IT202000004528A1 - Self-assembling peptides - Google Patents

Description

DESCRIPTION

Of the Patent Application for Industrial Invention entitled:

? Self-assembling peptides?

Technical field

? Peptide-based hydrogels have been shown to be preeminent biomedical materials due to their high water content, stability. modular mechanics, excellent biocompatibility? and excellent injectability. The capacity peptide-based hydrogels to provide environments that mimic the extracellular matrix opens up opportunities. for their biomedical applications such as tissue engineering and regenerative medicine, drug delivery, three-dimensional tissue culture and hemostasis.

Self-assembling peptides (SAP) are short peptides (usually 8-16 residues) that spontaneously self-organize into woven nanofibers with diameters of 10-20 nm (Pugliese, R., Gelain, F. Peptidic biomaterials: from selfassembling to regenerative medicine. Trends Biotechnol. 2017; 35,145-158). This free energy driven process can be regulated by molecular chemistry (e.g. co-assembly molecules), assembly conditions (pH, temperature, or electrolytes) and assembly kinetics. The versatility? design of the peptide blocks, in combination with their ability? to adopt specific secondary structures (usually sheet structures?), offers a suitable strategy for designing nanomaterials with structural characteristics controlled at the nanoscale level. Furthermore, SAPs are bioabsorbable and non-toxic biomaterials.

Examples of such SAPs include sheet peptides? with alternating charged hydrophilic and hydrophobic amino acid residues. RADA16 (i.e. Ac-RADARADARADARADA-CONH2, SEQ ID NO: 18)? a well known autocomplementary ion peptide, which gives? well characterized SAP-based hydrogels (Zhang, S. et al. Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane. Proc. Natl. Acad. Sci. USA 1993; 90,3334-3338). However, RADA16 and other known SAPs have practical limitations. For example, RADA16 must be formulated at acidic pH to be solubilized, which can cause damage to cells and / or tissues. Are peptides entities? chemicals that are ubiquitous in nature and their use? exploited for many applications in different fields; namely peptide material, drug chemistry, nutraceuticals and agriculture. Although the concept of peptide as a material? accepted since the 1990s, blends of food-borne peptides, such as legume proteins, as reservoir systems of new peptide material are completely unknown and little investigated.

Legume proteins should be fractionated with solubility? differential in albumin, globulin, prolamine and gluteline. Legume globulins, which function primarily as storage proteins, can be further separated by ultracentrifugation or chromatography into two main components: legumins (11S) and vicilins (7S) and some other minor fractions. In soy, legumins (also referred to as glycinine) are derived from a protein precursor, and are broken down into an acidic polypeptide (chain) A (pI? 5) and a basic polypeptide (chain) B (pI? 8.2). These two peptides are linked by a disulfide bridge and are considered subunits. Is there a strong homology between the legumes of different legume species and the cleavage site of polypeptide A / B? highly conserved, although the variable regions are mainly found in peptide A (Arnoldi, A. et al. The Role of Grain Legumes in the Prevention of Hypercholesterolemia and Hypertension. Crit Rev Plant Sci 2015; 34.144-168).

Therefore, the need remains? to improve self-assembling peptides that can be used for applications dealing with cells and / or tissues.

Description

The present disclosure is based, at least in part, on the development of self-assembling ionic peptides having specific combinations of ionic polar amino acids, hydrophobic amino acids and non-ionic polar amino acids. The SAPs according to the present invention are used to produce improved hydrogels with advantageous physical characteristics under physiological conditions. For example, aqueous solutions of the SAPs can be produced which remain stable, fluid and injectable under physiological conditions. At the time of gelation, the SAPs according to the present invention exhibit surprising viscoelastic parameters and spontaneously form long and grouped nanofibers, properties useful in clinical, industrial and / or research applications.

Description of the figures

Figure 1: (A) crystal structure of glycinine G1 and the? Strand comprised in the same peptide; (B) index of hydropathy; (C) propensity to form stable? -Sheet structures outside the protein environment in which it is found; (D) residues accessible to solvents.

