ES2984175A1 - Nanoemulsions to inhibit the formation of bacterial biofilms - Google Patents

Abstract

Nanoemulsions for inhibiting bacterial biofilm formation. The present invention relates to nanoemulsions composed of an oily core of α-tocopherol and octadecylamine and a surfactant layer covered, in combination with an antimicrobial peptide, preferably the DJK5-C14 peptide (modified DJK5), and the method for obtaining them. Furthermore, the present invention relates to the in vitro use of nanoemulsions for inhibiting the formation of H. influenzae biofilms, as well as their use as a medicine for the treatment or prevention of diseases caused by Haemophilus influenzae. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

Nanoemulsions to inhibit the formation of bacterial biofilms

The present invention relates to nanoemulsions composed of an oily core of α-tocopherol and octadecylamine and a cover of a surfactant layer, in combination with an antimicrobial peptide, preferably the modified DJK5 peptide, and their use for the in vitro inhibition of the formation of Haemophilus influenzae biofilms, as well as their use as a medicine for the treatment or prevention of diseases caused by H. influenzae.

BACKGROUND OF THE INVENTION

Conventional antibiotic treatments for bacterial infections are increasingly ineffective, mainly due to the emergence of antibiotic-resistant pathogens. In addition, the development of bacterial resistance is much faster than the development of new antimicrobials. In 2014, the World Health Organization warned that by 2050 bacterial infections will be the leading cause of death worldwide if effective measures are not taken (Murray, C. et al. Lancet, 2022, 399, 629-655).

Bacteria living in biofilms contribute greatly to the emergence of this drug resistance. A biofilm is a group of bacteria attached to a surface embedded in an extracellular polymeric matrix (EPS) that they themselves produce. Collectively, bacterial biofilms are around 10 to 1000 times more resistant to antibiotics and account for more than 60% of bacterial infections in humans, leading to the emergence of persistent and chronic infections.

As an alternative to conventional antibiotics and in search of solving the resistance of bacterial biofilms to antimicrobials, the use of drug delivery nanosystems has been investigated in recent decades. Drug delivery nanosystems are technologies that allow the controlled transport of drugs. Due to their utility in modulating drug release, the protection of labile materials (e.g., peptides, DNA or mRNA) against degradation, and the ability to actively transport drugs to specific sites, many efforts are being directed to produce nanoparticles as vehicles for active ingredients in various treatments, especially now with the new generation of lipid nanoparticle-based vaccines.

Lipid nanosystems (LN) include all those nanomaterials whose main component is a lipid. LN have attracted increasing attention during the last three decades as an alternative to other types of nanoparticles due to a number of advantages: i) High biocompatibility, since in most cases the lipids used have a very good toxicity and biodegradability profile; ii) possibility of incorporating both lipophilic and hydrophilic drugs; iii) greater bioavailability for some administration routes (oral, dermal) (Mishra, D. et al., Nanomedicine Nanotechnology Biol. Med., 2018, 14, 2023-2050).

The applicability of nanotechnology-based lipid systems is presented as an innovative tool in improving the treatment of bacterial infections. Important studies involving the use of liposomes, solid lipid nanoparticles, nanostructured lipid carriers, nanoemulsions, microemulsions and lipid nanocapsules have been carried out from 2000 until now, showing the potential of these systems as vehicles for active ingredients to attack bacterial biofilms (Ramos, MA. et al. International Journal of Pharmaceutics, 2021,603, 120706).

A few years ago, a series of synthetic protease-resistant peptides were described for the first time as active ingredients with anti-biofilm effect against different bacterial species. The synthetic peptide VQWRAIRVRVIR (where V is the D isomer), encoded as DJK5, was shown to prevent the intracellular accumulation of the second messenger ppGpp, which is key for the formation of bacterial biofilms (Pletzer, D. et al, Journal of Bacteriology, 2016, 198, 2572-8) (Pletzer, D. et al, Current Opinion in Microbiology, 2016, 33, 35-40).

The ability of the DJK5 peptide to inhibit bacterial biofilm formation has been tested in different bacterial species in its free form or in combination with other compounds, whether antimicrobial peptides, conventional antibiotics, or the chelating agent EDTA (Wang D. et al, Journal of Endotonics, 2018, 44, 1709-13) (Vuotto, C. et al., Drugs, 2019, 79, 1635-1655). The ability of the DJK5 peptide encapsulated in hyaluronic acid-based nanogels to prevent biofilm formation has also been studied (Klodzinska, SN. et al., Nanomedicine: Nanotechnology, Biology and Medicine, 2019, 20, 102022). These nanogels showed an improvement in the therapeutic efficacy of the peptide, with an encapsulation efficiency of between 30-60%, demonstrating the usefulness of using nanoparticle systems.

H. influenzae is a Gram-negative priority pathogen that is associated with the chronicity of different mucosal infections, such as chronic obstructive pulmonary disease, and other conditions, such as otitis media in children. Typeable H. influenzae conjugate vaccines have no effect on infections caused by non-typeable H. influenzae strains because these strains are not encapsulated (Murphy, TF. et al, Pediatric Infectious Disease Journal, 2009, 28, 43-8).

Currently, there is no approved therapy to prevent the formation of these biofilms, particularly against H. influenzae biofilms. Thus, there is a growing need to develop a treatment to prevent the formation of bacterial biofilms of H. influenzae and to provide delivery systems for active ingredients and, in particular, antibacterial peptides.

DESCRIPTION OF THE INVENTION

The present invention relates to nanoemulsions composed of a lipid mixture (oily core), preferably of α-tocopherol and octadecylamine, and a cover by a surfactant layer that allows its stabilization in water, in combination with an antimicrobial peptide comprising the sequence SEQ ID NO: 5, preferably the antimicrobial peptide DJK5 (SEQ ID NO: 2), DJK6 (SEQ ID NO: 3), or L1018 (SEQ ID NO: 4), and which also comprises an alkyl chain C < 14 > H < 28 > O < 2> attached at its NH < 2> end (DJK5-C14, DJK6-C14 or L1018-C14), more preferably the antimicrobial peptide DJK5 with an alkyl chain C < 14 > H < 28 > O < 2> attached at its NH < 2> end (DJK5-C14).

The inventors have demonstrated that these nanoemulsions present excellent physicochemical properties, with encapsulation efficiencies greater than 85% and high stability suitable in suspension at different temperatures, so they can be stored for long periods of time (Examples 1 to 5 and Figures 1 to 5).

The nanoemulsions were tested on several strains of the pathogen H. influenzae: one strain from a culture collection, strain R2866 (NCBI taxonomic ID number: 262728) and two clinical strains isolated from patients with chronic obstructive pulmonary disease, strain P605-7719 (also referred to herein as strain P605) and strain P639-3649 (also referred to herein as strain P639).

Furthermore, the inventors have observed that in none of the strains of the pathogen H. influenza tested (R2866, P639 and P605) with the peptide DJK5 or DJK5-C14, both in their free form, the formation of biofilm of H. influenzae is prevented, demonstrating that, contrary to what was expected, the activity of the peptide DJK5 to prevent the formation of biofilms of H. influenzae is null both in its free and unmodified form (Figure 7) and in its modified free form (DJK5-C14) (Figure 6).

Finally, the inventors have demonstrated that encapsulation of the DJK5, DJK6 and L1018 peptides, particularly the DJK5-C14, DJK6-C14 or L1018-C14 peptides, in nanoemulsions composed of an oily core, preferably of a-tocopherol and octadecylamine, and a surfactant shell significantly prevents bacterial biofilm formation by H. influenzae (Figure 6, 11 and 12).

Thus, in a first aspect, the present invention relates to nanoemulsions, hereinafter "the nanoemulsions of the invention", which comprise:

(a) an oily core comprising at least one liquid lipid and octadecylamine, (b) a shell of at least one surfactant, and

(c) the peptide comprising or consisting of the amino acid sequence SEQ ID NO: 5.

The term "nanoemulsions" as used in the present invention refers to lipid systems or lipophilic active ingredient carriers consisting of a liquid lipid, water and one or more surfactants, with sizes ranging from 10 to 1000 nm. According to their structure, nanoemulsions can be classified into three types: oil-aqueous (O/W), water-oil (W/O) and multiple emulsion in which microdomains of oily and aqueous phases are intercalated (W/O/W and O/W/O).

