EP3973550B1

EP3973550B1 - Peptide ionogel composition, methods and uses thereof

Info

Publication number: EP3973550B1
Application number: EP20732663.8A
Authority: EP
Inventors: Ana Cecília AFONSO ROQUE; Carina Alexandra MARQUES ESTEVES; Rein VINCENT ULIJN
Original assignee: NovaIdFct Associacao Para A Inovacao E Desenvolvimento Da Fct; Universidade Nova de Lisboa; Research Foundation of City University of New York
Current assignee: NovaIdFct Associacao Para A Inovacao E Desenvolvimento Da Fct; Universidade Nova de Lisboa; Research Foundation of City University of New York
Priority date: 2019-05-20
Filing date: 2020-05-20
Publication date: 2024-01-31
Anticipated expiration: 2040-05-20
Also published as: EP3973550A1; EP3973550C0; WO2020234806A1

Description

TECHNICAL FIELD

The present disclosure relates to unprotected tripeptide ionogel compositions, respective production processes and characterization with application in a variety of technological fields.

BACKGROUND

Hydrogels are materials typically composed of more than 90% (w/w) of water. Peptides can self-assemble into hydrogels, representing a promising class of soft biomaterials that has received increasing attention during the last decades. The process of peptide self-organization occurs at a molecular level in a 'bottom-up' approach where the simplest biomolecules, such as peptides or amino acids, interact with each other in aqueous solutions in a coordinated manner. In this process, one-dimensional peptide aggregates evolve to form fibrils that coil into larger fibres, crosslinking to form a gel.
These supramolecular self-assembled gels are highly dynamic structures where the molecular self-assembly process, mediated by non-covalent interactions such as hydrogen bonds, ionic bonds (electrostatic interactions), hydrophobic or van de Waals interactions, can be triggered and reversed by several external stimuli, such as pH, temperature, ionic strength, or gelator concentration [1-4]. Several protected and unprotected short peptides (2 to 4 amino acids) have been shown to form hydrogels [5,6]. The capability of hydrogelation of unprotected peptides at physiological pH has also been previously reported [7-9].
One of hydrogels weakness is the low stability in non-aqueous environments. When exposed to air, for example at ambient room conditions, hydrogels rapidly lose moisture, shrink and turn brittle due to water evaporation. On the other hand, to turn hydrogels into conductive materials there is usually the need to incorporate dopants in the hydrogel formulation [10].
The total or partial substitution of water by non-evaporating solvents, as ionic liquids, can lead to air-stable conducting ionogels. Ionic liquids are molten salts composed by at least one organic salt of composition X⁺ and Y^-, wherein X⁺ represents the cation of the salt and Y^- represents the anion of the salt. The melting point of ionic liquids is below 100°C, and they show very low volatility. Ionic liquids are designer solvents in the sense that the anion and cation can be varied and tuned to different purposes. Ionic liquids are considered green solvents with very interesting properties, namely high ionic conductivity, large electrochemical window, low vapour pressure, excellent thermal, electrochemical and chemical stability, non-flammability, non-toxicity, biocompatibility and biodegradability. Interestingly, ionogels retain the interesting features of the ionic liquids they are composed of, but the immobilization of ionic liquids to yield advanced functional materials is still challenging [11]. The entrapment of ionic liquids within biological matrices following a designed "bottom-up" approach is challenging and largely unexplored.
As opposed to hydrogels, where water represents more than 90% (w/w) of the final gel formulation, in ionogels the water content is greatly reduced as ionic liquids totally or partially replace water.
Low molecular weight gelators (LMWGs) are small molecules with molecular weight less than 2000 Da. The physical gelation of low-molecular- weight compounds results from non-covalent bonds, such as hydrogen bonds. Typically, during a gelation process, gelator molecules are first self-assembled, producing fibrous assemblies. Then, these fibrous assemblies form a three-dimensional network structure, and gelation occurs by trapping solvent in the networks.
Short peptide sequences, including unprotected tripeptides, fall within this category of LMWGs. Research studies on ionogels prepared by supramolecular self-assembly of LMWGs, and more specifically biological molecules such as amino acid-based LMWGs, are still scarce. Hanabusa and co-workers reported two cyclic peptide amphiphile molecules based on aspartame, cyclo(L-β-3,7-dimethyloctylasparaginyl-L-phenylalanyl) and cyclo(L- β -2-ethylhexylasparaginyl-L- phenylalanyl)), as efficient supramolecular gelators for a wide range of ionic liquids between imidazolium, pyridinium, piperidinium, pyrazolidinium and ammonium-based families [12]. Moreover, they were the first to show that supramolecular gelation does not affect the ionic conductivity of the ionic liquids, being slightly influenced by the concentration of the gelator. In other example, Dutta and co-workers reported LMW amino acid-based molecules as ionogelators [13]. These were prepared from a precursor scaffold of hydrogelators and organogelators, an amino acid/dipeptide moiety at the head of an amphiphilic molecule with a free amine group at the N-terminus and a long hydrophobic chain at C-terminus. In order to understand ionogelation process's structure-property correlation, systematic variations of amino acids and protective groups were performed. The developed ionogels were exploited as adsorbing agents in environmental clean-up and as templates in the synthesis of TiO₂ nanoparticles. Other examples report the development of conductive, soft, elastic ionogels with countless applications in chemocatalysis and biocatalysis; metal recovery; pharmaceuticals; electrochemical devices such batteries, capacitors and solar cells; electrolytes; semi-solid lubricants; magnetorheological fluids and electrochemical devices; adsorbing agents; or solid electrolytes [14-18]. In all these above-mentioned examples of ionogels made by the gelation of ionic liquids by LMWGs, there is no report on the employment of unprotected short peptide sequences as ionogelators. Typically, when employing amino acid or peptide-based gelators, these are chemically modified with other chemical groups that promote the self-assembly process, which reduces the easiness of production of the low molecular weight gelator, and also reduces the sustainability, biodegradability and biocompatibility of the final gel. Ishioka and co-workers provide an example of the use of supramolecular gelators based on benzenetricarboxamides to produce ionogels of imidazolium ionic liquid [24].
These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

GENERAL DESCRIPTION

The present disclosure relates to a peptide ionogel composition (or formulation), wherein unprotected tripeptides are used as gelators. The tripeptide ionogels result from the gelation of ionic liquids with unprotected tripeptides that present self-assembly propensity.
Typically, protected peptides with self-assembly propensity are those chemically modified with functional moieties at one or more different sites, namely the C-terminus, the N-terminus or at the amino acid side chains. The protecting groups used for the modification have self-assembling properties usually due to the hydrophobicity and aromaticity, and include the 9-fluorenylmethyloxycarbonyl (Fmoc) group, or any groups containing aromatic tails, naphtyls, lipids, fatty acids, cyclic compounds, sugars, carbamide, or aliphatic tail-moieties [19]. Unprotected peptides are those that are not modified at the C-terminus, N-terminus or side chains with protecting groups presenting self-assembling propensity, as listed above.
The tripeptide ionogels here described are supramolecular peptide assemblies in ionic liquids. They are stable at ambient conditions, have thermal, electrochemical and chemical stability in a wide range of temperature and humidity conditions, are conducting, soft, self-healing, thermoreversible, have a large electrochemical window, are non-flammable, non-toxic, biocompatible and biodegradable materials. These materials can further encapsulate other stimuli-responsive elements, as for example optical probes made of liquid crystals, including liquid crystals presenting nematic, smectic or chiral phases.
In an embodiment, the major advantages and unique properties of the formulations and production methods of tripeptide ionogels described in the present disclosure are:

