Nanostructured lipid carriers for inhibiting the formation of bacterial biofilms

Abstract

The present invention relates to nanostructured lipid carriers comprising a lipid core of liquid and solid lipids, particularly a triglyceride having a fatty acid chain of 12 to 16 carbon atoms and a mixture of triglycerides of medium-chain saturated fatty acids and covered by a surfactant layer, in which at least one of said surfactants is phosphatidylcholine. Said nanostructured lipid carriers are characterised in that they do not comprise any active ingredient or agent. The invention also relates to the (non-therapeutic) use of the nanostructured lipid carriers for inhibiting the formation of biofilms, as well as their therapeutic use as a medicament for treating or preventing diseases caused by biofilm-forming bacteria.

Description

[0001] NANOSTRUCTURED LIPID CARRIERS TO INHIBIT THE FORMATION OF BACTERIAL BIOFILM

[0002] DESCRIPTION

[0003] The present invention relates to nanostructured lipid transporters composed of a lipid core of a combination of solid and liquid lipids, and a coating of a surfactant layer, and their use for the ex vivo inhibition of bacterial biofilm formation, without the addition of any active agent or principle, as well as their use as a medicament for the treatment or prevention of diseases caused by biofilm-forming bacteria.

[0004] BACKGROUND OF THE INVENTION

[0005] Treatments for bacterial infections with conventional antibiotics are becoming increasingly ineffective, primarily due to the emergence of antibiotic-resistant pathogens. Furthermore, the development of bacterial resistance is far outpacing the development of new antimicrobials. In 2014, the World Health Organization warned that by 2050 bacterial infections would be the leading cause of death worldwide if effective measures were not taken (Murray, C. et al. Lancet, 2022, 399, 629-655).

[0006] Bacteria living in biofilms contribute significantly to the emergence of drug resistance (Hoiby, N. et al., Int J. Antimicrob. Agents, 2010, 35, 322-332). A biofilm is a group of bacteria attached to a surface and embedded in an extracellular polymeric matrix (EPS) that they themselves produce (Flemming, H. et al., Nat. Rev. Microbiol., 2016, 14, 563-575). Collectively, bacterial biofilms are approximately 10 to 1000 times more resistant to antibiotics and account for more than 60% of bacterial infections in humans, leading to persistent and chronic infections.

[0007] As an alternative to conventional antibiotics and in the quest to overcome antimicrobial resistance in bacterial biofilms, the use of drug delivery nanosystems has been investigated in recent decades. Drug delivery nanosystems are technologies that allow for the controlled transport of drugs. Due to their usefulness in modulating drug release, protecting labile materials (e.g., peptides, DNA, or mRNA) from degradation, and their ability to actively transport drugs to specific sites, significant efforts are being directed toward producing nanoparticles as vehicles for active ingredients in various treatments, especially now with the new generation of vaccines based on lipid nanoparticles.

[0008] Lipid nanosystems (LNs) include all nanomaterials whose major component is a lipid. LNs have attracted increasing attention over 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 routes of administration (oral, dermal) (Mishra, D. et al., Nanomedicine Nanotechnology Biol. Med., 2018, 14, 2023-2050).

[0009] Three main types of nanomaterials (NMs) can be distinguished according to their structure: emulsion-based, vesicular, and custom systems. Nanoemulsions belong to the first type and are colloidal dispersions obtained from oil, water, and surfactants. They have generated considerable interest for their application in the pharmaceutical and food industries as carriers of lipophilic active ingredients. The quintessential vesicular systems are liposomes, which are primarily composed of phospholipid bilayers enclosing an aqueous compartment and can incorporate both lipophilic and hydrophilic active ingredients. Despite their simple composition, the drawback of these two types of NMs is their limited stability.

[0010] These systems are considered an evolution of nanoemulsions and liposomes with improved stability properties (Muller, et al., Adv. Drug Deliv. Rev., 2002, 54, S131-S155). Solid lipid nanoparticles (SLNs) are composed of lipids that are solid at room and body temperature, stabilized by a surfactant in an aqueous dispersion, and have a colloidal size between 50 and 1000 nm. Despite greater control over drug release compared to oily phases, some limitations have emerged in their application related to the highly ordered crystalline structure of the lipids, resulting in low drug incorporation and premature drug release (Naseri, N. et al., Adv. Pharm. Bul., 2015, 5, 305-313).

[0011] For this reason, a second generation of lipid nanoparticles, called nanostructured lipid carriers (NLCs), was designed, where a fraction of the solid lipids is replaced by liquid lipids to form a more amorphous matrix, which improves drug incorporation and overcomes other limitations of SLNs (Jaiswal, P. et al., Artif. Cells, Nanomedicine, Biotechnol., 2016, 44, 27-40).

[0012] The applicability of nanotechnology-based lipid systems is presented as an innovative tool for improving the treatment of bacterial infections. Regarding the use of nanoplastic cells (NLCs) for this purpose, most studies use them as carriers of traditional antibiotic molecules (dos Santos, M., et al., Int. J. Pharm., 2021, 603, 120706).

[0013] The effect of various monoacylglycerols with increasing fatty acid chain length (monocaplin, monocapna, monolaunin, monomystin, monopalmitin, monosteain, monoarachidin, monobehenin) has been tested on different bacterial strains. The results showed that the antibacterial effect of these lipids increased with increasing fatty acid chain length, although there was a strong dependence on the specific bacterium. In this study, monolaunin was the most effective lipid against Streptococcus mutans and Xanthomonas oryzae (Ham, Y. and Kim, T., Springerplus, 2016, 5, 1526).

[0014] The presence of bacterial biofilms is associated with the chronicity of various mucosal infections, such as hidradenitis suppurativa, infections associated with medical devices like prosthetic implants (Su Y., et al, Adv. Sci. 2022, 9, 2203291), and recurrent respiratory infections associated with other pathologies such as chronic obstructive pulmonary disease and cystic fibrosis (Kolpen M, et al., Thorax 2022;77:1015-1022). Some pathogens responsible for these infections include nontypeable Haemophilus influenzae (NTHi), Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter baumannii, and Burkholderia cenocepacia, among others. Currently, there is no approved therapy to prevent the formation of these biofilms. Therefore, there is a growing need to develop a treatment to prevent biofilm formation in pathogenic bacteria.

[0015] DESCRIPTION OF THE INVENTION

[0016] The present invention relates to nanostructured lipid carriers (NLC) composed of a lipid mixture (lipid core) of solid and liquid lipids and a coating composed of a surfactant layer that allows its stabilization in water.

