walls, medical devices, surfaces for food preparation, and the like; non-woven textile materials, for example as wound-dressing material, where breathability and water resistance are required to obtain the ideal environment for proper healing; packaging materials, e.g. for food and pharmaceuticals, where the use of natural materials with effective water vapor barrier properties is highly desired as moisture can impair and accelerate product degradation, as well as separation and / or filtering membranes.

[0072] EXPERIMENTAL SECTION The present invention will be better illustrated in the following examples, which are for illustrative and non-limiting purpose only.

[0073] Materials and Methods

[0074] Nanofibrillated cellulose (CNF) was provided by VTT Technical Research Centre (Espoo, Finland) and obtained from bleached birch pulp, which has been Na-washed and fluidized six times to obtain nanofibrillated cellulose. The final CNF aqueous suspension was characterized by a dry matter content of 1 .6 wt%.

[0075] The amino acids used to synthetize the peptides according to the present invention were purchased from Fluorochem or TCI.

[0076] Electro-spray ionization mass spectrometry (ESI-MS)

[0077] Electro-spray ionization mass spectrometry (ESI-MS) was performed using a Bruker Esquire 3000+ instrument equipped with an electro-spray ionization source and a quadrupole ion trap detector (QITD).

[0078] Transmission Electron Microscopy (TEM)

[0079] TEM images were collected using Philips CM200 field emission microscope operated at 200 kV in bright field mode with Omega-type Zero-loss energy filter. The samples were prepared placing on a 200-mesh carbon film-coated grid 10 pL of each hydrogel (previously diluted 1 :10) and the exceeding water was removed with filter paper after 1 minute to prevent aggregation effects promoted by drying. potential measurements

[0080] Zeta ( potential measurements were performed with a Malvern Zetasizer Nano ZS diluting ten times the initial samples with MilliQ water. Average potential at the slipping plane was obtained using the Smoluchowski correlation. The temperature was kept at 25°C and the sample was left equilibrating 60 second before each measurement. Each sample was analysed three times, and the results were averaged.

[0081] Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy (ATR-FTIR)

[0082] FT-IR measurements were performed on lyophilized samples in attenuated total reflectance (ATR) mode by using a Nicolet iS50 FT-IR spectrometer equipped with an ATR device. Spectra were collected in the medium IR region (64 scans, 4000-400 cm-1). All the spectra were measured with a resolution of ± 1 cm-1and corrected for the air background.

[0083] Rheological measurements

[0084] Rheological properties of the hydrogels obtained both with CNF alone and with the adducts according to the present invention were evaluated with a Malvern Panalytical KINEXUS PRO+ rheometer equipped with a cone-plate geometry (diameter: 40 mm). The upper geometry cone was lowered until it was in conformal contact with the top surface of the hydrogel, corresponding to gap distances of 1 .0-1 .5 mm. Samples were pre-formed, directly transferred on the bottom plate, 72 hours after preparation, and homogenized by application of a mild shear rate (y = 10 s-1 ) for 30 seconds. Dynamic moduli and the ability of gels to provide an elastic response under oscillatory stresses were assessed through a combination of time sweep (frequency = 1 Hz, strain = 0.1 %), strain sweep for the estimation of the Linear Viscoelastic Region (LVER, frequency = 1 Hz, strain = 0.01 % to 100%) and frequency sweep (frequency = 0.1 Hz to 100 Hz, constant stress chosen within the LVER). The shear rate dependence of viscosity was studied adopting a shear rate ramp (y = 0.1 -100 s-1 ). Small deformation oscillatory rheology analyses were performed on an Anton Paar MCR 302 equipped with a parallel plate geometry (diameter: 25 mm) and operated at a gap distance of 1 mm. A constant shear strain of 0.1 % was employed, with other parameters selected in accordance with the analyses required, as reported above. All measurements were repeated at least three times and carried out at a constant temperature of 25°C.

[0085] Elemental mapping

[0086] Elemental analysis was performed by Energy Dispersive X-ray spectroscopy (EDS) on a scanning electron microscope Zeiss EVO 50 EP. Hydrogels were freeze dried prior to deposition on carbon tapes, whereas films were deposited without further treatments. All samples were sputter coated with a thin Au layer and analysed at an operating voltage of 20 kV.