The term "liquid lipid" as used herein refers to any substance of lipid nature that is liquid at room temperature. Examples of liquid lipids include but are not limited to, medium chain triglycerides, mineral oils, vegetable oils, partial glycerides and their derivatives, or tocopherols.

Medium chain triglycerides (MCT) are molecules consisting of three saturated fatty acids with a length of 6 to 12 carbon atoms esterified with glycerol. Examples of medium chain triglycerides include caproic acid triglycerides (C6:0), caprylic acid triglycerides (C8:0), capric acid triglycerides (C10:0), and lauric acid triglycerides (C12:0), as well as mixtures thereof.

Mineral oils are liquid by-products of petroleum distillation. The term “mineral oils” as used herein refers to white oils. These are highly refined mineral oils composed essentially of saturated aliphatic and alicyclic non-polar hydrocarbons. Examples of mineral oils include, but are not limited to, light white mineral oil or heavy white mineral oil.

Vegetable oils are triglycerides extracted from plants and contain in their composition a variable proportion of unsaturated or polyunsaturated fatty acids with a length of more than 12 carbon atoms esterified with glycerol. Examples of vegetable oils include, but are not limited to, olive oil, sunflower oil, soybean oil, cottonseed oil, castor oil, evening primrose oil or borage oil.

Partial glycerides and their derivatives are those compounds in which glycerol is partially esterified with one or two fatty acid chains, regardless of the length of the fatty acid chain. In addition, they can be modified by the attachment of a propylene glycol chain. Examples of partial glycerides and their derivatives include, but are not limited to, glycerol monolinoleate, glycerol monooleate or propylene glycol monolaurate.

The term “tocopherols” defines several organic compounds composed of various methylated phenols, which form a class of chemical compounds of which several act as Vitamin E. Examples of tocopherols include, but are not limited to, a-tocopherol, ptocopherol, and y-tocopherol.

In a preferred embodiment of the nanoemulsions of the invention, the liquid lipid is glycerol monolinoleate, propylene glycol monolaurate or a-tocopherol, preferably a-tocopherol.

Preferably, the nanoemulsions of the invention are formed by an oily core, where said oily core is composed of a lipid mixture, particularly of a-tocopherol and octadecylamine.

The term “a-tocopherol” used in the present invention refers to a naturally occurring antioxidant of the tocopherol family, practically insoluble in water and whose structure is represented below by formula I:

Formula I. Chemical structure of a-tocopherol

The term “octadecylamine” used in the present invention refers to a primary amine where the aliphatic chain is formed by a stearic acid of 18 carbon atoms.

To allow stabilization in water, the nanoemulsions of the invention have a shell formed by a layer of surfactant.

The term "surfactant" as used herein refers to substances that reduce the surface tension at the interface between two phases (e.g., two liquids insoluble in each other) by adsorbing these molecules at the interface. The term surfactant is equivalent to surfactant. Examples of surfactants include, but are not limited to, polysorbate 20, polysorbate 60, polysorbate 80, poloxamer 188, poloxamer 127, polyoxyethylene (40) stearate, propylene glycol stearate, macrogol lauryl ether, and phosphatidylcholine.

In a preferred embodiment of the nanoemulsions of the invention, the surfactant is polysorbate 20, polysorbate 80, poloxamer 188, polyoxyethylene (40) stearate and/or phosphatidylcholine.

In another more preferred embodiment of the nanoemulsions of the invention, the surfactant is polysorbate 20 and/or polysorbate 80, preferably it is polysorbate 80.

The inventors have demonstrated that the encapsulation of the peptide DJK5 (with amino acid sequence SEQ ID NO: 2), DJK6 (with amino acid sequence SEQ ID NO: 3) or L1018 (with amino acid sequence SEQ ID NO: 4), in the nanoemulsions of the invention; particularly of the peptide DJK5-C14 (peptide with sequence SEQ ID NO: 2 with alkyl chain C < 14 > H < 28 > O < 2 > attached at its NH < 2 > end), DJK6-C14 (peptide with sequence SEQ ID NO: 3 with alkyl chain C < 14 > H < 28 > O < 2 > attached at its NH < 2 > end) or L1018-C14 (peptide with sequence SEQ ID NO: 4 with alkyl chain C < 14 > H < 28 > O < 2 > attached at its NH < 2 > end), significantly prevents the formation of bacterial biofilms by H. influenzae (Figures 6, 11 and 12).

The antimicrobial peptide of the nanoemulsions of the invention comprises a sequence of between 6 and 24 amino acids, preferably 12 amino acids. In addition, said peptide comprises: (i) between 2 and 9 arginine (R) amino acid residues, preferably 4, 5, 6, 7 or 8 and (ii) a proportion of hydrophobic residues between 40 and 70%, preferably between 55 and 67%.

The term “hydrophobic residue” as used herein refers to nonpolar amino acid residues containing aromatic or aliphatic R groups. Examples of hydrophobic amino acid residues include proline, valine, tryptophan, leucine, isoleucine, alanine, tyrosine, phenylalanine, glycine and methionine.

The 3 peptides DJK5, DJK6 and L1018 are based on a common consensus sequence where said sequence comprises the amino acid sequence SEQ ID NO: 5. As a person skilled in the art would understand, the nanoemulsions of the invention comprising a peptide of sequence SEQ ID NO: 5 prevent the formation of bacterial biofilms by H. influenzae.

The amino acid sequence SEQ ID NO:5

SEQ ID NO:5

VX<2>X<3>X<4>X<5>X<6>X<7>X<8>X<9>X<10>X<11>R, where:

X<2>is glutamine (Q) or arginine (R),

X<3> is tryptophan (W) or leucine (L),

X<4>is arginine (R) or isoleucine (I),

X<5>is alanine (A) or arginine (R) or valine (V),

X6is isoleucine (I) or alanine (A),

X<7>is arginine (R) or valine (V),

X8is arginine (R) or valine (V),

X<9>is arginine (R) or tryptophan (W) or isoleucine (I),

X<10> is valine (V) or tryptophan (W) and

X<11>is isoleucine (I) or arginine (R).

In a preferred embodiment of the invention, the peptide comprises or consists of the amino acid sequence SEQ ID NO:1

SEQ ID NO: 1

VQWRX<5>IRVX<9>VIR, where

X<5> is alanine (A) or arginine (R) and

X<9> is arginine (R) or tryptophan (W).

In another preferred embodiment of the invention, the peptide is the DJK5 peptide comprising or consisting of the amino acid sequence SEQ ID NO: 2.

SEQ ID NO: 2

VQWRAIRVRVIR

In another preferred embodiment of the invention, the peptide is the DJK6 peptide comprising or consisting of the amino acid sequence SEQ ID NO: 3.

SEQ ID NO: 3

VQWRRIRVWVIR

In another preferred embodiment of the invention, the peptide is the L1018 peptide comprising or consisting of the amino acid sequence SEQ ID NO: 4.

SEQ ID NO: 4

VRLIVAVRIWRR

Additionally, the inventors have observed that the incorporation of a saturated alkyl chain of between 12 and 18 carbons covalently linked to the NH<2>terminal end of the antimicrobial peptides, preferably of 14 carbon atoms (C<14>H<28>O<2>), increases the lipophilicity of the peptides, facilitating the incorporation of the modified peptide into the nanoemulsion.

Thus, in another preferred embodiment of the nanoemulsions of the invention, the peptide comprises or consists of the amino acid sequence SEQ ID NO: 5 and wherein said sequence further comprises an alkyl chain selected from: C<12>H<24>O<2>, C<13>H<26>O<2>, C<14>H<28>O<2>, C<15>H<30>O<2>, C<16>H<32>O<2>, C<17>H<34>O<2> and C<18>H<36>O<2>, wherein said alkyl chain is covalently linked to the NH<2> end of the peptide of SEQ ID NO: 5.