Figure 2: Properties biomechanics of (A) RIES12; (B) RIES16; (C) SEIR12 assembled peptide scaffolds at 1% (w / v).

Figure 3: Properties biomechanics of RIES12 at different concentrations. Figure 4: evaluation of injectability of the peptide RIES12.

Figure 5: Supramolecular organization of 1% (w / v) RIES12 peptide hydrogel. (A) The RIES12 hydrogel exhibits a CD pattern comprising a negative peak near 218 nm and a positive peak at 195 nm characteristic of the? -Sheet conformation; (B) The ThT test showed a typical amyloid-binding emission signal (peak at ~ 490 nm) confirming the rich nature of? of the RIES12 hydrogel.

Figure 6: Atomic force microscopy (AFM) images of (A) RIES12; (B) SEIR12; (C, comparative purposes) RIES16; and (D, comparative purposes) 1% (w / v) HydroMatrix peptide hydrogel.

Figure 7: stability thermo-mechanics of the RIES12 peptide along a temperature ramp of 25-37? C.

Figure 8: vitality cell of (A) Caco-2 and (B) HepG2 cells on HydroMatrix, RIES12 0.5% or RIES121% (w / v) (bar 100? m).

In a first embodiment, the authors surprisingly describe the use of peptides extracted from natural products, preferably from legumes, even more so. preferably from legume globulins, in obtaining a new material.

The authors of the present invention have surprisingly demonstrated the propensity of the RIES peptide, belonging to glycinine G1, a soy protein, to spontaneous self-assembly.

From said surprising observation, in some respects, the disclosure provides self-assembling peptides (SAPs), in which the SAPs comprise a sequence of natural and / or synthetic amino acid residues, chemically modified amino acids and synthetic compounds which have properties known in the art to be characteristic of an amino acid, in which said sequence? as indicated in Formula I:

[XYZSer] n [SerZYX] m [XYZSer] o [SerZYX] p Formula I,

where each (X)? independently a positively charged amino acid, in which each (Y)? independently a non-polar amino acid with hydrophobic side chains, where each (Z)? independently a negatively charged amino acid, where n = 0 or an integer of 1, 2, 3 or 4, m = 0 or an integer of 1, 2, 3 or 4, o = 0 or an integer of 1, 2, 3 or 4, p = 0 or an integer of 1, 2, 3 or 4, where m n or p? an integer of 2, 3, or 4.

In one embodiment, the amino acid residues are natural and in said Formula I each (X)? independently a positively charged amino acid, each (Y)? independently a non-polar amino acid with hydrophobic side chains, each (Z)? independently a negatively charged amino acid.

In a preferred embodiment, in said Formula I each (X)? independently selected from the group consisting of Arg, Lys and His, each (Y)? selected independently from the group consisting of Ile, Leu, Val and Ala each (Z)? independently selected from the group consisting of Asp and Glu.

In some embodiments, the self-assembling peptide comprises an N-terminal functional group, a C-terminal functional group, or both.

In some embodiments, the N-terminal functional group? an acetyl, a formyl, pyroglutamyl (pGlu), biotin, polyethylene glycol (PEG), urea, alkylamine, carbamate, sulfonamide, dansyl, 2,4-dinitrophenyl, fluorescein, acetic acid 7-methoxy-coumarin, 9-fluorenylmethyloxycarbonyl, palmitic acid , succinyl, chloroacetyl, maleimide, benzyloxycarbonyl, bromoacetyl, nitrilotriacetyl, tertzboxxycarbonyl acid, 4-hydroxyphenylpropic acid, glycyl-triclic acid, butyl-triclic acid, butyl-butyl acid, tryl-butyl, butyl-tricylic acid -butyl, tryl-butyl, In some embodiments, the C-terminal functional group? or an amide, an N-alkyl amide, an aldehyde, an ester, an alcohol, para-nitroanilide (pNA), 7-amino-4-methylcoumarin (Amc), a hydrazide, hydroxamic acid, chloromethylketone, pnitroaniline, para-nitrophenol, hydroxysucinimide ester, fluoromethylketone, cysteamide, 9-fluorenemethyl ester (Fm), allyl ester, 2,4-dimethoxybenzyl ester, 2-phenylisopropyl ester, p-nitrobenzyl ester, 2-chlorotrityl ester.