[0017] The inventors have shown that these nanostructured lipid transporters exhibit excellent physicochemical properties and high stability in suspension at different temperatures for at least 70 days and a number of suspended particles, which remained around 90-100% of the initial value, so they can be stored for long periods of time (Example 4 and Figures 4 and 5).

[0018] Furthermore, the inventors have observed that, surprisingly, the use of said NLCs that do not comprise any active agent or principle prevents biofilm formation of nontypeable Haemophilus influenzae, particularly at a concentration of 12.5 pg / mL (Example 9 and Figure 11), and in Pseudomonas aeruginosa and Chromobacterium violaceum, particularly at a concentration of 41.25 pg / mL (Examples 12 and 14 and Figures 14 and 16), even completely inhibiting biofilm formation of the 3 bacterial pathogens at a concentration of 50 pg / mL.

[0019] Thus, in a first aspect, the present invention relates to nanostructured lipid transporters, hereinafter referred to as “the nanostructured lipid transporters of the invention” or the “NLCs of the invention”, comprising:

[0020] (a) a lipid core comprising at least one solid lipid and at least one liquid lipid, characterized in that the solid lipid is a triglyceride with a fatty acid chain of between 12 and 16 carbons, and

[0021] (b) a coating of at least two surfactants, characterized in that one of the surfactants is phosphatidylcholine; characterized in that said nanostructured lipid transporters do not comprise any active agent or principle.

[0022] The term “nanostructured lipid carriers” or “NLCs,” as used in the present invention, refers to lipid systems or colloidal systems that transport lipophilic actives, composed of solid and liquid lipids (in a physiological state) and at least two surfactants, with a size of less than 1000 nm, and which are stabilized in aqueous suspensions due to the presence of the surfactants. The mixed composition of the core, achieved by combining solid and liquid lipids, allows for the formation of an amorphous matrix that overcomes the limitations of other types of NLCs, such as liposomes or solid lipid nanoparticles. Preferably, the proportion of liquid lipid will be between 30% and 60% (w / w) with respect to the total lipid that makes up the core.

[0023] The nanostructured lipid transporters of the invention are formed by a lipid core, where said lipid core is composed of a lipid mixture, particularly of solid lipids and a liquid lipid, and are characterized in that they do not comprise any active agent or principle.

[0024] The term “solid lipid” used in the present invention refers to any substance of a lipid nature that is solid at room temperature. Examples of solid lipids include, but are not limited to, triglycerides, diglycerides, monoglycerides, fatty acids, spheroids, and waxes. Preferably, the lipid core of the NLCs of the present invention is characterized in that the solid lipid is a triglyceride with a fatty acid chain of between 12 and 16 carbons (inclusive), preferably trilaurin (C12), trimyristin (C14), or palmitin (C16), and its partial glycerides.

[0025] Thus, in a preferred embodiment of the NLCs of the present invention, the solid lipid is trilaurin (C12), trimyristin (C14) and / or thpalmitin (C16).

[0026] The term “trilaurin” used in the present invention refers to the triglyceride of laureth acid (C12:0), i.e., the molecule formed by three saturated fatty acids with a length of 12 carbon atoms sterilized with glycerol, as shown in formula I.

[0027] Formula I. Chemical structure of trilaurin

[0028] The term “trimyristin” used in the present invention refers to the triglyceride of myristic acid (C14:0), i.e., the molecule formed by three saturated fatty acids with a length of 14 carbon atoms esterified with glycerol, as shown in formula II.

[0029] Formula II. Chemical structure of trimyristin

[0030] The term “tripalmitin” used in the present invention refers to the triglyceride of palmitic acid (C16:0), i.e., the molecule formed by three saturated fatty acids with a length of 16 carbon atoms esterified with glycerol, as shown in formula III.

[0031] Formula III. Chemical structure of tripalmitin

[0032] In another more preferred embodiment of the nanostructured lipid transporters of the invention, the molar concentration of solid lipid (triglyceride with fatty acid chain between 12 and 16 carbons) is between 1 and 4 mM (final concentration in the NLC), preferably between 1.5 and 3.5 mM, more preferably between 1.87 and 3.20 mM.

[0033] In another more preferred embodiment of the nanostructured lipid transporters of the invention, the final concentration of the solid lipid in the NLC is between 2.5 and 3 mM, preferably 2.90 mM.

[0034] The term “liquid lipid” as used in the present invention refers to any substance of a 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.

[0035] In another embodiment of the NLCs of the present invention, the liquid lipid is selected from the list consisting of: medium-chain triglyceride (MCT), glycerol monolinoleate ? mineral oil and propylene glycol monolaurate.

[0036] The term “medium-chain triglycerides” or “MCTs” used in the present invention refers to glycerol esters with saturated fatty acids having a carbon chain length of 6 to 10 atoms. Examples of medium-chain triglycerides include caproic acid triglycerides (C6:0), caprylic acid triglycerides (C8:0), and capric acid triglycerides (C10:0), as well as combinations thereof.

[0037] Thus, in another preferred embodiment of the NLCs of the present invention, the liquid lipid is a mixture of saturated fatty acid triglycerides, preferably caprylic acid (C8:0) and capric acid (C10:0).

[0038] In another preferred embodiment of the nanostructured lipid transporters of the invention, the percentage on the total mass of the liquid lipid in caprylic acid (C 8:0) is between 30 and 80% w / w, and in capric acid (C 10:0) is between 20 and 60% w / w.

[0039] In another more preferred embodiment of the nanostructured lipid transporters of the invention, the percentage of caprylic acid (C8:0) by mass of the total liquid lipid is between 40 and 70% w / w, more preferably 55% w / w, and capric acid (C10:0) is between 30 and 50% w / w, more preferably 45% w / w. In another more preferred embodiment of the nanostructured lipid transporters of the invention, the concentration of liquid lipid is between 1 and 3.5 mM, preferably between 1.5 and 3 mM, more preferably between 1.6 and 2.73 mM (final concentration in the NLC).

[0040] In another more preferred embodiment, the final concentration in the NLC of liquid lipid is between 2 and 2.5mM, preferably 2.48mM.

[0041] To allow stabilization in water, the nanostructured lipid transporters of the invention have a coating formed by a surfactant layer.