i) easy production of tripeptides, without the need to protect the tripeptide or to chemically modify the tripeptide with functional groups yielding self-assembling properties, as observed in all previous examples of small-peptide based low-molecular weight ionogelators;
ii) since ionic liquids are designer solvents, it is possible to tune the ionic liquid to the particular tripeptide of interest;
iii) chemical diversity and functionality of tripeptide ionogels is greater than in tripeptide hydrogels. In hydrogels, chemical functionality arises solely from the tripeptide sequence, whereas in ionogels both components (tripeptide and ionic liquid) confer chemical and functional richness to the system;
iv) tripeptide ionogels present faster gelation times when compared to equivalent tripeptide hydrogels, making them suitable for additive manufacturing;
v) when compared to equivalent tripeptide hydrogels, the resulting tripeptide ionogels remain chemically and physically unaltered when stored at ambient conditions of temperature (18-27°C), pressure (atmospheric pressure, 1013.25hPa) and relative humidity (30-80%), due to the low volatility of the ionic liquid employed;
vi) when compared to equivalent tripeptide hydrogels, the resulting tripeptide ionogels present ionic conductivity over time in the same range as the ionic liquid used to produce the ionogel, which is not possible to observe in hydrogels where conductivity is typically given by dopants;
vii) tripeptide ionogels are thermally stable and thermoreversible;
viii) tripeptide ionogels are self-healing materials;
ix) tripeptide ionogels retain the physico-chemical and functional features of the tripeptides and of the ionic liquids they are composed of;
x) tripeptide ionogels represent a way of immobilizing ionic liquids in stable formulations and gel matrices using very mild processing conditions;
xi) the tripeptide ionogel formulations and resultant materials withstand several processing and fabrication techniques including spin coating, flow-coating (doctor blade), electro- and blow-spinning, 3D-printing, patterning, metal depositions, to give rise to a variety of structures;
xii) tripeptide ionogel formulations and resultant materials can be processed into different formats including thin films - spread on flexible or rigid substrates with or without patterning and with or without chemical functionalization - or other structures with different 1D, 2D and 3D geometries (e.g. fibres, particles) to yield stable materials with distinct geometries;
xiii) it is possible to use mould casting or film coating methods to produce a layer of tripeptide ionogel material on top of a surface modified with interdigitated electrodes, silicon wafers, or other surfaces typically used in micro- and nano-fabrication;
xiv) tripeptide ionogels can incorporate other elements, as for example liquid crystals which self-assemble within the ionogel in a stimuli-responsive manner and which could further contribute as opto-electrical transducers to chemical and physical changes;
xv) the intrinsic properties of tripeptide ionogels and their potential to provide electrical and optical signals in the presence of chemical analytes in gas or liquid phases, or as a response to physical changes (e.g. temperature, pressure, electromagnetic field), allows its utilization as sensitive films, able to detect analytes and physical changes through different transduction principles (optical, electrical, opto-electric, piezoelectric). Several combinations of these sensitive films can be arranged due to the wide range of components available for the films, allowing the optimization of response patterns for a variety of applications;
xvi) the ease of fabrication of the ionogels, the numerous possible variations in their composition and the possibility of combining ionogels with different formulations, confer sensitivity and selectivity to the different technical applications;
xvii) the formulation and production methods of tripeptide ionogels have very low production costs;
xviii) the formulation components and production method have reduced environmental impact;
xix) the production method of tripeptide ionogels is scalable and compatible with mass production.

An aspect of the present disclosure relates to an ionogel composition comprising:

at least one ionic liquid selected from imidazolium ionic liquids, ammonium-based ionic liquids, or mixtures thereof; and
at least one unprotected tripeptide as a gelator, preferably ionogelator, wherein the tripeptide has self-assembly propensity and is selected from the following list: Lysine-Tyrosine-Phenylalanine, Lysine-Phenylalanine-Phenylalanine, Arginine-Phenylalanine-Phenylalanine, Aspartic acid-Phenylalanine-Tyrosine, and Tyrosine-Phenylalanine-Aspartic acid; or
10-700 mM of at least one unprotected tripeptide as a gelator, wherein the unprotected tripeptide comprises at least a phenylalanine.

In the present disclosure, a gelator is a substance capable of forming a gel.
In the present disclosure, an ionogelator is defined as a molecule responsible for the gelation of ionic liquids. Gelation is defined as a process in which the immobilization of a given solvent leads to the formation of soft materials.
In an embodiment, the tripeptide concentration ranges from 10 - 700 mM.
In an embodiment, the tripeptide concentration ranges from 20 - 600 mM, preferably 100 - 400 mM, more preferably 50 - 400 mM.
In an embodiment, the tripeptide concentration ranges from 20 -200 mM, preferably 20 - 100 mM.
In an embodiment, the unprotected tripeptides have an amide at the C-terminal, preferably Aspartic acid-Phenylalanine-Tyrosine, and Tyrosine-Phenylalanine-Aspartic acid peptides.
In an embodiment, the unprotected tripeptides comprise a molecular weight equal or inferior to 2000 g/mol, preferably between 270-540 g/mol.
In an embodiment, the ionic liquid concentrations range from 20-98 % (w/w).
In an embodiment, the ionic liquid is selected from phosphate-containing ionic liquids, chloride-containing ionic liquids, dicyanamide-containing ionic liquids, quaternary ammonium salts, or mixtures thereof.
In a further embodiment, the imidazolium-based ionic liquids are selected from a list comprising: 1-butyl-3-methylimidazolium dicyanamide, 1-ethyl-3-methylimidazolium dicyanamide, 1-butyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazolium chloride, 1 or mixtures thereof.
In an embodiment, the ammonium-based ionic liquids are selected from a list comprising: choline chloride, choline hydroxide, choline phosphate buffer, 2-hydroxyethylammonium formate, choline dihydrogenphoshate, dicholine monohydrogenphosphate, or mixtures thereof.
In an embodiment, the choline phosphate ionic liquid buffer system (choline phosphate buffer) is produced by the addition in equimolar amounts of the ionic liquids choline dihydrogenphosphate and dicholine monohydrogenphosphate. The water content of choline phosphate buffer ranges from 16-22% wt.
In an embodiment, the tripeptides ionogelation is triggered by changes on the pH, increasing the pH to 7-9, preferentially 7.5-8.5.
In an embodiment, the pH is increased by addition of sodium hydroxide, choline hydroxide or 1-butyl-3-methylimidazolium hydroxide.
In an embodiment, the tripeptides ionogelation is triggered by changes on the temperature or a buffer composition.
In an embodiment, the ionogels' water concentration is below 90% (w/w), preferably between 2-75% (w/w).
In an embodiment, the molecular self-assembly of the tripeptide occurs by non-covalent interactions such as hydrogen bonding, hydrophobic, electrostatic, van der Walls, and π-stacking, π-cation, π-sulphur, dipole-dipole, ionic, or metal-chelating interactions.
In an embodiment, the ionogel composition may further comprise an additive selected from the following list: citric acid, tartaric acid, ascorbic acid, glucose, sucrose, xylose, arginine, histidine, proline, urea, sorbitol, xylitol, glycerol, monoethylene glycol, menthol, lactic acid, betain, or mixtures thereof.
In an embodiment, ionogel composition might comprise choline dihydrogenphosphate:citric acid, choline dihydrogenphosphate:tartaric acid, choline dihydrogenphosphate:ascorbic acid, choline dihydrogenphosphate:glucose, choline dihydrogenphosphate:sucrose, choline dihydrogenphosphate:xylose; choline dihydrogenphosphate:arginine; choline dihydrogenphosphate:histidine; choline dihydrogenphosphate:proline; choline dihydrogenphosphate:sorbitol; choline dihydrogenphosphate:xylitol; choline dihydrogenphosphate:monoethylene glycol; choline dihydrogenphosphate:lactic acid; dicholine monohydrogenphosphate: citric acid; dicholine monohydrogenphosphate:tartaric acid; dicholine monohydrogenphosphate:ascorbic acid, dicholine monohydrogenphosphate:glucose, dicholine monohydrogenphosphate:sucrose; dicholine monohydrogenphosphate:xylose; dicholine monohydrogenphosphate:arginine; dicholine monohydrogenphosphate:histidine; dicholine monohydrogenphosphate:proline; dicholine monohydrogenphosphate:sorbitol; dicholine monohydrogenphosphate:xylitol; dicholine monohydrogenphosphate:monoethylene glycol; dicholine monohydrogenphosphate:lactic acid. In a further embodiment, the molar ratio (mol/mol) between the ionic liquid and the additive ranges from 1:1 to 1:10, preferably 1:1 to 1:2.
In an embodiment, the ionogel is stable at ambient conditions (temperature - 18-27°C, pressure - atmospheric, 1013.25hPa, relative humidity - 30-80%).
In an embodiment, the ionogel is soft, self-healable, thermoreversible, stimuli-responsive, non-flammable, non-toxic, biocompatible and biodegradable.
In an embodiment, the ionogel composition may further comprise a stimuli-responsive component.
In an embodiment, the stimuli-responsive component is a liquid crystal.
In an embodiment, the ionogel composition may be obtainable by moulding, casting, spin-coating, film coating, spinning, electrospinning, microfluidic, 3D-printing, ink-jet printing, patterning or metal deposition method.
Another aspect of the present disclosure relates to a disc, film, fiber, sphere, cube, mesh, channel or patterned structure comprising the ionogel composition of the present disclosure.
Another aspect of the present disclosure relates to an article comprising the ionogel composition of the present disclosure.
In an embodiment, the article is a sensing device, electronic device, opto-electronic device, flexible device, wearable device, conducting device, coating material, electrochemical device, electrolyte, semi-solid lubricant, adsorbent agent; pharmaceutical formulation, food product, or cosmetic product.
Another aspect of the present disclosure relates to use of the ionogel composition described in the present disclosure for additive manufacturing, chemocatalysis or biocatalysis, encapsulation, drug delivery and/or cell assays.
Another aspect of the present disclosure further relates to a kit or a reagent comprising the ionogel composition of the present disclosure.
Another aspect of the present disclosure further relates to a method for preparing the ionogel composition of the present disclosure, comprising the following steps:

obtaining at least one unprotected tripeptide, selected from the following list: Lysine-Tyrosine-Phenylalanine, Lysine-Phenylalanine-Phenylalanine, Arginine-Phenylalanine-Phenylalanine, Aspartic acid-Phenylalanine-Tyrosine with the C-terminus capped with an amide, and Tyrosine-Phenylalanine-Aspartic acid with the C-terminus capped with an amide;
obtaining at least one ionic liquid selected from imidazolium ionic liquid, ammonium-based ionic liquids, or mixtures thereof;
dissolving the unprotected tripeptides in the ionic liquids;
promoting gelation by increasing the solution pH to pH 7- 9, preferably pH 7.5-8.5, or by heating.

In an embodiment, the gelation is promoted by heating the solution up to 40-110°C, preferably 40-80°C.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

Figure 1 depicts a schematic and simplified representation of the formulation and production process of tripeptide ionogels.
Figure 2 illustrates a schematic representation of the tripeptide structures used to produce the ionogels. The tripeptides are grouped in System I and System II, according to the gelation trigger identified in tripeptide hydrogels. Tripeptides belonging to System I self-assemble into hydrogels when the gelation trigger is pH-dependent. Tripeptides belonging to System II self-assemble into hydrogels when the gelation trigger combines buffer composition (typically phosphate dependent) and temperature.
Figure 3 shows a schematic representation of the experimental procedure and overall results of KYF ionogels in [Bmim][DCA] and [Emim][DCA]. It also compares the results obtained with the hydrogel, 40mM KYF in deionized water. Additionally, being produced in different laboratories, with different tripeptide's batch provided by different commercial houses, it also shows the reproducibility of this procedure.
Figure 4 illustrates the importance of water content (% w/w) in the formulations of KYF gels: when the water content in the formulation is 98.7% (w/w), the tripeptide concentration is 27mM (hydrogel photo); when the water content in the formulation is 71.3% (w/w), the tripeptide concentration is 50mM (ionogel photo); when the water content in the formulation is 26.9% (w/w), the tripeptide concentration is 86mM (ionogel photo); when the water content in the formulation is 11.31% (w/w), the tripeptide concentration is 257mM (ionogel photo). Photos depict inverted glass vials, to show the formation of self-supporting gels (inverted glass vial test).
Figure 5 shows an embodiment that illustrates the stability of the tripeptide ionogels at temperature, humidity and pressure of ambient conditions, 2 days after production: photographic images of 560 mM KYF ionogels prepared in [Bmim][DCA] and 40 mM KYF hydrogels prepared in deionized water.
Figure 6 schematically shows an embodiment of the experimental procedure, and overall results of KFF ionogels in [Bmim][DCA] and [Emim][DCA]. It also compares the results obtained with the hydrogel, 40mM KYF in deionized water. Additionally, being produced in different laboratories, with different tripeptide's batch provided by different commercial houses, it also shows the reproducibility of this procedure.
Figure 7 illustrates an embodiment of optical microscopy images of produced KFF (146mM) ionogels in the ionic liquid [Bmim][DCA], final water content 12.0%. In these images, microstructures of self-assembled KFF are observed between 0 and 115.72 minutes after gel preparation.
Figure 8 shows photographic representations of gels made of 352 mM KFF in [Emim][DCA] (final water content: 15.25% (w/w)) and 191 mM KFF in the ionic liquid [Bmim][DCA], (final water content: 11.26% (w/w)) between day 1 and month 5.5 after ionogel production. Photos depict inverted glass vials, to show the formation of self-supporting gels (inverted glass vial test). The insets illustrate (A and B) bright field and (C and D) polarised optical microscopy images of KFF self-assembly in the ionic liquid [Bmim][DCA] when the ionogel formulation comprises 191 mM KFF in the ionic liquid [Bmim][DCA], (final water content: 11.26%) at day 1 and month 4 after production.
Figure 9 illustrates the ATR-FTIR transmission spectra of (a) KYF ionogels in (i) [Bmim][DCA] and (ii) [Emim][DCA] and (b) KFF ionogels in (i) [Bmim][DCA] and (ii.) [Emim][DCA]. ATR-FTIR spectra of the ionic liquids [Bmim][DCA] and [Emim][DCA] are also shown.
Figure 10 illustrates an embodiment of the conductivity of tripeptide ionogels, giving as an example the conductivity of KYF ionogel in [Bmim][DCA] (320 mM KYF in [Bmim][DCA], final water content: 13.43% (w/w)). In A) it is shown a schematic of the apparatus to demonstrate the ion conduction of the tripeptide ionogel by lightening up a light-emitting diode (LED); in B) a photograph of the real set-up is shown. Similar results are observed for KYF in [EMIM][DCA], KFF in [BMIM][DCA] and KFF in [EMIM][DCA].
Figure 11 illustrates the chemical structure of the ionic liquids choline dihydrogenphosphate (ChDHP) and dicholine monohydrogenphosphate (DChHP) used to produce the choline phosphate buffer and the additive citric acid (CA) used on the nonconventional solvent DChHP:CA.
Figure 12 illustrates an embodiment of gels obtained with distinct formulations using System II tripeptides. The hydrogel presented for RFF has 30 mM tripeptide in sodium phosphate buffer 0.1 M pH 8.0, the hydrogel presented for DFY-NH₂ has 20 mM tripeptide in sodium phosphate buffer 0.1 M pH 8.0 and the hydrogel presented for YFD-NH₂ has 20 mM tripeptide in sodium phosphate buffer 0.1 M pH 8.0. For the presented ionogels, the compositions are as follows: 30mM RFF ionogel in choline phosphate buffer, 16% (w/w) water and 20 mM DFY-NH₂ in choline phosphate buffer, 16% (w/w) water. Moreover it is also presented an embodiment of the self-supported tripeptide ionogel composed of RFF:dicholine monohydrogenphosphate:citric acid with the following molar ratios: 0.008:1:1. Photos depict inverted glass vials, to show the formation of self-supporting gels (inverted glass vial test).
Figure 13 schematically shows an embodiment of the experimental procedure and overall results of RFF in [Bmim][DCA] and its successful gelation in choline phosphate buffer.
Figure 14 schematically shows an embodiment of the experimental procedure and overall results of DFY-NH₂ in [Bmim][DCA] and in choline phosphate buffer.
Figure 15 illustrates the ATR-FTIR transmission spectra of the 20 mM hydrogels (in 0.1 M D₂O sodium phosphate buffer pH 8.0) and 20 mM ionogels (in D₂O choline phosphate buffer, apparent pH 7.1) using the tripeptides DFY-NH₂ and YFD-NH₂.
Figure 16 illustrates A) an embodiment of the mechanical properties of DFY gels, hydrogel and ionogel. Storage (G') and loss modulus (G") as a function of the angular frequency of the gels formed by DFY (30 mM in choline phosphate buffer at an apparent pH 7.1 and sodium phosphate buffer at pH 8.0). B) Photographic image showing that despite being a gel, it is still possible to be pipetted.
Figure 17 illustrates A) a transmission electron microscopy image (TEM) for an embodiment of 30 mM DFY-NH₂ ionogel prepared in choline phosphate buffer, apparent pH 7.1. In B) it is presented a histogram showing the fibers diameter measured for 232 fibers.
Figure 18 shows an embodiment of the ionic conductivity of 30 mM DFY-NH₂ ionogel at day 1 and day 50 after production, at 20°C. At 1 kHz, ionic conductivity of DFY-NH₂ ionogel was 1.0×10^-3 S.cm^-1. Choline phosphate buffer is also presented. The inset presents ionic conductivity of 30 mM DFY-NH₂ ionogel 1 hour after production.
Figure 19 illustrates A) an embodiment of the stability over time of DFY gel (30 mM in choline phosphate buffer at an apparent pH 7.1 and sodium phosphate buffer at pH 8.0), as compared to DFY hydrogel when exposed to environmental conditions; and B) the variation of ionogel weight over time.
Figure 20 illustrates an embodiment of the thermoreversibility of 30 mM DFY-NH₂ ionogels in choline phosphate buffer, upon heating the ionogels from room temperature to 75°C, and then cooling down back to room temperature.
Figure 21 illustrates optical microscopy images of thin films (produced by film coating method) of 30 mM and 50 mM DFY-NH₂ ionogel formulations in choline phosphate buffer. At day 3, a scratch was inflicted in the gel and at day 6 the scratch is no longer visible, indicating the self-healing properties of the ionogel materials.
Figure 22 illustrates the conductivity of tripeptide ionogels, giving as an example conductivity of 20 mM DFY-NH₂ ionogel prepared in choline phosphate buffer, apparent pH 7.1, 16% (w/w) water content (determined using a Karl Fischer). In A) a schematic representation of an embodiment of the apparatus used to demonstrate the conduction of the tripeptide ionogel by lightening up a light-emitting diode (LED) is shown; in B) a photograph of the real set-up is shown. In C) it is shown an embodiment of a thin film of choline dihydrogenphosphate:citric acid (1:1) conducting material mediating electron transfer into a LED.
Figure 23 shows an embodiment of the humidity effect on the electrical properties of 50mM DFY-NH₂ ionogel thin films. Figure 23A presents a schematic illustration of an in-house built electronic nose used to perform the experiment. In B) the electrical signals of DFY-NH₂ ionogel thin films when exposed to cycles of humidification and drying between 0% and different relative humidity (RH) levels (i.) 58%, ii.) 50%, iii.) 44% and iv.) 25%) are observed. The variation of the amplitude of the electrical response as a function of %RH is shown in figure 23C.
Figure 24 shows an embodiment of polarised optical microscopy images of 517 mM KYF ionogels in [Bmim][DCA], 23.70% (w/w) water content and 560 mM KFF ionogels in [Bmim][DCA], 17.58% (w/w) water content. Both ionogels incorporate the liquid crystal 5CB (4-Cyano-4'-pentylbiphenyl), as stimuli-responsive optical probes, preferentially droplets within radial configuration.