Thus, in another more preferred embodiment of the nanoemulsions of the invention, the peptide comprises or consists of the amino acid sequence SEQ ID NO: 1 and wherein said sequence further comprises an alkyl chain selected from: C<12>H<24>O<2>, C<13>H<26>O<2>, C<14>H<28>O<2>, C<15>H<30>O<2>, C<16>H<32>O<2>, C<17>H<34>O<2> and C<18>H<36>O<2>, wherein said alkyl chain is covalently linked to the NH<2> end of the peptide of SEQ ID NO: 1.

Thus, in another even more preferred embodiment of the nanoemulsions of the invention, the peptide comprises or consists of an amino acid sequence SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and wherein said sequence further comprises an alkyl chain selected from: C<12>H<24>O<2>, C<13>H<26>O<2>, C<14>H<28>O<2>, C<15>H<30>O<2>, C<16>H<32>O<2>, C<17>H<34>O<2> and C<18>H<36>O<2>, wherein said alkyl chain is covalently linked to the NH<2> end of the peptide of sequence SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

Furthermore, in another preferred embodiment the nanoemulsions of the invention comprise the DJK5-C14 peptide, where said peptide comprises or consists of the amino acid sequence SEQ ID NO: 2, and where said sequence further comprises an alkyl chain C<14>H<28>O<2> covalently linked at its NH<2> end.

The nanoemulsions of the present invention have a reduced size with diameter less than 500 nm. The size of the nanoemulsions is mainly influenced by the composition and the formation conditions and can be measured using standard procedures known to the person skilled in the art. Examples of methods of measuring the size of nanoemulsions include, but are not limited to, dynamic light scattering (DLS), transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TNR).

In another preferred embodiment of the invention, the nanoemulsions have a size less than 200 nm.

In another more preferred embodiment of the invention, the nanoemulsions have a size between 100nm and 190nm, preferably between 140nm and 170nm.

In another even more preferred embodiment of the invention, the nanoemulsions have a size of 145nm, 146nm, 147nm, 148nm, 149nm, 150nm, 151nm, 152nm, 153nm, 154nm, 155nm, 156nm, 157nm, 158nm, 159nm, 160nm, 161nm, 162nm, 163nm, 164nm or 165nm.

The nanoemulsions of the invention may also comprise at least one pharmacological agent or active ingredient.

Thus, in another preferred embodiment of the invention, the nanoemulsions also comprise a pharmacological agent or active ingredient.

As used herein, the term “active ingredient” or “active agent” means any component that has the potential to provide pharmacological activity or other distinct effect in the diagnosis, cure, mitigation, treatment, or prevention of a disease. The term includes those components that promote a chemical change in the manufacture of the drug and are present in the drug in an intended modified form that provides the specific activity or effect. Examples of active ingredients include, but are not limited to, 2-(nitrophenyl) (phenyl)methanol, N-(2-phenylethylamino)-sulfanylidenemethylbenzamide, S-nitrosoglutathione, cinnamaldehyde, 2-nonenal, decenone, carvacrol, vanillinacetone, or combinations thereof.

In another embodiment of the nanoemulsions of the invention, the agent or active ingredient is lipophilic or hydrophilic in nature.

The term “lipophilic nature” refers to compounds, molecules or substances that tend to dissolve in other lipophilic substances such as lipids or fats.

The term “hydrophilic nature” refers to compounds, molecules or substances that tend to dissolve in water and other hydrophilic substances.

As understood by a person skilled in the art, the nanoemulsions of the invention may be comprised in a composition.

Therefore, another aspect of the invention is a composition comprising the nanoemulsions of the invention, hereinafter the “composition of the invention”.

Nanoemulsions have been described hereinbefore and apply equally to this aspect of the invention, as well as to all preferred embodiments thereof (alone or in combination).

Particularly, the composition of the invention may be a pharmaceutical composition for use as a medicament, for example, for use in the treatment or prevention of diseases caused by H. influenzae. Thus, in a preferred embodiment, the composition of the invention is a pharmaceutical composition.

The term “pharmaceutical composition” refers to a set, mixture, combination of components or substances comprising the nanoemulsions of the invention in any concentration. The pharmaceutical composition may be for human use. The term “pharmaceutical composition for human use” refers to a composition, substance or combination of substances with properties for use as a medicine, particularly as a medicine in the treatment or prevention of diseases caused by H. influenzae, or that can be used in humans or administered to humans in order to restore, correct or modify physiological functions by exerting a pharmacological, immunological or metabolic action, or to establish a medical diagnosis.

In another preferred embodiment, the pharmaceutical composition further comprises at least one pharmacologically acceptable vehicle and/or excipient.

The term "vehicle" or "carrier" refers to a substance, preferably an inert substance, that facilitates the incorporation of other compounds, which allows for better dosage and administration or improves the consistency and form of the pharmaceutical composition for use as a medicine in the treatment or prevention of diseases caused by H. influenzae. Therefore, the vehicle is a substance that is used in the medicine to dilute any of the components of the pharmaceutical composition to a certain volume or weight; or that, even without diluting said components, is capable of allowing for better dosage and administration or giving consistency and form to the medicine. When the presentation form is liquid, the pharmaceutically acceptable vehicle is the diluent.

The term "excipient" refers to a substance that helps the absorption of any of the components of the pharmaceutical composition, stabilizes said components, modifies their organoleptic properties or determines the physical-chemical properties of the pharmaceutical composition and its bioavailability. Thus, excipients could have the function of keeping the components together, such as starches, sugars or celluloses; sweetening function; coloring function; protection function of the medicine, such as, for example, to isolate it from air and/or humidity; filling function of a tablet, capsule, pill or any other form of presentation such as, for example, dibasic calcium phosphate; disintegrating function to facilitate the dissolution of the components; without excluding other types of excipients not mentioned in this paragraph.

Furthermore, as understood by those skilled in the art, the excipient and the vehicle must be pharmaceutically acceptable. The term "pharmaceutically acceptable" refers to the fact that the vehicle or the excipient must allow the activity of the compounds of the pharmaceutical composition, in particular of the particle of the invention, that is, it must be compatible with said components, so that it does not cause damage to the organisms to which it is administered.

The pharmaceutical composition may be presented in any clinically permitted form of administration and in a therapeutically effective amount. For example, it may be in a form adapted to oral, sublingual, nasal, intrathecal, bronchial, lymphatic, rectal, transdermal, intravenous, intraperitoneal, orogastric, intracolonic, inhaled or parenteral administration.

Another aspect of the invention is a method for obtaining the nanoemulsions of the invention, hereinafter "the method of the invention", which comprises:

(i) dissolving at least one liquid lipid, at least one surfactant, octadecylamine and the peptide comprising or consisting of the amino acid sequence SEQ ID NO: 5 in an alcoholic solvent, where the liquid lipid concentration is between 30 and 100 mM, the surfactant concentration is between 5 and 20 mM, the octadecylamine concentration is between 5 and 20 mM and the peptide concentration is between 0.1 and 1 mM, obtaining an organic phase;

(ii) injecting the organic phase obtained in (i) onto an aqueous phase under continuous stirring at a speed between 700 and 1000 rpm for at least 10 minutes, where the aqueous phase:organic phase ratio is at least 4:1, obtaining a solution with the nanoemulsions; and

(iii) filtering the solution obtained in (ii) using size exclusion chromatography columns or tangential filtration membranes, obtaining the nanoemulsions;

where the method is carried out at 25°C and 1 atmosphere of pressure.

The terms “nanoemulsions”, “liquid lipid”, “octadecylamine”, “peptide of sequence SEQ ID NO: 5” and “surfactant” have been described hereinbefore and apply equally to this aspect of the invention, as well as to all its preferred embodiments (alone or in combination).

The term "alcoholic solvent" as used herein refers to an alcoholic solution, dissolution, substance or component. In a preferred embodiment, the alcoholic solvent is ethanol or isopropanol, preferably ethanol.

The term "aqueous phase" as used in the present invention refers to a solution, dissolution, mixture formed by water, as well as its combination of other components or water-soluble substances. In a preferred embodiment the aqueous phase is water.

In a preferred embodiment of the method of the invention, the liquid lipid of step (i) is glycerol monolinoleate, propylene glycol monolaurate or a-tocopherol, preferably a-tocopherol.