[0042] The term “surfactant” used in the present invention refers to substances that reduce surface tension at the interface between two phases (for example, two immiscible liquids) by adsorbing these molecules onto the interface. The term surfactant is equivalent to surface-active agent. Examples of surfactants include, but are not limited to, phosphatidylcholine, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, poloxamer 407, polyoxyethylene (40) stearate (PEG 40 stearate), propylene glycol stearate, or macrogol lauryl ether.

[0043] The term “phosphatidylcholine” used in the present invention refers to the phospholipid composed of a glycerol molecule to which two fatty acids, palmitic acid (C16:0) and oleic acid (C18:1), and a phosphate group are attached, which in turn is linked by a phosphodiester bond to other molecules, generally containing nitrogen, such as choline, as shown in Formula IV. In the present invention, a soybean phospholipid with a phosphatidylcholine content of not less than

[0044] Formula IV. Chemical structure of phosphatidylcholine

[0045] In a preferred embodiment of the nanostructured lipid transporters of the invention, the surfactant is phosphatidylcholine, polysorbate 20, polysorbate 80, poloxamer 188, poloxamer 407 and / or PEG 40 stearate.

[0046] In another more preferred embodiment of the nanostructured lipid transporters of the invention, the surfactant is phosphatidylcholine, PEG 40 stearate and / or poloxamer 188.

[0047] In another, even more preferred embodiment of the nanostructured lipid transporters of the invention, the surfactant is the combination of phosphatidylcholine, PEG 40 stearate, and poloxamer 188.

[0048] In another preferred embodiment of the nanostructured lipid transporters of the invention, the surfactant concentration in %m / v is between 0.2 and 0.32% m / v (final concentration in the NLC).

[0049] In another, even more preferred embodiment of the NLCs of the invention, the surfactants are the combination of phosphatidylcholine, PEG 40 stearate and poloxamer 188 and the concentration of phosphatidylcholine is between 0.06% and 0.10% w / v, the concentration of PEG 40 stearate is between 0.11% and 0.19% w / v and the concentration of poloxamer 188 is between 0.01% and 0.025% w / v, preferably the concentration of phosphatidylcholine is 0.1% w / v, the concentration of PEG 40 stearate is 0.18% w / v and the concentration of poloxamer 188 is 0.02% w / v.

[0050] The nanostructured lipid carriers of the present invention have a reduced size with a diameter of less than 500 nm. The size of the nanostructured lipid carriers is primarily influenced by their composition and formation conditions and can be measured using standard procedures known to those skilled in the art. Examples of NLC size measurement procedures include, but are not limited to, dynamic light scattering (DLS), transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and tunable resistive pulse detection (TNR). The preferred NLC size measurement procedures are dynamic light scattering (DLS) and transmission electron microscopy (TEM).

[0051] In another preferred embodiment of the invention, the nanostructured lipid transporters have a size smaller than 200nm.

[0052] In another more preferred embodiment of the invention, the nanostructured lipid transporters have a size between 120nm and 200nm, preferably between 150nm and 190nm.

[0053] In another, even more preferred embodiment of the invention, the nanostructured lipid transporters have a size of 150nm, 151nm, 152nm, 153nm, 154nm,

[0054] 155nm, 156nm, 157nm, 158nm, 159nm, 160nm, 161nm, 162nm, 163nm, 164nm,

[0055] 165nm, 166nm, 167nm, 168nm, 169nm, 170nm, 171nm, 172nm, 173nm, 174nm,

[0056] 175nm nm, 176nm, 177nm, 178nm, 179nm, 180nm, 181nm, 182nm, 183nm, 184nm, 185nm, 186nm, 187nm, 188nm, 189nm or 190nm, preferably having a size of 175nm, 176nm, 177nm, 178nm, 179nm or 180nm.

[0057] As stated above, the nanostructured lipid transporters of the invention do not comprise any pharmacological agent or active ingredient.

[0058] As used herein, the term “active ingredient” or “active agent” means any component that potentially provides pharmacological activity or another 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 formulation of the drug and are present in the drug in an intended modified form that provides the specific activity or effect. In another embodiment of the nanostructured lipid carriers of the invention, they do not comprise agents or active ingredients of either a lipophilic or a hydrophilic nature.

[0059] The term “lipophilic nature” refers to compounds, molecules, or substances that tend to dissolve in other lipophilic substances such as lipids or fats.

[0060] The term “hydrophilic nature” refers to compounds, molecules, or substances that tend to dissolve in water and other hydrophilic substances.

[0061] As an expert in the field understands, the nanostructured lipid transporters of the invention can be comprised in a composition.

[0062] Therefore, another aspect of the invention is a composition comprising the nanostructured lipid transporters of the invention, hereinafter the “composition of the invention”.

[0063] Nanostructured lipid transporters have been described earlier in this document and apply equally to this aspect of the invention, as well as to all its preferred embodiments (alone or in combination).

[0064] In a preferred embodiment, the concentration of nanostructured lipid transporters in the composition of the invention is between 2 and 200 pg / mL, preferably between 5 and 50 pg / mL.

[0065] In particular, 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 bacterial pathogens, particularly H. influenzae (preferably non-typeable), or P. aeruginosa or C. violaceum. Thus, in a preferred embodiment, the composition of the invention is a pharmaceutical composition.

[0066] The term “pharmaceutical composition” refers to any assembly, mixture, or combination of components or substances comprising the nanostructured lipid carriers of the invention at 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 medicament, particularly as a medicament in the treatment or prevention of diseases caused by bacterial pathogens, particularly biofilm-forming pathogens such as H. influenzae (preferably nontypeable), P. aeruginosa, or C. violaceum, which may be used in or administered to humans for the purpose of restoring, correcting, or modifying physiological functions by exerting a pharmacological, immunological, or metabolic action, or for establishing a medical diagnosis.

[0067] In another preferred embodiment, the pharmaceutical composition further comprises at least one pharmacologically acceptable vehicle and / or excipient.

[0068] The term “vehicle” or “carrier” refers to a substance, preferably an inert one, that facilitates the incorporation of other compounds, allows for better dosage and administration, or improves the consistency and form of the pharmaceutical composition for use as a medicinal product in the treatment or prevention of diseases caused by bacterial pathogens, particularly biofilm-forming pathogens such as H. influenzae (preferably nontypeable), P. aeruginosa, or C. violaceum. Therefore, a vehicle is a substance used in a medicinal product to dilute any of the components of the pharmaceutical composition to a specific volume or weight; or, even without diluting these components, it allows for better dosage and administration or provides consistency and form to the medicinal product. When the formulation is liquid, the pharmaceutically acceptable vehicle is the diluent.