DETAILED DESCRIPTION

The present disclosure relates to a new soft gel material, prepared through the gelation of ionic liquids using unprotected tripeptides.
The rationale to design the tripeptide ionogel formulations and respective production methods started by understanding the gelation trigger and gelation protocols of previously described unprotected tripeptide hydrogelators. The gelation trigger includes, for example, changes in pH, ionic strength or temperature [7-9]. Once the gelation trigger was understood, the ionic liquids were designed to mimic this trigger. Also, the ionogel production protocols were designed accordingly. With this approach, the aqueous solvent of the hydrogels was totally or partially substituted by rationally designed ionic liquids.
Understanding the gelation trigger is crucial in the development of new self-assembled biomaterials. Two sets of systems were identified, where tripeptides hydrogelation is triggered by pH (System I) or triggered by buffer composition and temperature (System II) (Figure 2).

In System I hydrogels, tripeptides with the following amino acid sequences Lysine(K)-Tyrosine(Y)-Phenylalanine(F) (KYF), Lysine(K)-Phenylalanine(F)-Phenylalanine(F) (KFF) have been described as hydrogelators by Frederix et al. [7]. When dissolved in deionized water, these unprotected tripeptides are capable of forming a transparent hydrogel only when the pH value is increased to 7.5 ± 1.0 by adding sodium hydroxide (1 M). Following the same approach, KYF has also shown the capability to form hydrogels when dissolved in other aqueous solvents such as sodium chloride (5 mM), addition of sodium hydroxide (1 M) for the final pH value, or sodium phosphate buffer solution 0.1 M pH 8 (Table 1), but only when the pH value is in the range of 7.5 ± 1.0. KFF did not self-assembly in sodium phosphate buffer (0.1 M pH 8.0).

Table 1: System I hydrogels, tripeptide hydrogelation triggered by pH.

Solvent (aqueous solutions)	Gelator
Solvent (aqueous solutions)	KYF	KFF
Deionized water (+NaOH)*	Gel (20 mM)	Gel (40 mM)
5 mM NaCl (+NaOH)*	Gel (30 mM)	Not tested
0.1M Sodium phosphate buffer, pH 8.0	Gel (40 mM)	Solution

NaCl, sodium chloride; *Add NaOH (1M) to raise the pH to 7.5±1. Figures in parenthesis are gelation concentrations.

In System II hydrogels, the tripeptides Arginine(R)-Phenylalanine(F)-Phenylalanine(F) (RFF) and the previously described [9] hydrogelators, Aspartic acid(D)-Phenylalanine(F)-Tyrosine(Y) with the C-terminus capped with an amide (DFY-NH₂) are capable of forming transparent and clear gels when dissolved by heat in sodium phosphate buffer 0.1 M pH 8.0 and after cooling down to room temperature; in these same conditions Tyrosine(Y)-Phenylalanine(F)-Aspartic acid(D) with the C-terminus capped with an amide (YFD-NH₂) forms an opaque gel [6,9] (Table 2). DFY-NH₂ was also able to form a transparent gel when dissolved by heat in 0.2 M Citrate-0.1 M Phosphate buffer pH 7.0. However, this tripeptide precipitates when the solvent is 0.1 M Citrate buffer pH 6.2. In System II, the gelation trigger seems to be mainly phosphate dependent.

Table 2: System II hydrogels, tripeptide hydrogelation triggered by buffer composition and temperature.

Solvent	Gelator
Solvent	RFF	DFY-NH₂	YFD-NH₂
Deionized water (+NaOH)*	Solution	Insoluble	Not tested
0.1 M Sodium phosphate buffer, pH 8.0	Gel (30 mM)	Gel (20 mM)	Gel (20 mM)
0.1 M Glycine-NaOH buffer pH 8.6	Solution	Not tested	Not tested
0.2 M Citrate -0.1 M Phosphate buffer pH 7.0	Gel (30 mM)	Not tested	Not tested
0.1M Citrate buffer pH 6.2	Precipitate (30 mM)	Not tested	Not tested

* Add NaOH (1M) to raise the pH to 7.5±1. Figures in parenthesis are gelation concentrations.