[0087] Contact angle measurements Contact angles of the films were determined using an OCA 15 PLUS instrument (Dataphysics) using droplet volumes of 1 pL for both Milli-Q water (ultrapure water) and dodecane to assess the hydrophobicity and lipophilicity respectively. The average contact angles (Elliptic method) were calculated from a series of at least three independent measurements by the SCA20 software.

[0088] Water uptake estimation

[0089] Water uptake (WU) measurements were performed in accordance with the Twenty-Four Hour Immersion method reported in ASTM D570-98. Samples were originally conditioned for 24 hours under vacuum in a desiccator with anhydrous calcium chloride. Dry films were then weighed and soaked in Mi II i- Q water maintained at a temperature of 23°C for 24 hours. At the end of the 24thhour, specimens were removed from water, wiped off with a dry cloth and weighed again using an electronic balance with a 0.01 mg accuracy. WU was estimated as the ratio between the mass change after soaking and the original dry mass: m

[0090] WU = _w- m w -dd mdwhere mwis the wet film mass and md is the dry film mass. Measurements were performed in triplicates and average results are reported.

[0091] Water vapor transmission rate estimation

[0092] Water vapor transmission rate was determined at 25°C and 0°C in accordance with ASTM E96 / E96M standard Water Method. In this test, a permeation chamber with a 7 mm inner diameter was filled with 400 pL of Milli- Q water. Samples were fixed on top of the permeation chambers and placed under vacuum in a dry desiccator with anhydrous calcium chloride, used to maintain 0% relative humidity (RH) in the desiccator. The water permeation through the films was assessed by measuring the weight change of the permeation chamber at fixed timepoints using an electronic balance with a 0.01 mg accuracy. In order to compensate for balance fluctuations between each measurement, blank samples without water in the permeation chamber were measured parallelly and mass variations of blanks were subtracted to the sample’s variations acquired at the same timepoint. The mass variation was plotted against time and the slope of the line allowed to calculate the water vapor transmission rate (WVTR) as follows:

[0093] Examples

[0094] Example 1 - Peptide synthesis: general procedure

[0095] Three peptides according to the present invention (chemical structures represented below) were synthesized according to the general procedure described below. Peptide DFNKF contains only non-modified amino acids, the peptide indicated herein as DF(I)NKF comprises a para-iodo-phenylalanine in position 2 of the sequence, the peptide indicated herein as DF(Fs)NKF comprises a penta-fluoro-phenylalanine in position 2 of the sequence.

[0096] Resin loading

[0097] 2-CTC resin (0.5 mmol / g loading) was swollen in CH2CI2 (dichloromethane, DCM) for 30 min. A solution of entering Fmoc-amino acid and DIEA (N,N- Diisopropylethylamine; 1.2:8 eq over resin loading) in CH2CI2 (3 mL) was added and the resin shaken at rt for 4 h. The resin was washed with DMF (Dimethylformamide; 2x3 mL) and capping was performed by treatment with acetic anhydride / DIEA in DCM (1 x 30 min). The resin was then washed with DMF (2x3 mL), CH2CI2 (2x3 mL), and DMF (2x3 mL). The resin was subsequently submitted to fully automated iterative peptide assembly (Fmoc- SPPS).

[0098] Peptide Assembly via Iterative Automated microwave Assisted SPPS

[0099] Peptides were assembled by stepwise microwave-assisted Fmoc-SPPS on a Biotage ALSTRA Initiator-*- peptide synthesizer, operating in a 0.1 mmol scale. Activation of entering Fmoc-protected amino acids (0.3M solution in DMF) was performed using 0.5M Oxyma (ethyl cyano(hydroxyamino)acetate) in DMF I 0.5M N,N'-Diisopropylcarbodiimide (DIC) in DMF (1 :1 :1 molar ratio), with 5 equivalents excess over the initial resin loading. Coupling steps were performed for 30 minutes at 50°C. Fmoc-deprotection steps were performed by treatment with a 20% piperidine solution in DMF at room temperature (1x 10 min). Following each coupling or deprotection step, peptidyl-resin was washed with DMF (4 x 3.5 mL). Upon complete chain assembly, resin was washed with DCM (5 x 3.5 mL) and gently dried under a nitrogen flow.