In another embodiment of the method of the invention, in step i) the liquid lipid concentration is between 40 and 80 mM, preferably between 50 and 75 mM, more preferably it is 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 64, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 mM.

In another embodiment of the method of the invention, in step i) the surfactant concentration is between 8 and 16 mM, preferably between 10 and 14 mM, more preferably it is 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5 or 14 mM.

In another embodiment of the method of the invention, in step i) the concentration of octadecylamine is between 8 and 16 mM, preferably between 10 and 14 mM, more preferably it is 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5 or 14 mM. In another embodiment of the method of the invention, in step i) the concentration of peptide of SEQ ID NO: 1 is between 0.2 and 0.7 mM, preferably it is 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6 or 0.65 mM.

In another embodiment of the method of the invention, in step i) the concentration of liquid lipid, preferably of α-tocopherol, is 69.7 mM; the concentration of surfactant, preferably of polysorbate 80, is 11.6 mM; the concentration of octadecylamine is 12.6 mM and the concentration of peptide is 0.45 mM.

In another preferred embodiment of the method of the invention, the surfactant of step (i) can be added to the aqueous phase of step (ii).

In another embodiment of the method of the invention, the surfactant of step (i) or (ii) is polysorbate 20, polysorbate 80, poloxamer 188, polyoxyethylene (40) stearate and/or phosphatidylcholine.

In another more preferred embodiment of the method of the invention, the surfactant of step (i) or (ii) is polysorbate 20 and/or polysorbate 80, preferably it is polysorbate 80.

As described above, the peptide of step (i) of the method of the invention may be a peptide with amino acid sequence SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

Thus, in a preferred embodiment of the method of the invention, the peptide comprises or consists of the amino acid sequence SEQ ID NO:1.

In another more preferred embodiment of the method of the invention, the peptide comprises or consists of the amino acid sequence SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably the peptide of sequence SEQ ID NO: 2.

As previously mentioned, the addition of a saturated alkyl chain of between 12 and 18 carbons covalently linked to the NH<2>terminal end of the peptide, preferably of 14 carbon atoms (C<14>H<28>O<2>), increases the lipophilicity of the DJK5 peptide, facilitating the incorporation of the modified peptide into the nanoemulsion.

Thus, in another preferred embodiment of the method of the invention, the peptide of step (i) comprises or consists of the amino acid sequence SEQ ID NO: 5 and wherein said sequence further comprises an alkyl chain selected from: C<12>H<24>O<2>, C<13>H<26>O<2>, C<14>H<28>O<2>, C<15>H<30>O<2>, C<16>H<32>O<2>, C<17>H<34>O<2> and C<18>H<36>O<2>, preferably the alkyl chain C<14>H<28>O<2>, wherein said alkyl chain is covalently linked to the NH<2>terminus of the peptide of SEQ ID NO: 5.

In another more preferred embodiment of the method of the invention, the peptide of step (i) comprises or consists of the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably the sequence SEQ ID NO: 2, and wherein said sequence further comprises an alkyl chain selected from: C<12>H<24>O<2>, C<13>H<26>O<2>, C<14>H<28>O<2>, C<15>H<30>O<2>, C<16>H<32>O<2>, C17H34O2 and C<18>H<36>O<2>, wherein said alkyl chain is covalently linked to the NH<2>terminus of the peptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

In an even more preferred embodiment of the method of the invention, the peptide of step (i) comprises or consists of the amino acid sequence SEQ ID NO: 2 and wherein said sequence further comprises an alkyl chain C<14>H<28>O<2> covalently linked at its NH<2> end.

In another embodiment of the method of the invention, step (i) further comprises a pharmacological agent or active ingredient, preferably wherein said agent or active ingredient is lipophilic or hydrophilic in nature.

The terms “pharmacological agent or active ingredient”, “lipophilic nature” and “hydrophilic nature” have been described hereinbefore and apply equally to this embodiment of the invention, as well as to all its preferred embodiments (alone or in combination).

The inventors, using the method of the invention described above, have developed nanoemulsions composed of a lipid mixture (oily core) of attocopherol and octadecylamine and a cover by a layer of surfactant that allows its stabilization in water, in combination with the antimicrobial peptide DJK5, DJK6 or L1018 (modified or not) to inhibit the formation of H. influenzae biofilms.

Therefore, another aspect of the invention relates to nanoemulsions obtained by the method of the invention.

The nanoemulsions of the invention significantly prevent the formation of bacterial biofilms by H. influenzae, allowing their use in vitro to inhibit the formation of biofilms of H. influenzae on implants or surfaces or as a medicine, particularly for use in the treatment or prevention of diseases caused by H. influenzae.

Thus, another aspect of the present invention relates to the in vitro use of the nanoemulsions of the invention, the composition of the invention or the nanoemulsions obtained by the method of the invention, to inhibit the formation of H. influenzae biofilms on implants or surfaces.

The term “H. influenzae biofilms” or “H. influenzae biofilms” used in the present invention refers to an organized microbial ecosystem, made up of H. influenzae, and associated with a living or inert surface, with functional characteristics and complex structures. This type of microbial conformation occurs when planktonic cells adhere to a surface or substrate, forming a community, which is characterized by the excretion of a protective adhesive extracellular matrix.

Another aspect of the present invention relates to the nanoemulsions of the invention, the composition of the invention or the nanoemulsions obtained by the method of the invention, for use as a medicine, hereinafter “the first medical use of the invention”.

The term "medicament" as used herein refers to any substance used for the prevention, diagnosis, alleviation, treatment or cure of diseases in a subject or which can be administered to the subject in order to restore, correct or modify its physiological functions by exerting a pharmacological, immunological or metabolic action. In the context of the present invention, the medicament comprises the nanoemulsions of the invention or alternatively a composition comprising them. For the purposes of the present invention, the terms "medicament" and "pharmaceutical composition" are used synonymously.

Another aspect of the present invention relates to the nanoemulsions of the invention, the composition of the invention or the nanoemulsions obtained by the method of the invention, for use in the treatment or prevention of diseases caused by H. influenzae, “the second medical use of the invention”.

The term "treatment" (or "treating") refers to processes that involve a slowing, stopping, suspending, controlling, arresting, ameliorating, or reversing the progression or severity of an existing symptom, disorder, condition, or disease but may not necessarily involve a total elimination of all symptoms related to the disease, conditions, or disorders associated with diseases caused by H. influenzae. Treatment of a disorder or disease may, for example, lead to an interruption in the progression of the disorder or disease (e.g., no worsening of symptoms) or a slowing of the progression of the disorder or disease (in a case where the interruption of progression is only transient in nature). "Treatment" of a disorder or disease may also lead to a partial response (e.g., improvement of symptoms) or a complete response (e.g., disappearance of symptoms) of the subject/patient suffering from the disorder or disease.

As used herein, the term "prevention" refers to the inhibition or delay of disease caused by H. influenzae.

The term “diseases caused by Haemophilus influenzae” refers to those diseases caused by a bacterium, particularly H. influenzae, which can cause various infections such as urinary tract infections, pneumonia or lung infection, skin infections, ear infection or otitis, diarrhea, or blood infections. Examples of diseases caused by H. influenzae include, but are not limited to, meningitis, epiglottitis, otitis, laryngitis, osteomyelitis, pneumonia, sepsis, conjunctivitis, sinusitis, bronchitis, cellulitis, infectious arthritis.

DESCRIPTION OF FIGURES

Figure 1. Size (A) and polydispersity index (PDI) (B) of nanoemulsions loaded with the DJK5-C14 peptide (U-DJK5-C14) and unloaded nanoemulsions (U). The bar graph represents the mean ± standard error of three independent experiments with three technical replicates (n=9).

Figure 2. Transmission electron microscopy images of (A) nanoemulsions loaded with the DJK5-C14 peptide (U-DJK5-C14) and (B) unloaded nanoemulsions (U).

Figure 3. Zeta potential of (A) nanoemulsions loaded with the DJK5-C14 peptide (U-DJK5-C14) and (B) unloaded nanoemulsions (U). The bar graph represents the mean ± standard error of three independent experiments with three technical replicates (n=9).