After understanding the hydrogelation triggers in Systems I and II, the rational design and application of ionic liquids in the tripeptide ionogel formulations and respective protocols was performed.
In the case of System I the gelation is triggered by increasing the pH. For this system, basic imidazolium-based ionic liquids and ammonium-based ionic liquids were selected. The imidazolium-based ionic liquids can be selected from a list comprising 1-butyl-3-methylimidazolium dicyanamide ([Bmim][DCA]), 1-ethyl-3-methylimidazolium dicyanamide ([Emim][DCA]), 1-butyl-3-methylimidazolium chloride ([Bmim][CI]), 1-ethyl-3-methylimidazolium chloride ([Emim][CI]), or mixtures thereof.. The ammonium-based ionic liquids considered were choline chloride, choline hydroxide, choline phosphate buffer, and 2-hydroxyethylammonium formate ([HEtNH₃]CO₂ ^-]). The choline phosphate buffer is an ionic liquid buffer system formed by the mixture of two ionic liquids (i) choline dihydrogenphosphate and (ii) dicholine monohydrogen phosphate in equimolar amounts, to which 16-17% (w/w) deionized water are added. The pH of the choline phosphate buffer is 7.1±1.0, and is called the apparent pH (Figure 11).
Imidazolium-based ionic liquids are defined as those where the cation is a charged heterocycle organic molecule, an imidazole ring where one of the nitrogen atoms is protonated such as dialkylimidazolium. By its turn, ammonium-based ionic liquids are composed by an ammonium cation, a positively charged or protonated amine or a quaternary ammonium cation of the basic structure NR₄ ⁺, where one or more hydrogen atoms can be replaced by alkyl groups and/or alkyl groups with hydroxy group attached (R). As example an alkylammonium.
In an embodiment for System I, tripeptides (KYF and KFF) were dissolved in ionic liquids at a given concentration in a glass vial at room temperature (18-27°C), by means of vortex and sonication, followed by pH increasing to 7.5 ± 1, by dropwise addition of alkaline solutions, such as, sodium hydroxide (1 M or 2 M), potassium hydroxide, [Bmim][OH], or choline hydroxide.
It was observed that both tripeptides KYF and KFF were ionogelators. KYF and KFF tripeptides, when dissolved in ionic liquids of imidazolium-family, [Bmim][DCA], [Bmim][CI], [Emim][DCA] and [Emim][CI], and after rising the pH to 7.5 ± 1.0 (by the addition of NaOH solutions (1 M, 2 M), [Bmim][OH] or choline hydroxide), formed self-supporting gels (Figures 3 and 6).
In an embodiment, the gelator KYF formed a transparent and clear gel when dissolved in [Bmim][DCA] and in [Emim][DCA] (Figure 3). For these systems, the molecular self-assembly occurred at 412 mM, a high gelation concentration when compared with the one needed for a hydrogel formation (20 mM) (Table 1). These results might be related with material's water content that is lower for the ionogel (11% (w/w)) than for the hydrogel (98% (w/w)). When ionogel's water content was raised to 26.9 % (w/w), the amount of KYF ionogelator needed dropped to 86 mM and when the water content was 71.3% (w/w), the amount of KYF needed was 50 mM. These observations suggest that hydrogen-bonding of water molecules plays an important role, favouring KYF self-assembly (Figure 4).
In another embodiment, the tripeptide KFF formed opaque gels with self-assembled structures with birefringence properties when dissolved in [Bmim][DCA] and [Emim][DCA] and after rising the pH to 7.5 ± 1.0 (by the addition of NaOH solutions (1 M, 2 M), [Bmim][OH] or choline hydroxide). The formation of self-assembled structures with birefringence properties was observed in real-time by optical microscopy (Figures 7 and 8) in a similar process to the one described by Wang et al.[20]. With time, the opaque gels, containing the birefringent structures, changed to transparent clear gels, suggesting the 'evolution' to fibrillary structures (Figure 8). A high gelation concentration was needed for the tripeptide KFF molecular self-assembly to occur: the gelation concentration for [Emim][DCA] was 352 mM while for [Bmim][DCA] was 191 mM (Table 3). Table 3: Examples of gelation formulations for the System I tripeptides KYF and KFF.

Ionic liquids Gelators

KYF KFF

[Bmim][DCA]* Gel (412 mM) Gel (191 mM)

[Emim] [DCA]* Gel (447 mM) Gel (352 mM)

* Add NaOH (1M) to raise the pH to ≈7.5. Figures in parenthesis are typical example of tripeptide concentrations yielding ionogels.
In an embodiment, after observing the formation of ionogels using the System I tripeptides and the [Bmim][DCA] and [Emim][DCA] ionic liquids, different ionic liquids of the [Bmim]⁺-based family were tested. Here, the cation component was constant ([Bmim]⁺) and the anion was varied. It was observed the formation of self-supporting ionogels by the vial inversion test for the tripeptides KFF and KYF in Bmim DCA, Bmim Cl, Emim DCA and Emim Cl.
In the case of System II tripeptides, the gelation trigger seems to be phosphate dependent. In this sense, in the design of an ionic liquid to be gelated by these System II peptides, the aqueous solvent was mimicked by an ionic liquid buffer system, the choline phosphate buffer [21]. The ionic liquid buffer, with an apparent pH of 7.1±1, has a water content of 16% - 17% (characterization performed by Karl Fischer) (Figure 11).
In an embodiment for System II tripeptides (RFF, DFY-NH₂ and YFD-NH₂), these were dissolved in ionic liquid, ionic liquid mixture or ionic liquid with additive, at the required concentration, in a glass vial by heating at 75 ± 35°C. The resulting mixtures were then vortex and cooled to room temperature, or simply cooled to room temperature.

RFF and DFY-NH₂ tripeptides formed self-supported gels when dissolved in choline phosphate buffer at concentrations of 30mM and 45mM, respectively. The tripeptide RFF formed an opaque gel with self-assembly microstructures, while the gelator DFY-NH₂ was capable of forming a transparent clear gel (Figure 12). It is noteworthy that, depending on the batch used, the gelation time for DFY-NH₂ in choline phosphate buffer was much faster than for the corresponding hydrogel (observed for the tripeptide batch within counter ion chloride), occurring instantaneously. In addition to the above-referred ionogels with System II peptides, it was also observed that RFF could form gels with ionic liquids from the choline family, namely when mixing choline dihydrogenophosphate or dicholine monohydrogenphosphate with additives capable of forming hydrogen bonds, as for example citric acid (Figure 12). Such ionic liquid composition and additive were designed to mimic the citrate phosphate buffer seen as a solvent for hydrogelation (Table 2). In Table 4 examples of formulations for System II tripeptide ionogels are given.

Table 4 - Examples of gelation formulations for the System II tripeptides.

Ionic Liquid	Gelator
Ionic Liquid	RFF	DFY-NH₂
Choline phosphate buffer	Gel (45 mM)	Gel (30 Mm)
Dicholine monohydrogenphosphate:citric acid	Gel (40 mM)	Not tested

* Add NaOH (1M) to raise the pH to ≈7.5. Choline phosphate buffer is composed by choline dihydrogenphosphate and dicholine monohydrogenphosphate (equimolar amounts) and 16-17% (w/w) deionized water. Figures in parenthesis are gelation concentrations.