In some embodiments, the N-terminal functional group? an acetyl. In some embodiments, the C-terminal functional group? an amide. In some embodiments, the N-terminal functional group? an acetyl and the C-terminal functional group? an amide.

In some embodiments, the SAP comprises at least one biologically active peptide motif (e.g., one, two, three, four or more). In some embodiments, at least one biologically active peptide motif? present at the end? N-terminal of the SAP. In some embodiments, at least one biologically active peptide motif? present at the end? C-terminal of the SAP. In some embodiments, at least one biologically active peptide motif is derived from laminin-1, collagen IV, fibronectin, elastin, bone marrow homing peptide 1, bone marrow homing peptide 2, or myelopeptide.

Except as otherwise defined herein, scientific and technical terms used in this disclosure will have meanings that are commonly understood by those of ordinary skill in the art.

The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general references and more. specifics that are mentioned and discussed in this description. The chemical terms used here are used according to conventional use in art, as exemplified by? The McGraw-Hill Dictionary of Chemical Terms ", Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

The term "self-assembly", as used in this document, refers to the capacity. of some peptides to spontaneously self-bind to higher order structures (e.g.? -sheet). In some embodiments, the interaction between the individual self-assembling peptides? reversible, so that the composition can change reversibly between a gel state and a solution state. The interactions between individual self-assembling peptides can be non-covalent interactions including hydrogen bonds, ionic interactions, electrostatic interactions, van der Waals forces, and hydrophobic interactions. In various embodiments, the self-assembled peptide nanostructure comprises peptides in? -Sheet. The nanostructure can? be a nanofiber or a nanofiber network.

As used herein, the term "amino acid residue" or "amino acids" includes natural and synthetic amino acid residues including amino acids D and L; alpha, beta and gamma amino acids; chemically modified amino acids; and chemically synthesized compounds that have properties? known in the art as characteristics of an amino acid.

As used herein, the term "hydrogel" refers to a composition comprising a three-dimensional network of self-assembling peptides. The term hydrogel can? be used to indicate a network of self-assembling peptides in the dry state (xerogel) or in the wet state. In the wet state, the hydrogel can? include a high water content (for example, between about 90% and about 99.9% water). Do hydrogels have many properties? desirable for biomedical applications. For example, hydrogels can be manufactured in such a way that they are non-toxic and compatible with tissues. Furthermore, hydrogels are generally highly permeable to water, ions, electrolytes and small molecules.

As used herein, the term "isolate" in reference to cells refers to a cell that? been mechanically separated from the environment from which it occurs in nature.

As used herein, the terms "proteins" and "peptides" are used interchangeably to refer to one polymer of amino acid residues linked to each other by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms "protein" and "peptide" include polymers with modified amino acid residues (e.g., phosphorylated, amidated or glycated amino acid residues) and amino acid analogs.

In one aspect, the disclosure provides SAPs and compositions comprising at least one SAP described herein. SAPs spontaneously assemble into higher order structures via intermolecular and intramolecular electrostatic interactions when present in an aqueous solution. These higher-order structures include? -Sheets, nanofiber structures, and three-dimensional network or mesh structures. These higher order structures in aqueous solutions can become hydrogels having a variety of properties. owned desirable.

In addition to SAPs, the compositions may also include other additives such as tonic agents, buffers, biomolecules and cells. Although SAPs and higher-order structures (e.g. scaffolds) made from it may be biodegradable, these higher-order structures preferably retain their integrity. structural for a period of time necessary for the intended use.