[0069] The term “excipient” refers to a substance that aids in the absorption of any of the components of a pharmaceutical composition, stabilizes these components, modifies their organoleptic properties, or determines the physicochemical properties of the pharmaceutical composition and its bioavailability. Thus, excipients may have the function of holding components together, such as starches, sugars, or cellulose; a sweetening function; a coloring function; a protective function for the drug, such as isolating it from air and / or moisture; a filling function for a tablet, capsule, pill, or any other dosage form, such as dibasic calcium phosphate; a disintegrating function to facilitate the dissolution of components; without excluding other types of excipients not mentioned in this paragraph.

[0070] Furthermore, as the expert in the field understands, the excipient and the vehicle must be pharmacologically acceptable. The term “pharmaceutically acceptable” means that the vehicle or excipient must allow the activity of the compounds in the pharmaceutical composition, particularly the particle of the invention, that is, that it is compatible with said components, so as not to cause harm to the organisms to which it is administered.

[0071] The pharmaceutical composition may be presented in any clinically permissible form of administration and in a therapeutically effective quantity. For example, it may be in a form adapted for oral, sublingual, nasal, intrathecal, bronchial, lymphatic, rectal, transdermal, intravenous, intraperitoneal, orogastric, intracolonic, inhaled, or parenteral administration.

[0072] As the expert in the field knows, there are several procedures for obtaining NLC, for example, high-pressure homogenization, microemulsification with high-speed stirring or ultrasound, emulsification by evaporation or solvent diffusion, double emulsification (w / o / w) and high-speed nanospheronization.

[0073] Another aspect of the invention is a method for obtaining the nanostructured lipid transporters of the invention, hereinafter referred to as “the method of the invention”, comprising:

[0074] (i) preparing an organic phase by dissolving at least one solid lipid, characterized in that the solid lipid is a triglyceride with a fatty acid chain of between 12 and 16 carbons, at least one liquid lipid, and at least one surfactant (1) characterized in that the surfactant (1) is at least phosphatidylcholine, in at least one solvent, preferably ethanol and / or isopropanol;

[0075] (i) preparing an aqueous phase by dissolving at least one other surfactant (2) in an aqueous solvent, preferably water, (iii) independently heating the phases obtained in i) and i) to the same or 5 e C above the melting temperature of the solid lipid,

[0076] (iv) inject the organic phase onto the aqueous phase, obtained in (iii), by continuous stirring at a speed between 600 and 1000 rpm, leave stirring with controlled temperature at a temperature equal to or 5 e C above the melting temperature of the solid lipid for at least 10 minutes, preferably between 10 and 20 minutes, more preferably between 10 and 15 minutes;

[0077] (v) maintain continuous stirring without heat at a speed between 600 and 1000 rpm until a temperature between 20 and 25 is reached e C and 1 atmosphere of pressure, and

[0078] (vi) purifying the dispersion obtained in step (v), preferably by means of size exclusion chromatography columns or tangential filtration membranes, to obtain the nanostructured lipid carriers of the invention free of solvents and excipients and / or non-incorporated active ingredient; characterized in that said nanostructured lipid carriers obtained do not comprise any active agent or ingredient.

[0079] In a preferred embodiment of the method of the invention, the solid lipid is a triglyceride with a fatty acid chain between 12 and 16 carbons, preferably trilaurin (C12), trimyristin (C14) and / or thpalmitin (C16).

[0080] In another preferred embodiment of the method of the invention, the concentration of solid lipid in the organic phase of i) is between 15 and 40mM, preferably between 20 and 35mM, more preferably between 26 and 32mM.

[0081] In another preferred embodiment of the method of the invention, the concentration of solid lipid in the organic phase of i) is between 26.1 and 31.9 mM, preferably 29.0 mM.

[0082] In another embodiment of the method of the invention, the liquid lipid is selected from the list consisting of: medium chain triglyceride (MCT), glycerol monolinoleate, mineral oil, and propylene glycol monolaurate, preferably medium chain triglyceride.

[0083] Thus, in another preferred embodiment of the method of the invention, the liquid lipid is medium-chain triglycerides, preferably the mixture of caprylic acid (C8:0) and capric acid (C10:0) triglycerides.

[0084] In another more preferred embodiment of the method of the invention, the concentration of liquid lipid in the organic phase of i) is between 15 and 35 mM, preferably between 20 and 30 mM, more preferably between 22 and 28 mM.

[0085] In another, even more preferred embodiment of the method of the invention, the concentration of liquid lipid in the organic phase of i) is between 22.3 and 27.3 mM, preferably 24.8 mM.

[0086] In another preferred embodiment of the method of the invention, the surfactant is phosphatidylcholine, polysorbate 20, polysorbate 80, poloxamer 188, poloxamer 407 and / or PEG 40 stearate, preferably phosphatidylcholine, PEG 40 stearate and poloxamer 188.

[0087] In another more preferred embodiment of the method of the invention, the concentration of phosphatidylcholine (surfactant (1)) in the organic phase of i) is between 0.85% and 1.10% w / v, preferably 0.95% w / v.

[0088] In another more preferred embodiment of the method of the invention, the concentration of PEG 40 stearate in the aqueous phase of i) is between 0.15% and 0.250%, preferably 0.2% w / v; and the concentration of poloxamer 188 in the aqueous phase of i) is between 0.01% and 0.05%, preferably 0.02% w / v.

[0089] The terms “nanostructured lipid carriers,” “trimyristin,” “MCT,” and “surfactant” have been described earlier herein and apply equally to this aspect of the invention, as well as to all its preferred embodiments (alone or in combination). The term “organic phase” as used herein refers to an organic or oily solution immiscible in water. However, the term “aqueous phase” refers to an aqueous solution.

[0090] In another preferred embodiment of the method of the invention, the organic phase:aqueous phase ratio is equal to or greater than 1:4, and preferably said ratio is 1:9.

[0091] In another preferred embodiment of the method of the invention, the solvent in step (i) is isopropanol.

[0092] In another preferred embodiment of the method of the invention, the surfactant in step (i) has a hydrophilic-lipophilic balance (HLB) value between 12 and 15.

[0093] In another preferred embodiment of the method of the invention, the surfactant in step (i) has an HLB value equal to 13.1.