Tripeptide ionogels of both systems, I and II, were characterized by several techniques. In an embodiment, self-supporting hydrogels and ionogels can be checked visually by the glass vial inversion test. The macroscopic manifestation of successful gelation is the absence of observable material flow in the vials wall upon inversion of the glass vial. Examples are shown in Figures 3, 4, 6 and 8 for system I, and in Figure 12-14 and 20 for system II.
Gel-like properties of biomaterials can also but determined through rheological measurements. In an embodiment, the rheological properties of DFY-NH₂ gels (system II) were measured with an Anton Paar MCR 302 rheometer with the temperature controlled at 25°C using a parallel sandblasted 10 mm plate. Amplitude sweep was performed to determine the critical strain. Frequency sweep was performed to measure G' and G" at a constant strain (0.5 and/or 0.1), in the angular frequency range 0.1-100 rad/s. The experiments were carried out approximately 24h after ionogel sample cooling. The values of the storage modulus (G') were about three to four times higher than the loss modulus (G") over the entire angular frequency range (0.1-100 rad/s) at 25ºC indicating that 30 mM DFY-NH₂ ionogel exhibited gel-like characteristics. The results obtained from ionogel's characterization can be seen in Figure 16.
To access the hydrogen-boding interaction of self-assembled nanostructures, attenuated total reflection Fourier transform infrared (ATR-FTIR) studies were performed. In an embodiment, ATR-FTIR spectra were recorded using a PerkinElmer FT-IR Spectrometer Spectrum Two, using the UATR Two module. Each sample was characterized using 25 scans at a spectral resolution of 1cm^-1 over the range 400cm^-1 to 4000cm^-1. Peptide solutions were prepared using (i) ionic liquid and deuterated sodium hydroxide solution or deuterated potassium hydroxide and (ii) deuterated choline phosphate buffer. ATR-FTIR spectra obtained for KYF in [Bmim][DCA], KYF in [Emim][DCA], KFF in [Bmim][DCA] and KFF in [Emim][DCA] showed peak formation in amide I region for tripeptide ionogels (Figure 9). The results suggested different interactions, and consequently self-assembly for KYF and KFF in ionic liquids as macroscopic and microscopically observed. The peak observed at 1672 cm^-1 is due to the presence of trifluoroacetic acid in the tripeptide sample. DFY-NH₂ ionogel, DFY-NH₂ hydrogel and YFD-NH₂ hydrogel have shown peaks in the amide I region which may suggest periodical organization either through helical or β-sheet structures (Figure 15). The morphology of the gels was characterized by microscopy. In another embodiment, the gels were characterized by different microscopy analysis, namely optical and polarised microscopy and by transmission electron microscopy (TEM). KFF ionogel (system I) samples were observed by optical microscopy using a microscope Zeiss Axio Observer.Z1/7 coupled with an Axiocam 503 color camera equipped with crossed polarizers. Microscope images showed the formation of birefringent microstructures associated to the self-assembly of KFF in [Bmim][DCA] or [Emim][DCA]. These microstructures are only observed for opaque gel and not for translucid KFF ionogels. Examples are shown in figures 7 and 8. DFY-NH₂ ionogel (system II) nanostructures were characterized by TEM. For this technique, carbon-coated grids were purchased from Electron Microscopy Sciences. In an embodiment, a drop (5µl) of the sample solution was applied to the carbon-coated grid and incubated for one minute. The sample was then washed with water to remove ionic liquids and then stained with 5µl of 2% (w/v) uranyl acetate solution for 30 seconds. After blotting the excess stain solution, the grid was left to air dry. The negatively stained sample was imaged in a FEI TITAN Halo TEM operating at 300kV. Images were recorded in the low-dose mode (20e^-Å^-2) on a FEI CETA 16M camera (4096 x 4096 pixels). DFY-NH₂ ionogels showed a dense three-dimensional nanofibrous network as can be seen of Figure 17. The average diameter of the individual fibers was about 8-12 nm.
In a further embodiment, the ionic conductivity of ionogels was measured by electrochemical impedance spectroscopy at environmental conditions (temperature 18-27°C, atmospheric pressure (1013.25hPa), and RH 30-80%), covering a frequency range from 100 kHz to 0.1 Hz. At 1 kHz, ionic conductivity of DFY-NH₂ ionogel (System II) was 1.0×10^-3 S.cm^-1, very similar to the one exhibited by its solvent choline phosphate buffer (Figure 18). As expected, ionogel formation does not affect the ionic conductivity of the ionic liquids and the same value ranges were observed in the corresponding ionogels (for example, at 1 kHz for [Emim][DCA] 18×10^-1 S.cm^-1, for [Bmim][DCA] 10×10^-1 S.cm^-1 and for dicholine monohydrogenphosphate_citric acid (1:1) 4.7×10^-5 S.cm^-1). Conductive properties of the tripeptide ionogels (Systems I and II) were shown by lightening up light-emitting diodes (LED) as shown in Figures 10 (System I) and 22 (System II). It was observed that the LED lights up when using the tripeptide ionogels, independently of its water content.
In an embodiment, the thermoreversibility of tripeptide ionogels is illustrated in Figure 20. For this, obtained ionogels were heated to 75±35°C for 10 minutes. Afterwards, they were left to cool down to room temperature (18-25°C). The gels were observed by the glass vial inversion test before heating, after heating and after cooling down. This is shown to System II tripeptides but the same is observed for System I.
The potential to process System I and System II tripeptide ionogel formulations and mould in different formats, as discs and thin films, is shown in Figures 5, 19 and 21. Figure 21 illustrates an embodiment of the potential of the ionogel formulations to be spread as thin films on a substrate (here for example spread on an unmodified glass slide). This Figure 21 also shows the self-healing properties of tripeptide ionogels. In another embodiment, in Figures 5 and 19 it is possible to see the processing of the tripeptide ionogel macroscopic structures, moulded by casting the formulation in a 3D printed mould.
Other noteworthy features of System I and System II tripeptide ionogels are the stability to room conditions, summarised in Figures 5 and Figure 19. As opposed to the equivalent tripeptide hydrogels, where water evaporation is very fast, tripeptide ionogels remain very stable over time (10 months, approximately) with minor weight variation (4-10%) and with no cracking or shrinking observed. Figure 24 shows an embodiment of the potential of tripeptide ionogels to further encapsulate liquid crystal molecules, which are known to be potent optical probes for sensing materials [22,23].
In an embodiment, the potential of tripeptide ionogels as gas and volatile organic compound sensing materials used in devices was explored. Variations on the electric properties of tripeptides ionogels were observed according with the concentration of an exposed volatile compound. As an example, thin films of 50mM DFY-NH₂ ionogels, previously film-coated into gold-patterned electrodes glass substrates, were exposed to different humidity rates. A schematic representation of the in-house built electronics and results can be seen in Figure 23. This application is shown to tripeptide ionogels of system II but the same is observed for system I tripeptide ionogels.
In an embodiment, the addition of the tripeptide and ionic liquid is made in defined orders and concentrations, at specific conditions of pH, temperature, sonication and agitation, and with added additives when required. Depending on the tripeptide sequence, the pH of the tripeptide dissolved in the ionic liquid is increased by the addition of NaOH, KOH, [Bmim][OH], or choline hydroxide.
In another embodiment, it is required the addition of an additive capable of hydrogen bonding. This additive can be citric acid, tartaric acid, ascorbic acid, glucose, sucrose, xylose, arginine, histidine, proline, urea, sorbitol, xylitol, glycerol, monoethylene glycol, menthol, lactic acid, betain, or mixtures thereof. In all cases, the final tripeptide ionogels contain variable amounts of water (always below the 90% (w/w) threshold value found in hydrogels; typically, in tripeptide ionogels the water content is found between 2-75%).
In yet another embodiment, it is also possible to add further components to the formulation to add additional stimuli-responsive properties, as for example through the incorporation of liquid crystals.
In an embodiment, the formulations and respective tripeptide ionogels can be processed by diverse techniques and moulded into different formats, yielding advanced functional materials. The formed supramolecular structures can be processed in different geometries selected from discs, films, fibers, spheres, cubes, mesh, channels or patterned structures, using different methods as moulding, casting, spin coating, film coating, spinning, electrospinning, microfluidics, 3D-printing, ink-jet printing, patterning, metal depositions and fabrication methods. They can be casted into several substrates such as glass slides, silicon wafers, interdigitated electrodes, with or without chemical or physical modification.
In another embodiment, the tripeptide ionogels are stable at ambient conditions, have thermal, electrochemical and chemical stability in a wide range of temperature and humidity conditions, are conducting, soft, self-healing, thermoreversible, have a large electrochemical window, are non-flammable, non-toxic, biocompatible and biodegradable materials. Furthermore, the ionogels are stimuli-responsive. They possess electric, ionic and/or protonic conductivity. They possess optical and piezoelectric properties. The conductivity, optical and piezoelectricity changes when in presence of chemical (e.g. analytes in gas or aqueous phases, cells) or physical stimuli (e.g. temperature, pressure) can be used as transducing signals to develop sensors. When adding to the tripeptide formulation optical probes, as for example liquid crystals, it is added another optical component to the material to be used as another transducing element.
The tripeptide ionogels may find application in a variety of technological fields, such as sensing devices; electronic devices; opto-electronic devices; flexible and wearable devices; conducting devices; materials for additive manufacturing; chemocatalysis and biocatalysis; coating materials; metal recovery; encapsulation, drug delivery and/or cell assays; pharmaceutical formulations; electrochemical devices such batteries, capacitors and solar cells; electrolytes; semi-solid lubricants; magnetorheological fluids and electrochemical devices; adsorbents; purification matrices; food; and cosmetics products.
The present disclosure will now be described using different embodiments, which should not limit the scope of protection of this application.