Figure 4. Effect of incubation of nanoemulsions loaded with the DJK5-C14 peptide (U-DJK5-C14) diluted in BHI medium on the particle size (A) and the polydispersity index (PDI) (B) at 37°C for 24 hours. The graph represents the mean ± standard error of two independent experiments with three technical replicates (n=6). FD: Dilution factor of the nanoparticles in BHI medium (2.5, 10, 50, 100 and 300).

Figure 5. Effect of incubation of uncharged nanoemulsions (U) diluted in BHI medium on particle size (A) and polydispersity index (PDI) (B) at 37°C for 24 hours. The graph represents the mean ± standard error of two independent experiments with three technical replicates (n=6). FD: Dilution factor of the nanoparticles in BHI medium (2.5, 10, 50, 100 and 300).

Figure 6. A. Effect of unloaded nanoemulsions (U), nanoemulsions loaded with DJK5-C14 (U-DJK5-C14) and the free modified peptide (DJK5-C14) on the prevention of biofilm formation in strain R2866 ( A), strain P605 (B) and strain P639 (C) ofH. influenzae. The bars correspond to the mean optical density (OD) 570 nm (proportional to the amount of dye adhered to the biofilm) ± standard error of three independent experiments with three technical replicates (n=9) for increasing concentrations of the Test substances expressed as total lipid concentration for the unloaded nanoemulsions (U), peptide concentration and corresponding total lipid concentration for the DJK5-C14 loaded nanoemulsions (U-DJK5-C14) and peptide concentration for the modified peptide ( DJK5-C14). (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, vs control).

Figure 7. Effect of free unmodified peptide (DJK5) in preventing biofilm formation in H. influenzae strain R2866 (A), strain P605 (B) and strain P639 (C). Bars correspond to the mean optical density (OD) at 570 nm (proportional to the amount of dye adhered to the biofilm) ± standard error of three independent experiments with three technical replicates (n=9) for increasing concentrations of free unmodified peptide (DJK5) expressed as peptide concentration. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, vs control).

Figure 8. A. Effect of unloaded nanoemulsions (U), nanoemulsions loaded with DJK5-C14 (U-DJK5-C14) and the free modified peptide (DJK5-C14) on the inhibition of planktonic growth on H. influenzae strain R2866 (A), strain P605 (B) and strain P639 (C). Bars correspond to the mean optical density (OD) at 600 nm ± standard error of three independent experiments with three technical replicates (n=9) for increasing concentrations of the tested substances expressed as total lipid concentration for unloaded nanoemulsions (U), peptide concentration and corresponding total lipid concentration for DJK5-C14-loaded nanoemulsions (U-DJK5-C14) and peptide concentration for the free modified peptide (DJK5-C14). (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, vs control).

Figure 9. Effect of free unmodified peptide (DJK5) on the inhibition of planktonic growth in H. influenzae strain R2866 (A), strain P605 (B) and strain P639 (C). Bars correspond to the mean optical density (OD) at 600 nm ± standard error of three independent experiments with three technical replicates (n=9) for increasing concentrations of free unmodified peptide (DJK5) expressed as peptide concentration. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, vs control).

Figure 10. Effect of increasing concentrations (0.98 mg/mL, 1.14 mg/mL, 1.3 mg/mL, 1.47 mg/mL and 1.63 mg/mL) of nanoemulsions loaded with the DJK5-C14 peptide (U-DJK5-C14) on survival in a zebrafish embryo (Danio rerio) animal model for 96 h. The graph represents the percentage of live animals with respect to the total number of animals introduced in the test performed in triplicate and for each concentration (n=3).

Figure 11. Effect of nanoemulsions loaded with DJK6-C14 (U-DJK6-C14) and the free modified peptide (DJK6-C14) in preventing biofilm formation in H. influenzae strain R2866. Bars correspond to the mean optical density (OD) at 570 nm (proportional to the amount of dye adhered to the biofilm) ± standard error of an experiment with three technical replicates (n=3) for increasing concentrations of the tested substances expressed as peptide concentration and corresponding total lipid concentration for nanoemulsions loaded with DJK6-C14 (U-DJK6-C14) and peptide concentration for the free modified peptide (DJK6-C14). (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, vs control).

Figure 12. Effect of nanoemulsions loaded with L1018-C14 (U-L1018-C14) and the free modified peptide (L1018-C14) in preventing biofilm formation in H. influenzae strain R2866. Bars correspond to the mean optical density (OD) at 570 nm (proportional to the amount of dye adhered to the biofilm) ± standard error of an experiment with three technical replicates (n=3) for increasing concentrations of the tested substances expressed as peptide concentration and corresponding total lipid concentration for nanoemulsions loaded with L1018-C14 (U-L1018-C14) and peptide concentration for the free modified peptide (L1018-C14). (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, vs control).

Figure 13. Effect of nanoemulsions loaded with DJK6-C14 (U-DJK6-C14) or with L-1018-C14 (U-L1018-C14) and the free modified peptides (DJK6-C14 and L1018-C14) on the inhibition of planktonic growth on the R2866 strain of H. influenzae. The graph represents the number of CFU counts in both the control and treated wells.

EXAMPLES

The invention will now be illustrated by means of tests carried out by the inventors, which demonstrate the effectiveness of the invention.

Procedure for obtaining nanoemulsions

The method for obtaining the nanoemulsions of the invention comprises:

(a) Prepare an organic phase by dissolving in ethanol 69.7 mM of attocopherol, 12.6 mM of octadecylamine, 11.6 mM of polysorbate 80 and 0.45 mM of peptide DJK5-C14.

(b) Quickly add the solution obtained in step (a) by injection with a syringe with a needle into ultrapure water in a 4:1 ratio with respect to the organic phase and under moderate magnetic stirring (700 rpm).

(c) Stir the dispersion obtained in step (b) for 10 minutes at room temperature.

(d) Filter the preparation obtained in step (c) by size exclusion chromatography column or tangential filtration to obtain the purified nanoemulsion.

This method is carried out at 25 °C and 1 atmosphere of pressure.

The nanoemulsions obtained are characterized by dynamic light scattering using a Zetasizer nano ZS90 (Malvern Panalitical, UK), which allows the determination of the diameter, polydispersity index and zeta potential. For the determination of the latter, the nanoemulsions are diluted 1:20 in a 1 mM NaCl solution. The purified nanoemulsion presents an average particle size of 150 nm, zeta potential >+30 mV and a polydispersity index of 0.12.

Furthermore, the size and morphology of the nanoemulsions are studied by transmission electron microscopy (TEM) using a JEOL JEM 1400 microscope operated at 100 kV equipped with a Gatan Orius Sc 200 CCD camera. The nanoemulsions are embedded on the surface of carbon-coated copper grids, negatively stained with 2% uranyl acetate.

Example 1. Particle size evaluation

The size and polydispersity index (PDI) of the nanoemulsions were studied by dynamic light scattering using a Zetasizer nano ZS90 (Malvern Panalitical, UK). Nanoparticles loaded with the DJK5-C14 peptide (U-DJK5-C14) showed small sizes (153.0 nm ± 2.5 nm) and homogeneous distributions (PDI: 0.133 ± 0.013). Unloaded nanoparticles (U) (used as control) presented very similar data (size: 163.0 nm ± 9.3 nm; PDI: 0.123 ± 0.023). Each sample (n=3) was analyzed in triplicate at room temperature (Figure 1).

This demonstrates that the incorporation of the peptide into the platform does not modify its physicochemical properties, demonstrating the versatility of the nanoemulsion proposed in the present invention. It is concluded that the nanoemulsions obtained present adequate physicochemical properties, with sizes <200 nm and a PDI <0.15.

Example 2. Evaluation of particle morphology

The size and morphology of the nanoemulsions were studied by transmission electron microscopy (TEM) using a JEOL JEM 1400 microscope operated at 100 kV equipped with a Gatan Orius Sc 200 CCD camera. The nanoemulsions were diluted (1:25) in ultrapure water and deposited on the surface of carbon-coated copper grids, negatively stained with 2% uranyl acetate.

The TEM images obtained show nanoemulsions with spherical morphology and size between 100-200 nm, corroborating the sizes previously obtained by dynamic light scattering (Figure 2).