[0094] The inventors, using the method of the invention described above, have developed nanostructured lipid transporters, which do not comprise any active agent or principle, which are composed of a lipid mixture and a coating of a surfactant layer that allows their stabilization in water, to inhibit the formation of biofilms of H. influenzae (preferably non-typeable), C. violaceum and P. aeruginosa.

[0095] Therefore, another aspect of the invention relates to nanostructured lipid transporters obtained by the method of the invention.

[0096] The nanostructured lipid carriers of the invention, which do not comprise any active agent or principle, surprisingly, significantly prevent the formation of bacterial biofilms, particularly by H. influenzae (preferably non-typeable), C. violaceum and P. aeruginosa, allowing their use in vitro or ex vivo to inhibit biofilm formation on implants or surfaces or as a medicament, particularly for use in the treatment or prevention of diseases caused by bacterial pathogens, preferably bacterial pathogens such as H. influenzae (preferably non-typeable), C. violaceum and P. aeruginosa.

[0097] Thus, another aspect of the present invention relates to the ex vivo (non-therapeutic) use of the nanostructured lipid transporters of the invention, the composition of the invention, or the nanostructured lipid transporters obtained by the method of the invention, to inhibit the formation of biofilms of gram-positive or gram-negative bacteria, preferably H. / 7if7í / enzae (preferably non-typeable), C. violaceum, and P. aeruginosa, on implants or surfaces.

[0098] Alternatively, another aspect of the invention relates to an in vitro (or ex vivo) method of inhibiting, preventing, or breaking down a biofilm on an implant or surface, comprising delivering the nanostructured lipid transporters of the invention, the composition of the invention, or the nanostructured lipid transporters obtained by the method of the invention, onto said implant or surface.

[0099] The term “biofilms” used in the present invention refers to an organized microbial ecosystem, preferably composed of Gram-negative bacteria, more preferably H. influenzae (preferably nontypeable), P. aeruginosa, or C. violaceum, and associated with a living or inert surface, with complex functional characteristics and structures. This type of microbial formation occurs when planktonic cells adhere to a surface or substrate, forming a community characterized by the secretion of a protective, adhesive extracellular matrix.

[0100] Another aspect of the present invention relates to the nanostructured lipid transporters of the invention, the composition of the invention, or the nanostructured lipid transporters obtained by the method of the invention, for use as a medicament, hereinafter referred to as “the first medical use of the invention.” Alternatively, another aspect of the invention relates to the use of the nanostructured lipid transporters of the invention, the composition of the invention, or the nanostructured lipid transporters obtained by the method of the invention, for the manufacture of a medicament.The term "medicinal drug," as used herein, refers to any substance used for the prevention, diagnosis, relief, treatment, or cure of diseases in a subject, or that may be administered to a subject to restore, correct, or modify their physiological functions by exerting a pharmacological, immunological, or metabolic action. In the context of the present invention, the medicinal product comprises the nanostructured lipid transporters of the invention or, alternatively, a composition comprising them. For the purposes of the present invention, the terms "medicinal drug" and "pharmaceutical composition" are used synonymously.

[0101] Another aspect of the present invention relates to the nanostructured lipid transporters of the invention, the composition of the invention, or the nanostructured lipid transporters obtained by the method of the invention, for use in the treatment or prevention of diseases caused by biofilm-forming bacterial pathogens, the second medical use of the invention.

[0102] Alternatively, another aspect of the present invention relates to the use of the nanostructured lipid transporters of the invention, the composition of the invention, or the nanostructured lipid transporters obtained by the method of the invention, for the treatment or prevention of diseases caused by biofilm-forming bacterial pathogens, preferably where said bacterial pathogens are gram-negative bacteria.

[0103] The term "treatment" (or "treating") refers to processes that involve slowing, interrupting, suspending, controlling, stopping, improving, or reversing the progression or severity of an existing symptom, disorder, condition, or disease, but may not necessarily involve the complete elimination of all symptoms related to the disease, conditions, or disorders associated with diseases caused by biofilm-forming bacterial pathogens, such as H. influenzae (preferably nontypeable), C. violaceum, and / or P. aeruginosa. Treatment of a disorder or disease may, for example, lead to an interruption in the progression of the disorder or disease (e.g., without worsening of symptoms) or a delay in the progression of the disorder or disease (in cases where the interruption of progression is only transient). "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.

[0104] As used herein, the term "prevention" refers to the inhibition or delay of diseases caused by biofilm-forming bacterial pathogens.

[0105] The term “diseases caused by biofilm-forming bacterial pathogens” refers to diseases caused by gram-positive or gram-negative bacteria that can cause various infections such as urinary tract infections, pneumonia or lung infections, skin infections, ear infections or otitis, diarrhea, or blood infections, and where these bacterial pathogens are also biofilm producers. Examples of diseases caused by biofilm-forming bacterial pathogens include, but are not limited to, meningitis, epiglottitis, otitis, laryngitis, osteomyelitis, pneumonia, sepsis, conjunctivitis, sinusitis, bronchitis, cellulitis, and infectious arthritis.

[0106] In another preferred embodiment, the second medical use of the invention relates to the nanostructured lipid transporters of the invention, the composition of the invention, or the nanostructured lipid transporters obtained by the method of the invention, for use in the treatment or prevention of diseases caused by biofilm-forming bacterial pathogens, where said bacterial pathogens are preferably gram-negative bacteria.

[0107] In another preferred embodiment, the second medical use of the invention relates to the nanostructured lipid transporters of the invention, the composition of the invention, or the nanostructured lipid transporters obtained by the method of the invention, for use in the treatment or prevention of diseases caused by biofilm-forming bacterial pathogens, where said bacterial pathogens belong to the genera Haemophilus, Pseudomonas, Vibrio, Escherichia, Salmonella, Listeria, Enterococcus, Streptococcus, Staphylococcus, and Mycobacterium.

[0108] In another preferred embodiment, the second medical use of the invention relates to the nanostructured lipid carriers of the invention, the composition of the invention, or the nanostructured lipid carriers obtained by the method of the invention, for use in the treatment or prevention of diseases caused by biofilm-forming bacterial pathogens, wherein said bacterial pathogens belong to the species selected from the list consisting of H. influenzae (preferably nontypeable), P. aeruginosa, C. violaceum, Escherichia coli, Acinetobacter baumannii, Streptococcus pneumoniae, and / or Staphylococcus aureus.

[0109] Another aspect of the present invention relates to the method of treating diseases caused by biofilm-forming bacterial pathogens, comprising administering to a subject a therapeutically effective amount of the nanostructured lipid transporters of the invention, the composition of the invention, or the nanostructured lipid transporters obtained by the method of the invention.