In some embodiments, the SAP comprises a sequence of amino acids as indicated in:

[XYZSer] n [SerZYX] m [XYZSer] o [SerZYX] p Formula I,

where each (X)? independently a charged positive amino acid selected from the group consisting of Arg, Lys and His, in which each (Y)? independently a non-polar amino acid selected from the group consisting of Ile, Leu, Val and Ala in which each (Z)? independently a negative charged amino acid selected from the group consisting of Asp and Glu, where n = 0 or an integer of 1, 2, 3 or 4, m = 0 or an integer of 1, 2, 3 or 4 where or = 0 or an integer of 1, 2, 3 or 4; p = 0 or an integer of 1, 2, 3 or 4; m n or p? an integer of 2, 3, or 4.

In some embodiments, said X? Arg, called Y? selected independently from the group consisting of Ile, Leu, Val and Ala and called Z? independently selected from the group consisting of Asp, Glu.

In some embodiments, said Y? Ile, called X? independently selected from the group consisting of Arg, Lys, His and called Z? independently selected from the group consisting of Asp, Glu.

In some embodiments, said Z? Glu, called X? selected independently from the group consisting of Arg, Lys, His and called Y? selected independently from the group consisting of Ile, Leu, Val and Ala. In some embodiments, said X? Arg, called Y? Is Ile called Z? Glu. In a preferred embodiment, m n or p = 3. In some embodiments, called n = 1, called m = 2, o = 0, p = 0, or called n = 2, called m = 1, o = 0, p = 0. In another aspect, called n = 3 and called m, o, p = 0. In another aspect, called n, o, p = 0 and called m = 3.

In some embodiments, the SAP includes or? consisting of a sequence of amino acids as indicated in SEQ ID NO: 1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, 15.

Sample SAPs are provided in Table 1 below.

Table 1:

SEQ ID NO: Peptide sequence

1 AcN-RIESRIES-CONH2

2 AcN-RIESRIESRIES-CONH2

3 AcN-RIESRIESRIESRIES-CONH2

7 AcN-SEIRSEIRSEIR-CONH2

8 AcN-RIESSEIRSEIR-CONH2

9 AcN-RLESRLESRVDS-CONH2

10 AcN-KIESKIESRVDS-CONH2

11 AcN-HIESHIESHIES-CONH2

12 AcN-HVDSRIDSKLDS-CONH2

13 AcN-KLESHLDSRLDS-CONH2

14 AcN- KLDSHLDSRIDS -CONH2

15 AcN-SDLKSEIRSEIR-CONH2

16 AcN-KLDSSELHSDIR-CONH2

17 AcN-RIESRIESSEIR-CONH2

In a further embodiment, a composition comprising at least one SAP according to the present invention is described here.

In a preferred embodiment, said at least one SAP? present in said composition in a concentration between 0.1 and 10% (w / v) or between 0.5 and 5% (w / v).

In one embodiment, in said composition at least about 95% or at least about 99% of said SAPs are identical in length and sequence. In one embodiment, said composition? an aqueous solution. In one embodiment, the use of said SAP? in vitro, that is? for cell culture experiments.

The following examples are not intended to limit the invention, where the invention? understood as defined in the following claims.

Example 1: characterization of soy proteins

Was total soy protein, after extraction, digested using pepsin and total hydrolyzate? characterized with HPLC-MS / MS (Lammi et al. Soybean Peptides Exert Multifunctional Bioactivity Modulating 3-Hydroxy-3-Methylglutaryl-CoA Reductase and Dipeptidyl Peptidase-IV Targets in Vitro J. Funct. Foods 2019; 55,135-145). Overall, more? of 70 sequences of peptides and the corresponding parent proteins and, among these, the peptide LKPDNRIESEGGL (SEQ ID NO: 19), which belongs to glycinine G1 (PDB ID 1UCX), highlighted why? in the whole peptide, a? -strand, corresponding to the RIES sequence,? including (Figure 1A). Through bioinformatics resources (ie GRAVY and ExPASy) the properties have been analyzed? physical and chemical characteristics of the RIES peptide. Using the GRAVY and Hphob methods. / Kyte & Doolittle, the hydropathy index? was estimated to be -1.075 (Figure 1B). Also, using the? -Sheet / Chou & Fasman? It has been estimated that the peptide has a good propensity to form stable? -sheet structures outside the protein environment in which it is found (Figure 1C). In the end, ? has it been shown that the amino acids of the RIES peptide have good accessibility? solvents that can guarantee the start of the self-assembly process (Figure 1D). The data surprisingly demonstrated the propensity of the RIES peptide to spontaneous self-assembly due to its secondary structure and physical-chemical behavior.