Example 3. Evaluation of the zeta potential

The surface charge of the nanoemulsions (U-DJK5-C14, U) was estimated by electrophoretic light scattering (ELS) using a Zetasizer Nano ZS90 (Malvern Instruments, UK). The synthesized nanoemulsions in ultrapure water were diluted (1:20) in 1 mM sodium chloride before analysis. Each sample (n=3) was analyzed in triplicate at room temperature.

The nanoemulsions showed positive zeta potentials in all cases, with values above 30 mV at room temperature (Figure 3). Nanoparticles with a zeta potential value higher than -30 mV or 30 mV are generally considered to have excellent colloidal stability. This confirms the physicochemical stability predisposition of the developed nanoemulsions.

Example 4. Stability studies in brain heart infusion (BHI) culture medium

The colloidal stability of the nanoemulsions loaded with the modified DJK5-C14 peptide (U-DJK5-C14) in culture medium was determined by size measurement (Figure 4A) and PDI (Figure 4B) at pre-set times of 3, 6, 9, and 24 hours. The nanoemulsions were incubated at 37°C in brain heart infusion (BHI) medium, which is a general-purpose medium suitable for the cultivation of a wide variety of organism types, including bacteria, yeast, and filamentous fungi. The nanoemulsions were kept under continuous agitation for the duration of the assay. In addition, the effect of dilution on the stability of the nanoemulsions in BHI medium was studied. Dilution factors of the nanoemulsions (DF) of 2.5, 10, 50, 100, and 300 in BHI medium were studied.

The colloidal stability of the uncharged nanoemulsions (U) was also determined by size measurement (Figure 5A) and PDI (Figure 5B) following the same protocol. Each sample (n=2) was analyzed in triplicate.

The size of the nanoemulsions did not generally change over time regardless of the dilution factor (Figure 4A, Figure 5A). Only a slight increase in the size of the unfilled nanoemulsion (U) was observed at the highest dilution (Figure 5A). The nanoemulsions also maintained a homogeneous distribution (Figure 4B, Figure 5B). It is concluded that the nanoemulsions do not generally present an alteration of their colloidal and chemical properties when introduced into a culture medium, being able to remain stable for at least 24 h at a temperature of 37°C. This is key to being able to evaluate their potential biological activity.

Example 5. Encapsulation efficiency of the DJK5-C14 peptide in the nanoemulsion

The capacity of the nanoemulsion to incorporate the DJK5-C14 peptide was determined indirectly after purification of the nanoemulsion by ultrafiltration using 100 KDa membranes (Amicon Ultra 0.5, Merck). This washing process was repeated 3 times to ensure complete removal of the non-encapsulated peptide, and then the free peptide present in the filtrate was quantified by HPLC-UV (Agilent).

For this purpose, an Eclipse Plus C18 reversed-phase column (4.6 x 150mm, 5pm) was used with a working flow rate of 1.5 mL/min. Mobile phases A and B were 0.05% aqueous trifluoroacetic acid solution and acetonitrile, respectively, and the gradient used was from 5% to 100% phase B in 20 minutes. Detection was carried out at 220 nm. A calibration curve was made with increasing concentrations (5-50 pg/mL) of DJK5-C14 in methanol. The filtrate water was analyzed directly using these chromatographic conditions, and the area obtained was used to calculate the corresponding concentration according to the calibration curve. Analysis of 6 independent samples revealed an encapsulation efficiency of 87.7% ± 5.9%.

It is concluded that nanoemulsions allow a high encapsulation of the DJK5-C14 peptide. This is key to being able to evaluate the potential biological activity of the peptide integrated into the lipid nanoplatform.

Example 6. Evaluation of DJK5 in preventing biofilm formation

The prevention of biofilm formation by DJK5-C14 loaded nanoemulsions (U-DJK5-C14), unloaded nanoemulsions (U), free modified peptide (DJK5-C14) and free unmodified peptide (DJK5) was studied by crystal violet (CV) assay.

H. influenza was grown on a petri dish containing Mueller Hinton agar medium supplemented with factor X (hemin or hematin), factor V (nicotinamide adenine dinucleotide, or NAD), and yeast extract (HTM medium) at 37 °C for 12 h under 5% CO<2>. Subsequently, 100 µL of BHI medium supplemented with hemin (10% v/v) and NAD (1% v/v) (sBHI) was added into 96-well polystyrene microtiter plates. The concentration of the bacterial suspension was then adjusted to an optical density (OD) of 0.5 McFarland and diluted 1:100 in sBHI medium to transfer 100 µL of this solution to each well. The nanoemulsions (U and U-DJK5-C14), the free modified peptide (DJK5-C14) and the free unmodified peptide (DJK5) were diluted in sBHI to reach a final nanoemulsion concentration of 376 pg/mL expressed as total lipid concentration and 9 pg/mL of peptide respectively. The plates were then treated with 1/2 serial dilutions from these solutions.

After 24 h of incubation at 37°C and 5% CO<2>, the supernatant, containing the planktonic cells, was gently removed from the microplate wells. The wells were washed three times with 150 pL of ultrapure water. After washing, 200 pL of 1% CV was added and the suspension was shaken for 20 min at room temperature. The plate was then rinsed 3 times by adding 200 pL of ultrapure water to each well and the CV was dissolved by adding 200 pL of 96% ethanol to each well. The plate was shaken for 20 min at room temperature. The minimum biofilm inhibitory concentration (MBIC), defined as the lowest concentration of an antimicrobial agent required to inhibit biofilm formation, was determined by measuring the optical density (OD) at 570 nm of the microplate wells.

As a positive control for the nanoemulsion assays, bacterial cultures without the addition of nanoemulsions were used. As a negative control, only sBHI culture medium (microtiter wells containing uninoculated sBHI medium) was used in all assays. The assay was performed in triplicate with three independent batches. The biological activity of the nanoemulsions, the free modified peptide (DJK5-C14) and the free unmodified peptide (DJK5) was studied on three biofilm-forming H. influenzae strains: one strain from a culture collection, strain R2866 (NCBI taxonomic ID number: 262728), and two clinical strains isolated from patients with chronic obstructive pulmonary disease, strain P605-7719 (also referred to herein as strain P605) and strain P639-3649 (also referred to herein as strain P639).

DJK5-C14-loaded nanoemulsions (U-DJK5-C14) potentially prevent bacterial biofilm formation by H. influenzae in strain R2866 (Figure 6A, U-DJK5-C14_R2866), strain P605 (Figure 6B, U-DJK5-C14_P605), and strain P639 (Figure 6C, U-DJK5-C14_P639). This demonstrates that the anti-biofilm effect of DJK5-C14-loaded nanoemulsions is not strain-dependent, demonstrating the versatility of these formulations as agents against H. influenzae bacterial biofilms.

However, the free modified peptide (DJK5-C14) (Figure 6A, DJK5-C14_R2866; Figure 6B, DJK5-C14-P605; Figure 6C, DJK5-C14_P639) did not show antibiofilm effect on any of the three strains tested.

Furthermore, the unmodified peptide (DJK5) in its free form did not inhibit biofilm formation in any of the three strains tested (Figure 7).

This peptide is described in the literature as an anti-biofilm peptide against Pseudomonas aeruginosa and Acinetobacter baumannii, both Gram-negative pathogens. However, its activity against H. influenzae biofilms has not been described to date. Here, it is demonstrated that the activity of the DJK5 peptide in preventing the formation of H. influenzae biofilms is similar to that of the DJK5 peptide. influenzaees null in its free modified form (Figure 6A, DJK5-C14_R2866; Figure 6B, DJK5-C14_P605; Figure 6C, DJK5-C14_P639) and unmodified form (Figure 7), therefore it is concluded that: 1) the addition of a 14-carbon chain at the NH<2>terminal end of the DJK5 peptide does not modify its biological activity and allows said peptide to be encapsulated in nanoemulsions and 2) that the nanoparticular system such as the nanoemulsions described here allow the DJK5-C14 peptide to have biological activity against the formation of H. influenzae biofilms.