[0110] In the present invention, "size" preferably refers to the hydrodynamic size or hydrodynamic diameter (HDD-Hydrodynamic Diameter).

[0111] DESCRIPTION OF THE FIGURES

[0112] Figure 1. (A) Mean particle size expressed as hydrodynamic diameter (HDD) and polydispersity index (PDI) and (B) zeta potential of thymine and MCT-based NLC (NLC-pO). The bar chart illustrates the reproducibility of the preparation method by showing the mean ± standard error of 35 independent experiments, in which each data point represents an individual batch.

[0113] Figure 2. Transmission electron microscopy images of NLC of tmminstin and MCT (NLC-pO).

[0114] Figure 3. Thermogram obtained by differential scanning calorimetry (DSC) of trimyristin and MCT NLCs (NLC-pO) in triplicate (NLC-pO-1, NLC-pO-2 and NLC-pO-3), compared with the thermogram of non-nanostructured trimyristin. Figure 4. Evolution of the hydrodynamic size and polydispersity index (PDI) of trimyristin and MCT NLCs (NLC-pO) stored in water at room temperature (RT) and 4 eC. The graph represents the mean ± standard error of three independent experiments with three technical replicates (n=9).

[0115] Figure 5. Evolution of the zeta potential of trimyristin and MCT NLCs (NLC-pO) stored in water at room temperature (RT) and 4 e C. The graph represents the mean ± standard error of three independent experiments with three technical replicates (n=9).

[0116] Figure 6. Effect on the mean size (hydrodynamic diameter) of trimyristin and MCT-based NLC (NLC-pO) of dilution and incubation at 37 °C for 24 hours in sBHI medium (A) and LB medium (B). The graph shows the mean ± standard error of two independent experiments with three technical replicates (n = 6). FD: Dilution factor of the nanoparticles in the respective media: sBHI medium: 10, 50, 100, and 500; LB medium: 10, 50, and 100.

[0117] Figure 7. (A) Hydrodynamic size and polydispersity index (PDI) and (B) zeta potential of NLC prototypes with different lipid and surfactant compositions with respect to the reference prototype (NLC-pO). The bar chart allows evaluation of the reproducibility of the preparation method by representing the mean ± standard error of 10 independent experiments.

[0118] Figure 8. Evolution of the hydrodynamic size and polydispersity index (PDI) of NLC prototypes with different lipid and surfactant compositions with respect to the reference prototype (NLC-pO) stored in water at room temperature (RT).

[0119] Figure 9. Evolution of the zeta potential of NLC prototypes with different lipid and surfactant composition with respect to the reference prototype (NLC- pO) stored in water at room temperature (RT).

[0120] Figure 10. Evolution of the mean size (hydrodynamic diameter) and PDI of trimyristin and MCT-based NLC with two different surfactant compositions, NLC-pO (A) and NLC-p11 (B), after dilution (1:10) and incubation at 37 °C in PBS for 30 days. The graph shows the mean ± standard error of an independent experiment with three technical replicates (n = 3).

[0121] Figure 11. Effect of trimyristin and MCT NLCs (NLC-pO) on the prevention of in vitro biofilm formation in nontypeable H. influenzae strain R2866 (A), strain P605 (B), and strain P639 (C). Effect of tripalmitin and MCT NLCs (NLC-p2) on the prevention of in vitro biofilm formation in nontypeable H. influenzae strain R2866 (D), strain P605 (E), and strain P639 (F). The bars correspond to the mean optical density (OD) 570 nm (proportional to the amount of dye attached to the biofilm) ± standard error of three independent experiments with three technical replicates (n=9) for increasing concentrations of the tested substances expressed as total lipid concentration (*p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 , vs control).

[0122] Figure 12. Effect of trimyristin and MCT NLCs (NLC-pO) on the in vitro inhibition of planktonic growth on non-typeable H. influenzae strain R2866 (A), strain P605 (B), and strain P639 (C). Effect of tripalmitin and MCT NLCs (NLC-p2) on the inhibition of planktonic growth on non-typeable H. influenzae strain R2866 (D), strain P605 (E), and strain P639 (F). Bars represent the mean optical density (OD) 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 (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, vs. control).

[0123] Figure 13. Effect of trimyristin and MCT NLCs (NLC-pO) on in vitro disruption of mature biofilms of nontypeable H. influenzae strain R2866 (A), strain P605 (B), and strain P639 (C). Preformed biofilms were incubated in the presence of the NLC-pOs for 6 hours at 37°C, 5% CO2, and colony-forming units / mL (CFU / mL) were enumerated after 24 h of incubation, as detailed in the text. Data represent the mean ± standard error of three independent experiments with three technical replicates (n=9) for increasing concentrations of the tested substances expressed as total lipid concentration. Figure 14. Effect of trimyristin and MCT NLCs (NLC-pO) on the prevention of in vitro biofilm formation in the PAO1 strain of P. aeruginosa.The bars correspond to the mean optical density (OD) 570 nm (proportional to the amount of dye attached to the biofilm) ± standard error of three independent experiments with three technical replicates (n=9) for increasing concentrations of the tested substances expressed as total lipid concentration (*p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 , vs control).

[0124] Figure 15. Effect of trimyristin and MCT NLCs (NLC-pO) on the in vitro inhibition of planktonic growth on the PAO1 strain of P. aeruginosa. The bars correspond to the mean optical density (OD) 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 (*p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 , vs control).

[0125] Figure 16. Effect of trimyristin and MCT NLCs (NLC-pO) on the prevention of in vitro biofilm formation in the ATCC 12472 strain of C. violaceum. Bars correspond to the mean optical density (OD) 570 nm (proportional to the amount of dye adhering to the biofilm) ± standard error of two independent experiments with two technical replicates (n=4) for increasing concentrations of the tested substances expressed as total lipid concentration (*p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 , vs control).

[0126] Figure 17. Effect of trimyristin and MCT NLCs (NLC-pO) on the in vitro inhibition of planktonic growth on the ATCC 12472 strain of C. violaceum. The bars correspond to the mean optical density (OD) 600 nm ± standard error of two independent experiments with two technical replicates (n=4) for increasing concentrations of the tested substances expressed as total lipid concentration (*p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 , vs control).