[0100] Resin-bound peptide was then treated with an ice-cold TFA (trifluoroacetic acid), TIS (Triisopropylsilane), water, thioanisole mixture (90:5:2.5:2.5 v / v / v / v, 4mL). After gently shaking the resin for 2 hours at room temperature, the resin was filtered and washed with neat TFA (2 x 4 mL). The combined cleavage solutions were worked-up as indicated below.

[0101] Finally, the cleavage mixture was concentrated under nitrogen stream and then added dropwise to ice cold diethyl ether (40 mL) to precipitate the crude peptide. The crude peptide was collected by centrifugation and washed with further cold diethyl ether to remove scavengers. Residual diethyl ether was removed by a gentle nitrogen stream and the crude peptide was purified by RP-HPLC as follows. Preparative RP-HPLC was performed on a Shimadzu HPLC Prominence system using a Gemini, Shimadzu, C18 column (10 micron, 21 .2 mm i.d. x 250 mm) using the following chromatographic method: 0% B to 90% B in 45 min; flow rate, 14 ml / min. Pure RP-HPLC fractions (>95%) were combined and lyophilized, and then analysed by ESI-MS, as described in the materials and methods section above. Example 2 - Preparation of nanofibril lated cellulose hydrogels (comparative)

[0102] Nanofibrillated cellulose (CNF) hydrogels were prepared by diluting an initial CNF aqueous suspension (concentration 1 .6 wt%) with Milli-Q water to a final concentration of 0.8 wt%.

[0103] After dilution, samples were agitated for 1 minute on a benchtop vortex, and tip sonicated for 1 min at 130 W, 100% amplitude, with a 3 mm probe head (Vibracell VCX 130PB, Sonics & Materials Inc) using an ice bath to prevent excessive heating.

[0104] Despite sonication being a widespread method to promote CNF separation, especially for surface-functionalized fibers, prolonged exposure of CNF to ultrasounds is known to potentially deteriorate it, leading to fibers fragmentation. However, neither fibers breakage nor crystallinity index variations were observed upon sonication, suggesting that the CNF morphology is mostly preserved after the ultrasonic treatment.

[0105] Example 3 - Preparation of hydrogels comprising the DFNKF peptides-CNF adducts according to the present invention

[0106] Hydrogels comprising the DFNKF peptides-CNF adducts according to the present invention were prepared with the following procedure.

[0107] At first, stock solutions of three different peptides according to the present invention and obtained as disclosed in Example 1 above, i.e. DFNKF, DF(I)NKF and DF(Fs)NKF, in Milli-Q water were prepared by sonication with an ultrasound bath (Bandelin Sonorex Super RK 100H) for 20 seconds followed by heating at 90°C until dissolution.

[0108] Then, nanofibrillated cellulose was diluted with the peptide stock solution to simultaneously achieve a CNF content of 0.8% w / w and a desired final peptide concentration.

[0109] After dilution, samples were agitated for 1 minute on a benchtop vortex, and tip sonicated for 1 min at 130 W and 100% amplitude with a 3 mm probe head (Vibracell VCX 130PB, Sonics & Materials Inc) using an ice bath to prevent excessive heating.

[0110] All samples obtained were stored at room temperature for three days before analysis.

[0111] The peptides solutions were used in peptide amounts varying from 0.1 % (1.25 mM) to 0.01 % (0.125 mM), namely with a CNF : peptide weight ratio between 8:1 to 80:1. Table 1 below shows the quali-quantitative compositions of the hydrogels obtained.