Example 2: synthesis of peptides

The peptides according to an embodiment of the present invention were synthesized according to a solid phase synthesis method Fmoc. The peptides were synthesized on Rink's 4-methyl-benzhydrylamine amine resin (0.5 mmol? G <-1> substitution) using an automated microwave synthesizer. The amino acids protected with Fmoc were dissolved at 0.2 M in DMF and the deprotection solution used for the removal of the Fmoc groups was a 20% v / v solution of 4-methylpiperidine in DMF. 0.5 M HBTU in DMF and 2 M DIEA in NMP were used as activator and activator base solution, respectively. Removal and cleavage of peptide side chains? was performed with 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% triisopropylsilane. The C terminus of peptides? been amidated after cleavage of the peptide sequence and the N terminus? was acetylated using a 20% v / v solution of Ac2O in DMF. The cleaved peptides were precipitated, washed three times with cold diethyl ether and dissolved in 25% acetonitrile solution before being lyophilized and stored at -20 ° C. The resulting crude peptide? was purified by binary HPLC (> 95%) on a Prep C18 column, using a gradient of acetonitrile in H2O with 0.1% TFA in 20 minutes. The molecular weight of the purified peptide? was identified by MALDI-TOF spectroscopy (4800 Applied Biosystems). The purified peptide powder? it was subsequently dissolved in a 0.1 M HCl solution to remove the presence of possible TFA salts.

Example 3: formation of peptide hydrogels

The peptides obtained according to example 1 were dissolved in deionized water at a final concentration of 0.5%, 1%, 3% and 5% (w / v) by sonication for 30 minutes. The formation of peptide hydrogels? was obtained by priming DPBS, HBSS and DMEM. The results are shown in Table 2.

Table 2: Properties physico-chemical and structural of the peptides, in which the compounds SEQ ID NO: 1, 2, 3 and 7 are according to the invention and the compounds SEQ ID NO: 4, 5 and 6 are for comparative purposes.

Training

SEQ Solubility? of the gel in

ID Peptide sequence ID in water DPBS, PM (g Net charge at NO: 1% (w / v) HBSS, mol-1) pH 7

DMEM

1 AcN-RIESRIES-CONH2 RIES8 - 1030.1

4 0 2 AcN-RIESRIESRIES- RIES12 1515.6

CONH2 7 0

AcN-3 RIESRIESRIESRIES- RIES16 / - 2001.2

CONH 1 0

2

4 AcN-SIERSIERSIER-

CONH SIER12 - 1515.6

2 7 0

5 AcN-IRSEIRSEIRSE-

CONH IRSE12 - 1515.6

2 7 0

6 AcN-SERISERISERI- 515.6

CONH SERI12 - - 1

2 7 0

7 AcN-SEIRSEIRSEIR-

CONH SEIR12 1515.6

2 7 0

Example 4: rheological tests

The properties rheological characteristics of the peptides of the present invention were studied with an AR-2000ex rheometer (TA Instruments). ? a truncated cone geometry was used (truncated acrylic diameter 20 mm; truncation 34? m; angle 1%). All measurements were obtained at 25 ° C using a Peltier cell as the bottom plate of the instrument to control the temperature during each test.

The memory (G ') and loss (G ") modules were recorded as an angular frequency of function (0.1-1000 Hz) with a fixed deformation of 1%. The assembly of all peptides was activated adding DMEM laterally to the peptide solution positioned in the truncation space of the 34µm conical plate. The results obtained are reported in Table 3. Table 3: Viscoelastic parameters of 1% (w / v) peptides, in which the SEQ compounds ID NO: 1, 2, 3 and 7 are according to the invention and the compounds SEQ ID NO: 4, 5 and 6 are for comparative purposes.