[0127] Figure 18. Effect of trimyristin and MCT NLCs (NLC-pO) on in vitro violacein production on the ATCC 12472 strain of C. violaceum. Bars correspond to the mean percentage of violacein production relative to the total violacein produced by C. violaceum in the absence of treatment ± standard error of two independent experiments with two technical replicates (n=4) for increasing concentrations of the tested substances expressed as total lipid concentration (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, vs control).

[0128] Figure 19. Effect of trimyristin and MCT NLCs (NLC-pO) on the prevention of biofilm formation in the LESB58 and PA14 strains of P. aeruginosa using an ex vivo porcine lung model. Points correspond to the count of colony-forming units / mL (CFU / mL) per well ± standard error of independent experiments with three technical replicates (n=9) for a total lipid concentration of 1093 µg / mL (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, vs control).

[0129] Figure 20. Effect of increasing concentrations (0.16 mg / ml, 0.31 mg / ml, 0.62 mg / ml, 1.25 mg / ml and 2.5 mg / ml) of trimyristin and MCT-based NLC (NLC-pO) on in vitro survival of the HepG2 cell line. The graph shows the percentage of live cells compared to untreated cells (control), performed in triplicate for each concentration (n = 3) of total mass concentration.

[0130] Figure 21. Effect of increasing concentrations (0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL and 1.5 mg / mL) of trimyristin and MCT NLCs (NLC-pO) on survival in a zebrafish embryo (Danio rerio) animal model for 96h. The graph represents the percentage of live animals with respect to the total number of animals introduced into the test performed in triplicate and for each concentration (n=3).

[0131] EXAMPLES

[0132] The invention will then be illustrated by means of tests carried out by the inventors, which demonstrate the effectiveness of the invention.

[0133] The procedure for obtaining the nanostructured lipid transporters, particularly NLC-pO, was obtained by: (a) preparation of an organic phase by dissolving trimyristin (29 mM), MCT liquid lipid (24.8 mM) and phosphatidylcholine (0.95% w / v) in 0.5 mL isopropanol,

[0134] (b) preparation of an aqueous phase by dissolving 0.20% w / v PEG 40 stearate and 0.022% w / v poloxamer 188 in 4.5 mL of ultrapure water, this being the volume required for the organic phase: aqueous phase ratio to be 1:9,

[0135] (c) heating the phases obtained in a) and b) to a temperature equal to or 5 degrees Celsius higher than the melting temperature of trimyristin (55 e C),

[0136] (d) Inject the organic phase drop by drop onto the aqueous phase, allowing diffusion of the organic solvent into it, under continuous stirring at a speed between 600 and 1000 rpm and leave stirring with a controlled temperature of 55 e C for at least 10 minutes,

[0137] (e) leave in continuous stirring at a speed between 600 and 1000 rpm at room temperature for the time required for the temperature to be between 20 and 25 e C, and

[0138] (f) purify the dispersion obtained in step (iv) using size exclusion chromatography columns or tangential filtration membranes to obtain purified nanostructured lipid transporters, i.e., free of unincorporated components.

[0139] The obtained NLC-p0 was characterized by dynamic light scattering using a Zetasizer Nano ZS90 (Malvern Panalitical, UK), which allowed the determination of the diameter and polydispersity index, and by electrophoretic light scattering (ELS) using a Zetasizer Nano ZS90 (Malvern Instruments, UK) to determine the zeta potential. For the latter determination, the NLCs were diluted 1:20 in a 1 mM NaCl solution. The purified trimyristin and MCT NLCs (hereafter, NLC-p0) had a mean particle size of 180 nm, a zeta potential >-20 mV, and a polydispersity index of 0.160.

[0140] Furthermore, the size and morphology of the NLCs were studied by transmission electron microscopy (TEM) using a JEOL JEM 1400 microscope operated at 100 kV equipped with a Gatan Oñus Se 200 CCD camera. The NLCs were embedded in the surface of carbon-coated copper grids, negatively stained with 2% uranyl acetate. In the examples, a commercial mixture of MCTs with a carbon atom length of 6 to 10 atoms was used.

[0141] Example 1. Evaluation of the physicochemical characteristics of NLCs

[0142] NLC-pO were prepared with the final composition using the previously described procedure, which employs the organic-phase injection method in aqueous phase for synthesis. The HLB value obtained for the surfactant mixture used in the aqueous phase was 13.1. Once prepared and purified, their mean diameter and polydispersity index were measured by dynamic light scattering using a Zetasizer Nano ZS90 (Malvern Panalitical, UK), as well as their surface electrical charge (zeta potential) by electrophoretic light scattering (ELS), also using the Zetasizer Nano ZS90 (Malvern Instruments, UK). The NLC-pO showed small sizes (180.6 nm ± 10.1 nm) and homogeneous distributions (PDI: 0.162 ± 0.016), as well as a negative zeta potential (-22.8 ± 4.3 mV). 35 independent batches of NLC-pO were synthesized and each of them was analyzed in triplicate at room temperature.Furthermore, the preparation method was found to be robust and reproducible regardless of the person in charge of carrying out the synthesis (Figure 1).

[0143] Example 2. Evaluation of particle morphology

[0144] The size and morphology of the NLCs were studied by transmission electron microscopy (TEM) using a JEOL JEM 1400 microscope operated at 100 kV equipped with a Gatan Oñus Se 200 CCD camera. The pO-NLCs 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 indicated that the NLCs have a spherical morphology and a size between 100 and 200 nm, appearing as a homogeneous population, corroborating the sizes and PDI previously obtained by dynamic light scattering (Figure 2). Example 3. Evaluation of the physical state of the lipid core

[0145] The physical state of the lipid core was analyzed using differential scanning calorimetry (DSC), which allows for the determination of its degree of crystallinity. DSC studies showed the absence of crystalline lipid compared to non-nanostructured trimyristin, which exhibits a transition peak corresponding to its melting temperature (Figure 3), confirming that in pO-NLCs the solid lipid (trimyristin) has an amorphous structure, an intrinsic characteristic of NLCs.

[0146] Example 4. Evaluation of the stability of NLCs in storage at different temperatures

[0147] The NLCs underwent a stability study in water over time at 4 eC and ambient temperature (RT), during which their physicochemical characteristics were analyzed. As shown in Figures 4 and 5, the NLC-pO remained stable at both tested temperatures during the 70 days of the study, which is reflected in the maintenance of the initial size, PDI and zeta potential, as well as the number of suspended particles, which remained around 90-100% of the initial value, indicating that there is no aggregation and / or precipitation of the particles.