EXAMPLE 1- General formulation and production process of tripeptide ionogels

Unprotected tripeptides KYF, KFF, RFF, DFY-NH₂ and YFD-NH₂ were produced in-house by conventional peptide synthesis methods or obtained from commercial suppliers. The ionic liquids used were purchased to IoLiTec (Germany). The water content of all ionic liquids used was determined by Karl-Fischer titration using an 831 KF Coulometer with a generator electrode without diaphragm with Hydranal Coulomat AG as the analyte. The final value noted was the average of a minimum of three experiments.
In an embodiment, for System I tripeptides (KYF and KFF), these were dissolved in ionic liquids at a given concentration in a glass vial at room temperature, by means of vortex and sonication, followed by pH rise to 7.5±1, by dropwise addition of alkaline solutions, such as, sodium hydroxide (1 M or 2 M), potassium hydroxide, [Bmim][OH], or choline hydroxide. In the tripeptide ionogels, the final tripeptide concentration is in between 25-600 mM, and the water content is in between 2%-70% (w/w). For System II tripeptides (RFF, DFY-NH₂ and YFD-NH₂), these were dissolved in ionic liquid, ionic liquid mixture or ionic liquid with additive, at the required concentration in a tube by heating at 75±35 °C. The resulting mixtures were then vortex and cooled to room temperature, or simply cooled to room temperature. In the tripeptide ionogels, the final tripeptide concentration is in between 20-200 mM, and the water content is in between 2%-70% (w/w).
In both cases (Systems I and II), the formation of the gels was confirmed by the glass vial inversion test.

EXAMPLE 2 - Formulation and preparation of KYF tripeptide ionogels

The tripeptide KYF was dissolved in 1-butyl-3-methyl imidazolium dicyanamide (water content estimated by Karl Fisher as 0.5±0.5% (w/w)) by vortex and sonication, followed by increasing the pH to 7.5 ± 1 by dropwise addition of sodium hydroxide solution (NaOH) 2 M. The final peptide concentration range in the ionogel was ideally between 50 - 550 mM, with 2-72% (w/w) water. KYF ionogel can also be prepared in 1-ethyl-3-methylimidazolium dicyanamide (water content estimated by Karl Fisher as 0.5±0.5%(w/w)) following the experimental protocol described above, yielding a final peptide concentration ideally between 50-500 mM, with 2-72% (w/w) water. All characterization of the gels took place at least 24 hours after ionogel preparation. Samples for ATR-FTIR analysis were prepared with NaOD solution instead of NaOH. As an alternative, 1-butyl-3-methyl imidazolium chloride or 1-ethyl-3-methylimidazolium chloride can be used to prepare the ionogels. Examples of KYF ionogels or related characterization can be seen in Figures 3 - 5, 9, 10 and 24.

EXAMPLE 3 - Formulation and preparation of KFF tripeptide ionogels

KFF was dissolved in 1-butyl-3-methyl imidazolium dicyanamide (water content estimated by Karl Fisher as 0.5±0.5% (w/w)) by vortex and sonication, followed by increasing the pH to 7.5 ± 1 by dropwise addition of sodium hydroxide solution (NaOH) 2M. The final peptide concentration in the ionogel was ideally between 50-200 mM, with 2-75% (w/w) water. KFF ionogel can also be prepared in 1-ethyl-3-methylimidazolium dicyanamide (water content estimated by Karl Fisher as 0.5±0.5% (w/w)) following the experimental protocol described above, yielding a final peptide concentration ideally between 50-500 mM, with 2-75% (w/w) water. Characterization of the gels took place at least 24 hours after ionogel preparation. Samples for ATR-FTIR analysis were prepared using NaOD solution instead of NaOH. As an alternative, 1-butyl-3-methyl imidazolium chloride or 1-ethyl-3-methylimidazolium chloride can be used to prepare the ionogels. Examples of KFF ionogels or related characterization can be seen in Figures 6 - 9, 24.

EXAMPLE 4 - Preparation of designed choline phosphate ionic liquid buffer system

The choline phosphate ionic liquid buffer system is an ionic liquid hydrated buffer formed by the mixture of two ionic liquids (i) choline dihydrogenphosphate and (ii) dicholine monohydrogenphosphate in equimolar amounts, to which 16-17% (w/w) deionized water are added. The buffer is homogenized by means of vortex and sonication. Ionic liquid buffer water content was determined using a Karl Fischer; apparent pH was verified to be at 7.1±1. Chemical structures of choline dihydrogenphosphate and dicholine monohydrogenphosphate are presented in Figure 11.

EXAMPLE 5 - Formulation and preparation of tripeptide DFY-NH₂ ionogels

DFY-NH₂ was dissolved in choline phosphate buffer (apparent pH 7.1±1, 16%-17% (w/w) water content) heating at 75±35 °C following by vortex and cooling down to room temperature, or just cooling down to room temperature. The final peptide concentration ideally is between 20-100 mM, with 16-20 % (w/w) water. Characterization of the gels took place at least 24 hours after ionogel preparation. Samples for ATR-FTIR analysis, choline phosphate buffer was prepared using D₂O instead of H₂O. Examples of DFY-NH₂ ionogels or related characterization can be seen in Figures 12 and 14-23.

EXAMPLE 6 - Formulation and preparation of RFF ionogels

RFF was dissolved in choline phosphate buffer (apparent pH 7.1±1, 16%-17% (w/w) water content) heating at 75±35 ºC following by cooling to room temperature. The final peptide concentration in the ionogel ideally is between 20-100 mM, with 16-20 % (w/w) water. Characterization of the gels took place at least 24 hours after ionogel preparation. Samples for ATR-FTIR analysis, choline phosphate buffer was prepared using D₂O instead of H₂O. Examples of RFF ionogels can be seen in Figures 12, 13.

EXAMPLE 7 - Formulation and preparation of RFF ionogels with additive

RFF ionogel was prepared by heating and stirring method. This method consist in mixing the components of the gel: (i) ammonium quaternary salt: choline dihydrogenphosphate, dicholine monohydrogenphosphate or choline chloride (ii) an additive which has the capability of H-bond donor or receptor, such as citric acid, and (iii) an unprotected tripeptide, RFF according with the amounts calculated considering different molar ratios preferentially 1:1:0.5 - 1:1:0.005, ideally 1:1:0.1 - 1:1:0.005. The system was heated to 80±20ºC and stirred at 300 rpm, until a clear transparent viscous solution or gel is obtained. Examples of RFF ionogels with additive can be seen in Figure 12.

EXAMPLE 8 - Addition of liquid crystal to the peptide ionogel

The ionic liquid 1-butyl-3-methyl imidazolium dicyanamide (water content estimated by Karl Fisher as 0.5 ± 0.5%) and the liquid crystal 5CB (4-Cyano-4'-pentyibiphenyl) were mixed by magnetic stirring at 300rpm. The tripeptide KYF or tripeptide KFF was added to the mixture following by increasing the pH to 7.5 ± 1 by dropwise addition of sodium hydroxide solution (NaOH) 1M. Examples are shown in Figure 24.