[0112] Table 1

[0113] Comparative, obtained as described in Example 2

[0114] The obtained hydrogels were characterized as disclosed in the following examples. Overall, the data collected confirmed the formation of a stable adduct between the nanofibrillated cellulose and each of the tested peptides at each of the concentrations (therefore the CNF: peptide weight ratios) tested, resulting in thicker and stiffer hydrogels when compared to that obtained with CNF only (sample 1A) regardless of the peptide sequence used. Of note, although these peptides are known to possess strong self-assembly properties, gelation cannot be driven by peptides self-assembly at the concentrations tested, as the minimum gelation concentration of the best gelator in the series was reported to be around 0.2% w / w (Bertolani, A. et al., Nature communications 2015, 6, 1-9), thus between 2 and 20 times higher than those tested. Example 4 - Microscopic characterization

[0115] The microscopic characterization of the hydrogels obtained was carried out via Transmission Electron Microscopy (TEM) as disclosed above. Herein we report data collected on comparative sample 1A(0.8wt% CNF only), an exemplary hydrogel according to the present invention (sample 9, DF(I)NKF 0.05wt%), as well as on a sample of peptide DF(I)NKF taken alone at 0.05 wt% in water (not shown).

[0116] The analysis showed a long and well-dispersed network of individual CNF fibers with a size of 18.25 ± 8.32 nm (see Fig. 1 , panel A), which, upon peptide addition, appeared increased in thickness, and coated by an amorphous matrix (see Fig. 1 , panel B). Of note, the morphology of sample 9 was not observed in either the peptide or nanofibrillated cellulose alone, confirming that the interaction between the two components deeply affects the assembly pattern of the system, thus offering proof of the formation of an adduct between the peptide and the CNF. Overall, the aspect of the network turned less regular, with respect to the network composed of sole CNF, with the peptide matrix tightly binding to pre-existing fibers.

[0117] Example 5 - potential measurements

[0118] ^-potential measurements were carried out on comparative sample 1A, on samples of 1 mM aqueous solutions of the three peptides alone (DFNKF, DF(I)NKF, and DF(Fs)NKF), and on a number of exemplary hydrogels obtained in Example 3 (sample 2, samples 6-12 and sample 16). Results are shown in table 2 below.

[0119] Table 2

[0120] As can be seen from the table, sample 1Ashows a negative surface potential, which can be attributed to impurities, such as hemicellulose or pectin, leading to the presence of some sugar acids (e.g. uronic acids) on the CNF surface.

[0121] On the other hand, the peptides alone show highly positive values, owing to their Lys residue which are positively charged (isoelectric point (pl) = 6.8) under the conditions tested (pH = 5.7).

[0122] The results showed that by increasing the peptide content, the hydrogels tvpotential progressively increases from highly negative (t;=-30mV) to less negative values (t;=-15 / -20mV), confirming, despite the minimum quantity of peptide used, the electrostatic complexation between the peptide and the CNF.

[0123] Example 6 - IR characterization

[0124] To further confirm the formation of the adducts, IR analyses were carried out on samples 1A, 4, 9, 14, and on samples of the three peptides alone (Fig. 2). In the spectra, characteristic peaks can be seen for both the nanofibrillated cellulose and the peptides, indicating the presence of both uniformly distributed in the system.

[0125] In particular, the bands at 3337, 2900 and 1053 cm’1are associated to -OH, C-H, and C-O-C groups typical of nanofibrillated cellulose. The presence of peptides in the samples is instead confirmed by the presence of bands at 1600- 1700 cm’1and 1500 cm’1attributable to the amides present in the structure. Moreover, the comparison between FTIR spectra of samples 4, 9 and 14 with that of the respective peptides taken alone (Fig. 3) showed modifications in the hydrogen bonding network of the amine groups: in the adducts according to the invention, the N-H bands resulted to be blue-shifted of 7 cm’1, 10 cm’1and 16 cm’1, respectively, after complexation with CNF. Indeed, this offers proof that the protonated NH2 group of the Lys residue in the peptides may enable the interaction with carboxylate anions present on the nanofibrillated cellulose surface via hydrogen bonding.

[0126] Example 7- SEM imaging and elettromapping

[0127] The morphology of the samples according to the present invention slightly changed also in their dry state, where peptide addition led to the appearance of local bundles of densely fibri Hated patches, which could not be observed in pure CNF samples as evidenced by SEM images of comparative sample 1A(Fig. 4 panel A) and of an exemplary adduct according to the invention, sample 9 (Fig. 4 panel B).