SEQ ID NO: Peptide sequence ID G? (Pa) G? (Pa)

1 AcN-RIESRIES-CONH2 RIES8 NA NA

2 AcN-RIESRIESRIES-CONH2 RIES12 1285 182

3 AcN-RIESRIESRIESRIES-CONH2 RIES16 150 34

4 AcN-SIERSIERSIER-CONH2 SIER12 NA NA

5 AcN-IRSEIRSEIRSE-CONH2 IRSE12 NA NA

6 AcN-SERISERISERI-CONH2 SERI12 NA NA

7 AcN-SEIRSEIRSEIR-CONH2 SEIR12 2495 323

A comparison between G 'and G' 'for some exemplary compounds, RIES12, RIES16 and SEIR12,? shown in Figure 2. Hydrogel frequency sweep experiments were recorded as a function of angular frequency (0.1-100 Hz) at a fixed stress of 1%. In each assembled peptide, the elastic (G ', black circles) and loss modulus (G', white circles) trends showed typical hydrogel profiles, characterized by a predominantly solid elastic behavior (G ') with respect to the viscous component ( G "). Indeed, the values of G? they were generally of an order of magnitude greater than G. "

Figure 3, panels A and B, shows that increasing the concentration of RIES12 (from 0.5%, square, to 5%, diamond) increases the value of G ', indicating modularity? of the peptide for different application needs. To test the injectability? of the peptide solutions, the shear thinning tests were performed with a series of peak maintenance tests in which the velocities? were kept constant. First of all, ? a speed has been applied? cut of 0.01 s <-1> for 60 s, then? a speed has been applied? of cut of 5.3 s <-1> for 20 s, to simulate the speed? inside the barrel of the syringe. Subsequently, ? was applied a high speed? 1000 s <-1> cutoff for 20 s to simulate solution purge injection. Subsequently ? was again applied a speed? of cut of 5.3 s <-1> (20 s), imitating cos? the flow of the peptide solution out of the needle. In the end, ? was performed a speed? cut of 0.01 s <-1> to simulate the low cut condition of the solution during injection.

An illustrative result? shown in Figure 4, where the thixotropic test suggests a thinning behavior of RIES12 thanks to the rapid recovery of viscosity. initial after sham injection.

Example 5: circular dichroism (CD) and thioflavin T (ThT) spectroscopy assay

The CD spectra of peptides according to the present invention were recorded in the modality. continuous scanning (190-300 nm) at 25 ° C using the Jasco J-810 spectropolarimeter (Jasco Corp., Tokyo, Japan). Samples were prepared by diluting the peptide stock solutions in distilled water (1%) to a working concentration of 0.001%. All spectra were collected using a 1 mm path length quartz cell and averaged over three accumulations (velocity: 10 nm? Min <-1>). The reference spectrum of distilled water? been recorded and subtracted from each spectrum. The estimation of the secondary structure of the peptide? was obtained using a Russeau method.

Figure 5, panel A, shows an exemplary result observed with RIES12. The RIES12 CD model includes a negative peak close to 218 nm and a positive peak at 195 nm, characteristic of the? Sheet conformation.

ThT analysis of peptides? was then performed to assess the presence of fibril structures? -fsheet. The ThT stock solution (Sigma-Aldrich, T3516)? was prepared by adding 8 mg of ThT to 10 ml of phosphate buffer (10 mM phosphate, 150 mM NaCl, pH 7) and filtered through a 0.2 μm syringe filter. Shortly before analysis, 1 ml of ThT stock solution? been diluted in 50 ml of phosphate buffer (working solution). The peptides (1% w / v) were mixed with the working solution (1: 0.5 v / v) and stirred for 4 minutes. The intensity of ThT fluorescence? recorded using an Infinite M200 PRO (Tecan) plate reader with kex = 440 nm (5 nm band pass) and kem = 482 nm (10 nm band pass), over 60 seconds at 25 ° C. The exemplary results obtained with RIES12 are shown in Figure 5, panel B, which shows the typical amyloid-binding emission signal (peak at about 490 nm), reflecting the rich nature of? of the RIES12 hydrogel.