Example 7. Evaluation of DJK5 in the inhibition of planktonic growth

Briefly, H. influenza was grown on a petri dish containing Mueller Hinton agar medium supplemented with factor X (hemin or hematin), factor V (nicotine amide adenine dinucleotide, or NAD), and yeast extract (HTM medium) at 37 °C for 12 h under 5% CO<2>. Subsequently, 100 µl of BHI medium supplemented with hemin (10% v/v) and NAD (1% v/v) (sBHI) was added into 96-well polystyrene microtiter plates. The concentration of the bacterial suspension was then adjusted to an optical density (OD) of 0.5 McFarland and diluted 1:100 in sBHI medium to transfer 100 µl of this solution to each well. DJK5-C14 loaded nanoemulsions (U-DJK5-C14), unloaded nanoemulsions (U), free modified peptide (DJK5-C14) and free unmodified peptide (DJK5) were diluted in sBHI to reach a final nanoemulsion concentration of 376 pg/mL expressed as total lipid concentration and 9 pg/mL of peptide respectively.

Plates were then treated with 1/2 serial dilutions of these solutions. The minimum inhibitory concentration (MIC), defined as the lowest concentration of an antimicrobial agent required to inhibit planktonic cell growth, was determined by measuring the optical density (OD) at 600 nm. Bacterial cultures without nanoemulsions were used as a positive control for nanoemulsion assays. sBHI culture medium alone (microtiter wells containing uninoculated sBHI medium) was used as a negative control for all assays. The assay was performed in triplicate with three independent batches. The biological activity of the nanoemulsions, the free modified peptide (DJK5-C14) and the free unmodified peptide (DJK5) was studied on three biofilm-forming strains of H. influenzae (strains R2866, P605, and P639).

In none of the three strains was a bactericidal effect observed for nanoemulsions loaded with DJK5-C14 (U-DJK5-C14) (Figure 8A, U-DJK5-C14_R2866; Figure 8B, U-DJK5-C14_P605; Figure 8C, U-DJK5-C14_P639) and unloaded nanoemulsions (U) (Figure 8A, U_R2866; Figure 8B, U_P605; Figure 8C, U_P639). This corroborates the use of nanoemulsions as anti-virulence therapy. Anti-virulence therapy is based on blocking the virulence factors that make bacteria pathogenic without affecting their cell viability. There are several advantages that this type of therapy presents over classic antibiotic therapy; Among them, it stands out that it only interferes with the expression or activity of virulence factors that in most cases are not essential for bacterial survival, not affecting the patient's microbiota and reducing bacterial selection pressure, a factor that contributes to the development of resistance.

Here it is also demonstrated that the activity of the DJK5 peptide to inhibit the planktonic growth of H. influenzae is null in its free, modified (Figure 8A, DJK5-C14_R2866: Figure 8B, DJK5-C14_P605; Figure 8C, DJK5-C14_P639) and unmodified forms (Figure 9), so it is concluded that the addition of a 14-carbon chain at the NH<2>terminal end of the DJK5 peptide does not modify its biological activity.

Example 8. Evaluation of biocompatibility in vivo in a zebrafish embryo model (Danio rerio)

The zebrafish is considered a good in vivo model for toxicological and biocompatibility studies since it shows a concordance with mammalian models of between 65% and 100%. To evaluate the toxicity of the nanoemulsions, the harmonized OECD protocol for acute toxicity in fish embryos (FET) was used. This test is carried out on embryos between 120 h post-fertilization and 5 days post-fertilization by immersion exposure. Five nanoemulsion concentrations expressed as total lipid concentration were tested in triplicate: 0.98 mg/mL, 1.14 mg/mL, 1.3 mg/mL, 1.47 mg/mL, and 1.63 mg/mL, using a total of 35–40 embryos for each concentration, both of unloaded nanoemulsions (U) and of nanoemulsions loaded with DJK5-C14 (U-DJK5-C14). Mortality was measured every 24 h for a total of 96 h.

Unloaded nanoemulsions showed 100% survival at all tested concentrations, whereas a correlation between concentration and toxicity was observed for nanoemulsions loaded with DJK5-C14 (Figure 10). Thus, for U-DJK5-C14 the lethal concentration 50 (LC<50>) was determined by Probit regression, while the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC) were calculated by Fisher's exact test. Thus, in terms of total lipid concentration, the LC50 of U-DJK5-C14 was 1.45 mg/mL, while the LOEC was 1.3 mg/mL and the NOEC was 1.14 mg/mL.

These results demonstrate the low toxicity of the developed nanoemulsions and, therefore, their high biocompatibility, with the LC50 being 10 times higher than the concentration necessary to inhibit biofilm formation (Figure 6).

Example 9. Evaluation of DJK6 and L1018 in preventing biofilm formation

The prevention of biofilm formation by nanoemulsions loaded with DJK6-C14 (U-DJK6-C14) or L1018-C14 (U-L1018-C14), as well as by free modified peptides (DJK6-C14 and L1018-C14) was studied by crystal violet (CV) assay.

H. influenza was grown on a petri dish containing Mueller Hinton agar medium supplemented with factor X (hemin or hematin), factor V (nicotinamide adenine dinucleotide, or NAD), and yeast extract (HTM medium) at 37 °C for 12 h under 5% CO<2>. Subsequently, 100 µL of BHI medium supplemented with hemin (10% v/v) and NAD (1% v/v) (sBHI) was added into 96-well polystyrene microtiter plates. The concentration of the bacterial suspension was then adjusted to an optical density (OD) of 0.5 McFarland and diluted 1:100 in sBHI medium to transfer 100 µL of this solution to each well. U-DJK6-C14 and U-L1018-C14 nanoemulsions as well as free modified peptides (DJK6-C14 and L1018-C14) were diluted in sBHI to reach a final nanoemulsion concentration of 4.3 mg/mL expressed as total lipid concentration and 9 pg/mL of peptide respectively. Free peptides were diluted in the same proportion to reach a concentration of 9 pg/mL. Plates were then treated with 1/2 serial dilutions from these solutions.

After 24 h of incubation at 37°C and 5% CO<2>, the supernatant, containing the planktonic cells, was gently removed from the microplate wells. The wells were washed three times with 150 pL of ultrapure water. After washing, 200 pL of 1% CV was added and the suspension was shaken for 20 min at room temperature. The plate was then rinsed 3 times by adding 200 pL of ultrapure water to each well and the CV was dissolved by adding 200 pL of 96% ethanol to each well. The plate was shaken for 20 min at room temperature. The minimum biofilm inhibitory concentration (MBIC), defined as the lowest concentration of an antimicrobial agent required to inhibit biofilm formation, was determined by measuring the optical density (OD) at 570 nm of the microplate wells.

Bacterial cultures without nanoemulsions were used as positive controls for the nanoemulsion assays. As a negative control, sBHI culture medium alone (microtiter wells containing uninoculated sBHI medium) was used in all assays. The assay was performed in triplicate with one batch of each nanoemulsion. The biological activity of the nanoemulsions and the free modified peptides were studied in H. influenzae strain R2866.

Nanoemulsions loaded with DJK6-C14 (U-DJK6-C14) and with L1018-C14 (U-L1018-C14) potentially prevent the formation of bacterial biofilms by H. influenzaeen strain R2866 (Figure 11, U-DJK6-C14_R2866 and Figure 12, U-L1018-C14_R2866). The anti-biofilm inhibition in the tested strain observed with the nanoemulsions loaded with the peptides was much higher than that shown by the free modified peptides that only showed a slight anti-biofilm effect at the highest concentrations (Figure 11, DJK6-C14_R2866 and Figure 12, L1018-C14_R2866), which shows that the use of nanoemulsion enhances the antibiofilm effect of peptides DJK6-C14 and L1018-C14.

These peptides are described in the literature as anti-biofilm peptides against Pseudomonas aeruginosa and Acinetobacter baumannii, both Gram-negative pathogens. However, their activity against H. influenzae biofilms has not been described to date. Here it is demonstrated that the activity of the DJK6 and L1018 peptide in preventing H. influenzae biofilm formation is poor in its free and modified form, and that the use of a nanoparticular system such as the nanoemulsions described here enhances their ability to inhibit H. influenzae biofilm formation.