EXAMPLE 9 - Stability to ambient conditions

While warm, 30 mM of DFY-NH₂ ionogel in choline phosphate buffer were pipetted to a 3D-printed mould ring. The gel was weighted after 24 h and over a 10-month period. Example of results in figure 19.

EXAMPLE 10 - Evidence of conducting ionogels

Liquid/viscous samples were prepared by pipetting 100µl of the solution into an 0.5 ml Eppendorf previously fill with 100µl PDMS in order to decrease the amount of biomaterial need for the characterization. The gold coated electrodes (connection pines) were inserted in an in-house 3D printed adapter and position in the Eppendorf being partially submersed in the biomaterial. Gel samples were prepared using an in-house 3D printed o-ring with 6mm inner diameter and 0.6mm height. The system, ring - hydrogel, was sandwiched between two gold contacts.
Ionic conductivity of the gels and viscous/liquid samples was determined at environmental conditions by Electrochemical Impedance Spectroscopy using a potentiostat Gamry Instruments - Reference 3000 (measurement conditions: frequency range from 100 kHz - 0.1 Hz and Vpp 100mV (AC)). The conductivity was calculated using the following equation $σ = \frac{1}{R} (\frac{l}{A})$
where σ is the conductivity, R is the resistance; and I and A are the thickness and area of the samples, respectively. The results of ionic conductivity studies for 30 mM DFY-NH₂ ionogel in choline phosphate buffer are shown in Figure 18. Figures 10 and 22 demonstrate the ion conduction of the tripeptide ionogel by lightening up a light-emitting diode (LED).

EXAMPLE 11- Self-healing assay

30 mM and 50 mM DFY-NH₂ ionogels were prepared. While still warm they were pipetted into a glass slide previously cleaned with isopropanol. A 30 µm thin film was immediately prepared by using the automatic film applicator and quadruplex with predefined thickness. After three days the prepared thin film was scratch with a tip. Bright field microscopy characterization was performed during 2 weeks. Example of results in figure 21.

EXAMPLE 12 - Processing the formulation as a film

While still warm, 30 mM DFY-NH₂ in choline phosphate buffer was deposited onto an untreated glass slide, using an automatic film applicator equipped with a heated bed and a quadruplex to obtain the predefined thickness of 30 µm thin film. The DFY-NH₂ ionogel thin films were left at room conditions for 24 hours. An example is shown in figure 21.

EXAMPLE 13 - Processing the formulation by moulding

While still warm, 30 mM DFY-NH₂ in choline phosphate buffer was drop casted into a 3D printed ring. The DFY-NH₂ ionogel thin films were left at room conditions for 24 hours. Examples are shown in figures 5 and 19.

EXAMPLE 14 - Application of tripeptide ionogel thin films

While still warm, 7.5µl of 50mM peptide solution in choline phosphate buffer were deposited onto an untreated Gold-Titanium interdigitated electrode on glass substrate (18 parallel, 300µm in width, spaced by 300µm), using an automatic film applicator equipped with a heated bed and a quadruplex for a predefined thickness of 15µm. The thin films were left at room conditions for 24 hours before characterization.
Triplicates of DFY-NH₂ ionogel thin films were placed in the detection chamber of an associated in-house electronic nose (Figure 23A) and exposed to five humidification (the thin films were exposed to humidified nitrogen for 140s at 1.5slpm) and drying (the thin films were exposed to dry nitrogen for 140s at 1.5 slpm) periods, for four different RH levels (58%, 50%, 44%, 25%). The RH levels were generated by bubbling a nitrogen stream, control by a mass flow controller (MFC, MC-5SLPM-D/5M, Alicat Scientific Inc.) at 1.5slpm, in the different supersaturated salt solutions (distilled water, sodium chloride, sodium bromide and magnesium chloride) before entering the detection chamber. A humidity sensor was placed in the outlet of the detection chamber, in order to measure the variations of RH applied to the ionogel thin films when they are exposed alternately to humid and dry nitrogen. The results are shown in Figure 23. Similar results were obtained when exposing the thin films to volatile organic compounds.
The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.
The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above described embodiments are combinable.
The following claims further set out particular embodiments of the disclosure. The claims define the scope of the invention.
The project leading to this invention has received funding from the European Research Council through the grant reference SCENT-ERC-2014-STG-639123 and from Fundação para a Ciência e Tecnologia through the PhD scholarship SFRH/BD/113112/2015.

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Claims

An ionogel composition comprising:
at least one ionic liquid selected from imidazolium ionic liquid, ammonium-based ionic liquids, or mixtures thereof; and

at least one unprotected tripeptide as a gelator, wherein the tripeptide is selected from the following list: Lysine-Tyrosine-Phenylalanine, Lysine-Phenylalanine-Phenylalanine, Arginine-Phenylalanine-Phenylalanine, Aspartic acid-Phenylalanine-Tyrosine, and Tyrosine-Phenylalanine-Aspartic acid; or
10-700 mM, preferably 20 - 600 mM, of at least one unprotected tripeptide as a gelator, wherein the unprotected tripeptide comprises at least a phenylalanine.
The ionogel according to the previous claim wherein the unprotected tripeptides have an amide at the C-terminal, preferably the Aspartic acid-Phenylalanine-Tyrosine, and Tyrosine-Phenylalanine-Aspartic acid tripeptides.
The ionogel according to any of the previous claims wherein the unprotected tripeptides comprise a molecular weight equal or inferior to 2000 g/mol, preferably between 270-540 g/mol.
The ionogel according to any of the previous claims wherein the ionic liquid concentrations range from 20-98 % (w/w).
The ionogel according to any of the previous claims, wherein the ionic liquid is selected from phosphate-containing ionic liquids, chloride-containing ionic liquids, dicyanamide-containing ionic liquids, quaternary ammonium salts, or mixtures thereof.
The ionogel according to any of the previous claims wherein the imidazolium-based ionic liquids are selected from a list comprising: 1-butyl-3-methylimidazolium dicyanamide, 1-ethyl-3-methylimidazolium dicyanamide, 1-butyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazolium chloride, or mixtures thereof, or wherein the ammonium-based ionic liquids are selected from a list comprising: choline chloride, choline hydroxide, choline phosphate buffer, 2-hydroxyethylammonium formate, choline dihydrogenophosphate, dicholine monohydrogenphosphate, or mixtures thereof.
The ionogel according to any of the previous claims wherein the tripeptides hydrogelation is triggered by changes on the temperature, buffer solution or pH, preferably increasing the pH to 7-7.5, wherein the pH is increased by addition of sodium hydroxide, choline hydroxide or 1-butyl-3-methylimidazolium hydroxide.
The ionogel according to any of the previous claims further comprising an additive selected from the following list: citric acid, tartaric acid, ascorbic acid, glucose, sucrose, xylose, arginine, histidine, proline, urea, sorbitol, xylitol, glycerol, monoethylene glycol, menthol, lactic acid, betain, or mixtures thereof.
The ionogel according to the previous claim wherein the molar ratio between the ionic liquid and the additive ranges from 1:1 to 1:10, preferably 1:1 to 1:2.
The ionogel according to any of the previous claims wherein the ionogel is soft, self-healable, thermoreversible, stimuli-responsive, non-flammable, non-toxic, biocompatible and biodegradable.
The ionogel according to any of the previous claims further comprising a stimuli-responsive component, preferably the stimuli-responsive component is a liquid crystal.
The ionogel according to any of the previous claims obtainable by moulding, casting, spin-coating, film coating, spinning, electrospinning, microfluidic, 3D-printing, ink-jet printing, patterning or metal deposition method.
Disc, film, fiber, sphere, cube, mesh, channel or patterned structure comprising the ionogel composition described in any of the previous claims.
An article comprising the ionogel composition described in any of the previous claims, preferably wherein the article is a sensing device, electronic device, opto-electronic device, flexible device, wearable device, conducting device, coating material, electrochemical device, electrolyte, semi-solid lubricant, adsorbent agent, pharmaceutical formulation, food product, or cosmetic product.
Kit or reagent comprising the ionogel composition described in any of the previous claims.