From the CD and ThT tests, the secondary structure of SEQ ID NO: 2? been estimated and the results are shown in Table 5.

Table 5:

SEQ ID NO: Peptide sequence ID? -Sheet? -Turn Random coil

2 AcN- RIESRIESRIES-CONH2 RIES12 70% 12% 18%

Example 6: atomic force microscopy

Were the AFM images captured in mode? interception, using single beam silicon cantilever probes (Veeco RFESP MPP-21100? 10, cantilever f0, 59-69 kHz; constant force: 3 N? m <-1>). The peptides from stock solutions (1% w / v) were diluted to a working concentration of 0.01% (w / v) and deposited on a freshly cracked mica surface. 2 microliters of each solution were kept on the mica for 4 minutes at room temperature. The samples were subsequently rinsed with distilled water to remove loosely bound peptides, then dried at room temperature for 30 minutes. 100 different nanofibers of approximately 10 independent fields per sample were measured and characterized.

The exemplary images of AFM are shown in Figure 6, panel A, RIES12; panel B, SEIR12; panel C, RIES16 (comparative) and panel D HydroMatrix peptide hydrogel (comparative) at 1% (w / v). The graphs show the distribution of the width derived from the evaluation of three different fields for each sample. The results obtained using the peptides according to the present invention clearly show long and narrow nanofibers with a width of 5-12 nm and a height of 2-6 nm. When using SAP of the known art such as RIES16 or HydroMatrix are observed nanofibers pi? short with 1-6 nm in width and approximately 1 nm in height. Also, as shown in Graphs A and B versus Graphs C and D, the distribution of the width? bimodal when using the peptides according to the present invention, in which a Gaussian distribution is observed with the peptides of the known art.

Example 7: stability test thermal

The lyophilized SAPs of the present invention were dissolved in distilled water to reach a concentration of 1% w / v, according to example 2. Then the peptides were kept at 4 ° C and their stability was maintained at 4 ° C. thermomechanics? was evaluated immediately after solubilization of the peptide, after 1 week and 2 weeks after solubilization. To test the stability? thermo-mechanical,? a controlled temperature gradient as a function of G 'was recorded (Trate = 5? C min <-1>, deformation of 1%,? = 1 Hz). The exemplary results obtained with RIES12 are shown in Figure 7, which shows the maintenance of module G? in time. The same experiment, when repeated with other peptides according to the present invention, gave the same results (data not shown).

Example 8: test of vitality? cellular (MTT).