Example 10. Evaluation of DJK6 and L1018 in the inhibition of planktonic growth

H. influenza was grown on a petri dish containing Mueller Hinton agar medium supplemented with factor X (hemin or hematin), factor V (nicotine amide adenine dinucleotide, or NAD), and yeast extract (HTM medium) at 37 °C for 12 h and 5% CO<2>. Subsequently, 100 µL of BHI medium supplemented with hemin (10% v/v) and NAD (1% v/v) (sBHI) was added into 96-well polystyrene microtiter plates. The concentration of the bacterial suspension was then adjusted to an optical density (OD) of 0.5 McFarland and diluted 1:100 in sBHI medium to transfer 100 µL of this solution to each well. Nanoemulsions loaded with DJK6-C14 (U-DJK6-C14) and L1018-C14 (U-L1018-C14) peptides, and free modified peptides (DJK6-C14 and L1018-C14) were diluted in sBHI to reach a final nanoemulsion concentration of 4.3 mg/mL expressed as total lipid concentration and 9 pg/mL of peptide respectively. Free peptides were diluted in the same proportion to reach a concentration of 9 pg/mL.

Plates were then treated with 1/2 serial dilutions from these dilutions and incubated for 24 h at 37°C and 5% CO<2>. After incubation, the supernatant from the wells with the highest concentrations of formulation/free peptide was gently removed from the microplate wells. The minimum inhibitory concentration (MIC), defined as the lowest concentration of an antimicrobial agent required to inhibit the growth of planktonic cells, was determined using the following protocol: addition of 100 pL of each supernatant to 900 pL of 1x PBS, from which 5 serial dilutions were made. Subsequently, 100 pL of each dilution was added to HTM plates. Plates were incubated for 24 h at 37°C for 24 h and 5% CO<2>. The following day, colony forming units (CFUs) were counted.

The formulations/free peptides were tested on a single strain (R2866) and with a single batch. Three biological replicates were made.

No bactericidal effect was observed for nanoemulsions loaded with DJK6-C14 (U-DJK6-C14) or L1018 (U-L1018-C14), and neither for the free modified peptides (DJK6-C14 and L1018-C14), obtaining for all of them a number of CFU comparable to that of the untreated control (Figure 13). This result corroborates the use of nanoemulsions as anti-virulence therapy, as described in example 7.

Claims (27)

1. Nanoemulsions comprising: (a) an oily core comprising at least one liquid lipid and octadecylamine, (b) a shell of at least one surfactant, and (c) the peptide comprising or consisting of the amino acid sequence SEQ ID NO: 5. 2. Nanoemulsions according to claim 1, wherein the peptide comprises or consists of the amino acid sequence SEQ ID NO: 1. 3. Nanoemulsions according to claim 1 or 2, wherein the peptide comprises or consists of the amino acid sequence SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably the peptide comprising or consisting of the amino acid sequence SEQ ID NO: 2. 4. Nanoemulsions according to any one of claims 1 to 3, wherein the peptide further comprises an alkyl chain covalently linked at its NH<2> end, wherein the alkyl chain is selected from: C<12>H<24>O<2>, C<13>H<26>O<2>, C<14>H<28>O<2>, C<15>H<30>O<2>, C<16>H<32>O<2>, C17H34O2y C<18>H<36>O<2>, preferably C<14>H<28>O<2>. 5. Nanoemulsions according to any one of claims 1 to 4, wherein the peptide comprises or consists of the amino acid sequence SEQ ID NO: 2 and comprising the alkyl chain C<14>H<28>O<2> covalently linked at its NH<2> end. 6. Nanoemulsions according to any one of claims 1 to 5, wherein the liquid lipid is glycerol monolinoleate, propylene glycol monolaurate or α-tocopherol, preferably α-tocopherol. 7. Nanoemulsions according to any one of claims 1 to 6, wherein the surfactant is polysorbate 20 and/or polysorbate 80, preferably polysorbate 80. 8. Nanoemulsions according to any one of claims 1 to 7, which further comprise a pharmacological agent or active ingredient. 9. Nanoemulsions according to any one of claims 1 to 8, wherein the size of the nanoemulsions is less than 200nm, preferably between 140nm and 170nm. 10. A composition comprising the nanoemulsions according to any one of claims 1 to 9. 11. The composition according to claim 10, wherein the composition is a pharmaceutical composition. 12. The composition according to claim 11, further comprising a pharmaceutically acceptable carrier and/or excipient. 13. A method for obtaining nanoemulsions according to any one of claims 1 to 9 comprising: (i) dissolving at least one liquid lipid, at least one surfactant, octadecylamine and the peptide comprising or consisting of the amino acid sequence SEQ ID NO: 5 in an alcoholic solvent, where the liquid lipid concentration is between 30 and 100 mM, the surfactant concentration is between 5 and 20 mM, the octadecylamine concentration is between 5 and 20 mM and the peptide concentration is between 0.1 and 1 mM, obtaining an organic phase; (ii) injecting the organic phase obtained in (i) onto an aqueous phase under continuous stirring at a speed between 700 and 1000 rpm for at least 10 minutes, where the aqueous phase:organic phase ratio is at least 4:1, obtaining a solution with the nanoemulsions; and (iii) filtering the solution obtained in (ii) using size exclusion chromatography columns or tangential filtration membranes, obtaining the nanoemulsions; where the method is carried out at 25°C and 1 atmosphere of pressure. 14. Method according to claim 13, wherein the peptide of step (i) is a peptide comprising or consisting of the amino acid sequence SEQ ID NO: 1. 15. Method according to claim 13 or 14, wherein the peptide of step (i) is a peptide comprising or consisting of the amino acid sequence SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably a peptide comprising or consisting of the amino acid sequence SEQ ID NO: 2. 16. Method according to any one of claims 13 to 15, wherein the peptide of step (i) further comprises an alkyl chain covalently linked at its NH<2> end, wherein the alkyl chain is selected from: C<12>H<24>O<2>, C<13>H<26>O<2>, C<14>H<28>O<2>, C<15>H<30>O<2>, C<16>H<32>O<2>, C17H34O2y C<18>H<36>O<2>, preferably C<14>H<28>O<2>. 17. Method according to any one of claims 13 to 16, wherein the peptide of step (i) comprises or consists of the amino acid sequence SEQ ID NO: 2 and comprising the alkyl chain C<14>H<28>O<2> covalently linked at its NH<2> end. 18. Method according to any one of claims 13 to 17, wherein the alcoholic solvent is ethanol or isopropanol, preferably ethanol, and where the aqueous phase is water. 19. Method according to any one of claims 13 to 18, wherein the liquid lipid of step (i) is glycerol monolinoleate, propylene glycol monolaurate or α-tocopherol, preferably α-tocopherol. 20. Method according to any one of claims 13 to 19, wherein in step i) the concentration of liquid lipid, preferably of α-tocopherol, is 69.7 mM; the concentration of surfactant, preferably of polysorbate 80, is 11.6 mM; the concentration of octadecylamine is 12.6 mM and the concentration of peptide is 0.45 mM. 21. Method according to any one of claims 13 to 20, wherein the surfactant of step (i) can be added in the aqueous phase of step (ii). 22. Method according to any one of claims 13 to 21, wherein step (i) further comprises a pharmacological agent or active ingredient, preferably wherein said agent or active ingredient is lipophilic or hydrophilic in nature. 23. Method according to any one of claims 13 to 22, wherein the surfactant of step (i) or (ii) is polysorbate 20 and/or polysorbate 80, preferably it is polysorbate 80. 24. Nanoemulsions obtained by the method according to any one of claims 13 to 23. 25. Ex vivo use of the nanoemulsions according to any one of claims 1 to 9, the composition according to claim 10 or the nanoemulsions according to claim 24, to inhibit the formation of Haemophilus influenzae biofilms on implants or surfaces. 26. Nanoemulsions according to any one of claims 1 to 9, the composition according to any one of claims 10 to 12, or the nanoemulsions according to claim 24, for use as a medicament. 27. Nanoemulsions according to any one of claims 1 to 9, the composition according to any one of claims 10 to 12 or the nanoemulsions according to claim 24, for use in the treatment or prevention of diseases caused by Haemophilus influenzae.