[0128] To better probe the peptide presence within the nanofibrillated cellulose matrix, an analysis was carried out on exemplary samples of hydrogels according to the present invention using scanning electron microscopy (SEM) coupled with energy dispersive X-ray spectroscopy (EDS).

[0129] By selecting nitrogen, iodine, and fluorine as exclusive labels for the adducts CNF-DF(I)NKF (sample 9) and CNF-DF(F5)NKF (sample 14), respectively, elemental maps were obtained (Fig. 4, panels C-D) that showed an even peptide distribution throughout the whole sample, confirming a successful blending, namely a co-assembly of the two constituents in the systems. Similar results were obtained on the CNF-DFNKF adduct (sample 4, not shown).

[0130] The results were further corroborated by the nitrogen channel, utilized as a secondary label, which provided an almost superimposable map with respect to the halogen channel (not shown).

[0131] The lack of segregation or self-sorting in N, I, and F signals confirmed, once more, the inability of peptides to self-assemble into amyloid fibrils when incorporated in a nanofibrillated cellulose network, which instead is able to engage peptide monomers and small oligomers in electrostatic interactions and charge-assisted hydrogen bonds.

[0132] Example 8 - Rheological studies of nanofibrillated cellulose-peptide adducts Rheological properties of the hydrogels obtained in Examples 2 and 3 where firstly assessed via oscillatory rheology studies. Results are showed in Table 3 below.

[0133] Table 3 Sample 1Abehaved as a weak percolating network possessing single plateau region of the dynamic moduli, with the storage modulus (G1) dominating over the loss modulus (G"), indicative of a gelled / solid-like behavior, and a linear viscoelastic region (LVER) up to ~4 % strain, after which the structure starts to break down. The introduction of the peptides, even at the lowest concentration (0.01 % w / w, 0.125 mM), was able to improve the rheological response of the nanofibrillated cellulose network in an appreciable manner. At higher peptide concentrations, between 0.025wt% and 0.1wt% (0.5-1.25 mM), all the hydrogels comprising the adducts according to the present invention shared a more marked change with similar viscoelastic responses, showing both dynamic moduli rising up by one order of magnitude. Peptides inclusion did not affect the nanofibrillated cellulose crossover frequency (G1= G"), which remained constant (~30 Hz) at all the tested conditions and equal to the one of neat CNF, thus not impacting the CNF relaxation behavior.

[0134] Amplitude strain sweeps showed that the hydrogels according to the invention were able to withstand slightly smaller strains (LVER ~2 % strain), indicating more connections in the nanofibrillated cellulose network and an easily flowing system. Additionally, at lower peptide contents (< 0.025%, i.e. 0.36 mM) there was a rise in the G" in proximity of the yield point, indicating a temporary internal structuring prior to network collapse.

[0135] This rheological response is similar to what is typically observed when ionic solutes are added to cellulose networks: at increased ionic strength the repulsion between cellulose fibrils is screened, leading to aggregation and stronger networks. Similarly, in the hydrogels according to the invention, the nanofibrillated cellulose electrostatic screening occurs upon peptide addition, as evidenced by the gradual increase in the ^-potential value (see Example 5). The peptides, acting as physical crosslinks, enable stronger and more robust connections between cellulose fibers, resulting in enhanced rheological properties.

[0136] Aside from local interactions, the nanofibrillated cellulose-peptide interaction can also be favoured by the entropic gain resulting from the release of structured water molecules present on the nanofibrillated cellulose surface before peptide adsorption.

[0137] Overall, the observed rheological properties are direct indicators of a successful binding between the chosen peptides and cellulose nanofibrils. Such a binding leads to the formation of robust hybrid networks that can be leveraged in applications requiring high mechanical strength and stability, such as coatings or packaging materials.

[0138] Example 9 - Preparation of CNF-based and CNF-DFNKF-based films

[0139] Films derived from the hydrogels prepared according to Examples 2 and 3, and summarized in Table 1 above, were obtained according to the process described below.