The RIES12 peptide MTT assay? was performed in order to evaluate its potential cytotoxic effect on human intestinal Caco-2 and human liver HepG2 cells, respectively. For 2D cell culture on RIES12 hydrogel (0.5-1% w / v), Caco-2 and HepG2 cells at density? of 5? 10 <4> / well were seeded on the surface of the RIES12 hydrogel in clear 96-well plates and incubated for 5- and 3 days, respectively, at 37? C with 5% CO2 atmosphere. HydroMatrix (Sigma A6982)? been used as a control. Subsequently, the medium out of stock? was removed and 100 µl / well of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) was added. After 2 hours of incubation at 37 ° C in an atmosphere of 5% CO2, 0.5 mg / mL of solution was aspirated and 100? L / well of the lysis buffer (8 mM of HCl 0.5% NP- 40 in DMSO). After 5 minutes of slow stirring, the absorbance at 575 nm? was read using the Synergy H1 fluorescence plate reader (Biotek, Bad Friedrichshall, Germany). The data reported in Figure 8 indicate that the peptides according to the present invention are safe and effective. HydroMatrix (Sigma A6982), a state-of-the-art hydrogel,? included for comparison purposes. The RIES12 hydrogel? safe for both human intestinal Caco-2 cells (panel A) and human HepG2 liver cells (panel B), respectively. Both cell lines can easily grow on top of the hydrogel. Compared to HydroMatrix 1% w / v, the results obtained using the peptides according to the present invention suggest that Caco-2 and HepG2 cells can be grown on RIES12 hydrogel, without compromising their bioavailability. Pi? in detail, for Caco-2 cells, any cytotoxic effects and morphological changes were observed after 5 days of culture on 0.5% and 1% (w / v) of RIES12 vs HydroMatrix 1% (w / v) of hydrogel , respectively. Also, when did Caco-2 cells grow above RIES120.5% (w / v)? was a significant improvement in their vitality observed? of 29.7% and 21.2% compared to HydroMatrix and RIES12 at 1% (w / v), respectively (p <0.5), suggesting that based on the property biomechanics of the intestinal cell system, a bio-material pi? rigid can influence its growth. Cytotoxic effects and morphological changes were observed for HepG2 cells even after 3 days of culture on RIES12 hydrogel (1% w / v). When are human liver cells cultured on RIES12 hydrogel (1% w / v)? has been observed a significant increase in their vitality? 56.4% compared to HydroMatrix (1% w / v) (p <0.001). (bar 100 micron).

Claims (11)

CLAIMS 1. A self-assembling peptide (SAP) comprising a sequence of natural and / or synthetic amino acid residues, chemically modified amino acids and / or chemically synthesized compounds which have properties known in the art as characteristics of an amino acid, in which said sequence? as indicated in Formula I: [XYZSer] n [SerZYX] m [XYZSer] o [SerZYX] p Formula I, where is it each (X)? independently a positively charged amino acid; each (Y)? independently a non-polar amino acid with hydrophobic side chains; each (Z)? independently a negatively charged amino acid; n = 0 or an integer equal to 1, 2, 3 or 4; m = 0 or an integer equal to 1, 2, 3 or 4; o = 0 or an integer equal to 1, 2, 3 or 4; p = 0 or an integer equal to 1, 2, 3 or 4; m n or p? an integer equal to 2, 3, or 4. 2. Self-assembling peptide (SAP) according to claim 1, wherein each (X)? independently a positively charged amino acid selected in the group consisting of Arg, Lys and His; each (Y)? independently a non-polar amino acid selected from the group consisting of Ile, Leu, Val and Ala; each (Z)? independently a negatively charged amino acid selected from the group consisting of Asp and Glu. A self-assembling peptide (SAP) according to claim 1 or 2, wherein said (X)? Arg, said (Y)? independently selected from the group consisting of Ile, Leu, Val and Ala and said (Z)? independently selected from the group consisting of Asp, Glu. 4. Self-assembling peptide (SAP) according to claim 1 or 2, wherein said (Y)? Ile, said (X)? independently selected from the group consisting of Arg, Lys, His and said (Z)? independently selected from the group consisting of Asp, Glu. 5. Self-assembling peptide (SAP) according to claim 1 or 2, wherein (Z)? Glu, said (X)? independently selected from the group consisting of Arg, Lys, His and said (Y)? independently selected from the group consisting of Ile, Leu, Val and Ala. A SAP according to any one of claims 1-5, wherein m n or p = 3, or n = 1, m = 2, o = 0, p = 0 or n = 2, m = 1, o = 0, p = 0, or n = 3 and m, o, p = 0 or m = 3 and n, o, p = 0. A SAP according to any one of claims 1-6, the self-assembling peptide comprising an N-terminal functional group, a C-terminal functional group or both. A SAP according to any one of claims 1-7, comprising or consisting of a sequence of amino acids as indicated in SEQ ID NOs: 1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17. A composition comprising at least one SAP according to any one of claims 1-8. 10. Composition according to claim 9, wherein said peptides are in a concentration of between 0.1 and 10% (w / V) or between 0.5 and 5% (w / V). 11. Use of peptides extracted from natural products, preferably from legumes, to obtain a new class of bioinspired nanomaterials.