[0140] The hydrogels were left to rest for three days at room temperature and were then filtered under vacuum for 45 min at 20 mbar on a 2.1 cm diameter filter paper support. The diameter of the film was determined by the insertion of an O-ring. A 300 g load was applied on top of the setup to prevent wrinkling. At the end, filter paper was peeled off and self-standing films of 2 cm of diameter were obtained. Peptides inclusion in the final films was verified by IR spectroscopy, highlighted by the presence of distinguishable amide peaks at -1665 cm’1, -1635 cm’1and -1545 cm’1.

[0141] The presence and correct distribution of the peptide within the films was further verified by elemental analysis in combination with scanning electron microscopy (EDS-SEM, performed by Zeiss EVO EP at 20 kV) performed as detailed above on exemplary samples of a film comprising CNF only and one comprising a CNF-DF(Fs)NKF adduct according to the present invention. As shown by the SEM images and elemental map (Fig. 5), peptides addition did not alter significantly the nanofibrillated cellulose film morphology, resulting in a homogenous film with relatively low porosity.

[0142] Example 10 - Contact angle measurements

[0143] In order to evaluate the impact of peptide addition on nanofibrillated cellulose idrophobicity, films surface wettability was first investigated through contact angle measurements on the films obtained.

[0144] Table 4 below shows the samples used and the results obtained.

[0145] As expected, sample 17Ashowed a water contact angle (wCA) of around 41 ± 2°, confirming that nanofibrillated cellulose-based films are vulnerable to water. Upon peptides introduction, wCA averagely increased in all tested conditions as a result of the peptides adsorption on the nanofibrillated cellulose surface, partially hindering the interaction between nanofibrillated cellulose hydroxyl groups and water. Notably, a significant increase in wCA was observed for the samples at 0.1 wt% peptide concentration (samples 18, 23 and 28): in particular, sample 28 comprising a CNF-DF(Fs)NKF adduct, showed a wCA above 90°, thus achieving hydrophobic wCA values.

[0146] The observed trend is easily attributable to the different hydrophobicity of the slightly modified F residues, showing the great impact provided by the type, number, and position of halogen atoms used. Despite the possibility to further reduce the wettability of nanofibrillated cellulose-based systems with more common hydrophobic additives, it is essential to note that the overall peptide content added to these systems is remarkably low, with a maximum nanofibrillated cellulose: peptide ratio of 8:1 and an overall peptide content of milligram fractions.

[0147] Table 4 Complementarity to water contact angles, n-dodecane (C12) contact angle (dCA) measurements were performed. Dodecane contact angle progressively decreased by peptide addition until dropping to almost negligible values at sufficiently high contents of peptide resulting in nanofibrillated cellulose films with enhanced water resistance and increased affinity to oils. Example 11 - Water uptake and water vapor transmission rate measurements

[0148] Given the remarkable decrease of the CNF hydrophilic character observed in the films comprising the adducts between CNF and DF(Fs)NKF, as disclosed in Example 10, sample 28 was further studied in terms of water uptake and water vapor permeability (WVTR). The measurements were carried out as disclosed in the material and methods section.

[0149] Fig. 6a shows water uptake values after 24 hours of immersion of samples 17Aand 28. As can be seen, sample17Aabsorbed around 175 ± 17% of water after 24 hours of immersion, a value that decreased to 95 ± 19.9% for sample 28, demonstrating its reduced water uptake capacity.

[0150] Similarly, as shown in Fig. 6b, sample 28 exhibited a WVTR of 2046 ± 169 g rrr2day-1, which was twofold lower than the 5360 ± 1077 g nr2day-1observed for sample 17A. This value remained stable throughout the entire monitoring period (up to 24 hours), and above all sample 28 demonstrated a WTR value that was comparable to, if not superior to, other commonly used and industrially relevant cellulose materials, including cellulose acetate (CA), cellulose acetate propionate (CAP) or other fluorinated cellulose esters, as shown by the data reported in Fig. 8.

Claims

CLAIMS1 . Adduct between a nanocellulose and a peptide of sequence SEQ ID NO:1 DF*NKF*, wherein each of the phenylalanine F* residues, independently from one another, can be optionally substituted on the aromatic ring with from 1 to 5 halogen atoms selected from fluorine, iodine, bromine, chlorine, and mixtures thereof.

2. The adduct according to claim 1 , wherein said peptide is a peptide of sequence SEQ ID NO: 2 DF*NKF, wherein the phenylalanine F* residue in position 2 can be optionally substituted on the aromatic ring with from 1 to 5 halogen atoms selected from fluorine, iodine, bromine, chlorine, and mixtures thereof.

3. The adduct according to claim 2, wherein in said peptide of sequence SEQ ID NO: 2 DF*NKF the phenylalanine F* residue in position 2 can be optionally substituted in the para-position of the aromatic ring with one halogen atom selected from an iodine, a chlorine, a bromine and a fluorine atom; or wherein the phenylalanine F* residue in position 2 can be optionally substituted in positions 3 and 5 of the aromatic ring with two halogen atoms selected from iodine, chlorine, bromine, and fluorine atoms, and mixtures thereof; or wherein the phenylalanine F* residue in position2 can be optionally substituted on the aromatic ring with five halogen atoms selected from iodine, chlorine, bromine, and fluorine atoms, and mixtures thereof.

4. The adduct according to any one of claims 1 -3, wherein said peptide is selected from:• a peptide of sequence DFNKF (SEQ ID NO: 1 ),• a peptide of sequence SEQ ID NO: 2: DF*NKF wherein the phenylalanine F* residue in position 2 is substituted on the aromatic ring in the para-position with an iodine atom,• a peptide of sequence SEQ ID NO: 2 DF*NKF wherein the phenylalanine F* residue in position 2 is substituted on the aromatic ring in the para-position with a fluorine atom,• a peptide of sequence SEQ ID NO: 2 DF*NKF wherein thephenylalanine F* residue in position 2 is substituted on the aromatic ring in the para-position with a bromine atom,• a peptide of sequence SEQ ID NO: 2 DF*NKF wherein the phenylalanine F* residue in position 2 is substituted on the aromatic ring with two fluorine atoms in positions 3 and 5,• a peptide of sequence SEQ ID NO: 2 DF*NKF wherein the phenylalanine F* residue in position 2 is substituted on the aromatic ring with two bromine atoms in positions 3 and 5, and• a peptide of sequence SEQ ID NO: 2 DF*NKF wherein the phenylalanine F* residue in position 2 is substituted on the aromatic ring with five fluorine atoms.

5. The adduct according to any one of claims 1 -4, wherein said nanocellulose is a nanofibrillated cellulose.

6. The adduct according to any one of claims 1-5, wherein the weight ratio between said nanocellulose and said peptide is of from 2:1 to 100:1.

7. The adduct according to any one of claims 1-6, wherein said adduct is in the form of a hydrogel.

8. The adduct according to claim 7, wherein the concentration of the nanocellulose in said hydrogel is of from 0.2 wt% to 2 wt%.

9. The adduct according to any one of claims 7-8, wherein the concentration of the peptide in said hydrogel is of from 0.002 wt% to 1wt%.

10. Process for the preparation of an adduct between a nanocellulose and a DF*NKF* peptide (SEQ ID NO: 1 ) according to any one of claims 1 -9, the process comprising the following steps: a) providing an aqueous suspension of a nanocellulose; b) providing an aqueous solution of at least one peptide of sequence DF*NKF* (SEQ ID NO: 1 ) as defined in any one of claims 1 -4; c) adding the aqueous solution obtained in step b) to the aqueous suspension obtained in step a); and d) agitating and then sonicating the mixture obtained in step c) until formation of an adduct.

11. The process according to claim 10, wherein the sonication in step d) is carried out at 100-400 W.

12. Film based on an adduct between a nanocellulose and a peptide of sequence DF*NKF* (SEQ ID NO: 1 ) according to any one of claims 1 -9.

13. Process for the preparation of a film according to claim 12, the process comprising the steps of: i. providing a hydrogel comprising an adduct between a nanocellulose and a peptide of sequence DF*NKF* (SEQ ID NO: 1 ) as defined in any one of claims 1 -9; and ii. filtering under vacuum said hydrogel.

14. Use of the adduct according to any one of claims 1-9, or of the film according to claim 12, as a coating, as a non-woven textile material, as a packaging material, or as a separation and / or filtration membrane.

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