[0148] Example 5. Evaluation of the stability of NLCs in supplemented brain heart infusion (sBHI) and Luria Bertani (LB) culture media

[0149] The NLCs (NLC-pO) were subjected to a stability study in the culture media BHI supplemented with hemin and NAD (sBHI), and Luna Bertani (LB), used respectively for the growth of non-typeable H. influenzae and P. aeruginosa.

[0150] For this purpose, serial dilutions of the NLCs were prepared in both media to simulate the conditions of the biofilm inhibition assay. These dilutions were incubated at 37 e The samples were placed in an orbital shaker for 24 h, and their size was measured at predetermined intervals as an indicator of stability and to observe whether aggregation occurred. To evaluate stability in sBHI medium, the tested dilutions were 1:10, 1:50, 1:100, and 1:500. As shown in Figure 6A, all the tested NLC dilutions remained stable up to 10 h; however, at 24 h, they showed a slight sign of aggregation, as deduced from the increase in hydrodynamic size. The stability shown in this medium is sufficient for performing the biofilm inhibition assays in nontypeable H. influenzae.

[0151] To evaluate stability in LB medium, the tested dilutions were 1:10, 1:50, and 1:100. As shown in Figure 6B, all the tested NLC dilutions remained stable until the end of the study, indicating that their stability in this medium is suitable for performing biofilm inhibition assays in P. aeruginosa.

[0152] Example 6. Evaluation of different prototypes

[0153] In general, the composition of the NLC-pO prototype has the following characteristics: constant amount of phosphatidylcholine, constant molar amount of solid lipid (0.015 moles) and a hydrophilic-lipophilic balance (HLB) of the aqueous phase equal to 13.1.

[0154] The inventors carried out a comparative test by modifying the composition of the NLCs (Table 1), with respect to the reference composition (NLC-pO), and carried out by the same procedure described above, which were the following:

[0155] • The NLC-p1 prototype lacks phosphatidylcholine (PC) and was developed precisely with the aim of determining whether this component is essential.

[0156] • In the NLC-p2, NLC-p3, NLC-p6 and NLC-p7 prototypes, the solid lipid (trimyristin) was molarly replaced by another solid lipid.

[0157] • In the NLC-p4, NLC-p5 and NLC-p13 prototypes, the liquid lipid (MCT) was molarly replaced by another liquid lipid.

[0158] • In prototypes NLC-p8, NLC-p9, NLC-p10, NLC-p11, and NLC-p12, the surfactant mixture (poloxamer 188 and PEG-40 stearate) was modified with other surfactant mixtures that also provided an HLB value of 13.1. Table 1. Qualitative composition of the synthesized prototypes. TMyr: Trimyristin; TP: Tripalmitin; GML: Glycerol monolaurate; AL: Lauric Acid; TL: Trilaurine; LS75: Phosphatidylcholine. MCT: Medium-chain triglyceride; GMLin: Glycerol monolinoleate; AM: Mineral Oil; PML: Propylene glycol monolaurate. MS40: PEG-40 stearate; PF68: Poloxamer 188; PF127: poloxamer 407; T20: polysorbate 20; T80: polysorbate 80.

[0159] Table 1. Continued. The results obtained were:

[0160] • NLC-p1 did not form correctly, indicating that phosphatidylcholine is a fundamental component for NLC formation.

[0161] • NLC-p3 and NLC-p6 showed very different physicochemical characteristics from NLC-pO, leading to the conclusion that the solid lipid must be a triglyceride with fatty acid chains between 12 and 16 carbon atoms. In fact, NLC-p2 and NLC-p7, with the solid lipid tripalmitin (C16) and trilaurin (C12), presented physicochemical characteristics similar to those of NLC-pO (Figure 7 A and B).

[0162] The NLC-p3 and NLC-p6 formulations exhibited smaller particle sizes compared to the NLC-pO formulation (172 nm), with values ​​of 93 nm and 102 nm, respectively. Furthermore, NLC-p3 and NLC-p6 showed high PDI values ​​(>0.25), considerably higher than those of the other formulations obtained. This parameter indicates that the formulation is not homogeneous and contains various particle populations of different sizes, making it unsuitable for therapeutic application. These different physicochemical properties may be due to the solid lipid:liquid lipid (SL:LL) ratio, which for these NLC-p3 and NLC-p6 formulations is 1:1.2 (w / w) and 1:1.7 (w / w), respectively. The NLC-p3 and NLC-p6 formulations have less solid lipid compared to NLC-pO, resulting in the smallest particle size observed for these nanoplatforms.This decrease in size associated with the reduction in the amount of solid lipids could be attributed to the fact that a higher proportion of solid lipids affects the melting process and creates agglomerates during NLC production (Danaei et al., 2018).

[0163] • The remaining prototypes showed physicochemical characteristics comparable to NLC-pO.

[0164] Example 7. Evaluation of the stability of NLC prototypes with a new chemical composition developed under room temperature storage

[0165] The NLCs were subjected to a long-term stability study in water at room temperature (RT), during which their physicochemical characteristics were analyzed. As shown in Figures 8 and 9, most of the developed prototypes remained stable at the tested temperature for the 175 days of the study. This is reflected in the maintenance of the initial size, PDI, and zeta potential, as well as the number of suspended particles, which remained around 90-100% of the initial value, indicating that there was no aggregation and / or precipitation of the particles.

[0166] Example 8. Evaluation of the stability of NLC in phosphate-buffered saline (PBS) at 37 e C

[0167] Stability was evaluated at physiological pH and temperature. Two NLC prototypes based on thymine and MCT with different surfactant compositions, NLC-p0 and NLC-p11, were diluted (1:10) in PBS and subjected to a stability study over time at 37 e C, during which their physicochemical characteristics were analyzed. As shown in Figures 10A and 10B, the two prototypes remained stable at the tested temperature during the thirty days of the study, regardless of the composition of their surfactant, which is reflected in the maintenance of their initial hydrodynamic diameter and their PDI.

[0168] Example 9. Inhibition of in vitro bacterial biofilm formation in non-typeable H. influenzae using NLCs

[0169] The prevention of biofilm formation by NLCs (NLC-p0 and NLC-p2) was studied in vitro using the crystal violet (CV) assay. Nontypeable H. influenzae was cultured on a Petr plate with Mueller Hinton agar supplemented with factor X (hemin), factor V (nicotinamide adenine dinucleotide, or NAD), and yeast extract (HTM medium) at 37°C. eThe bacteria were heated to 12°C with 5% CO2. Subsequently, 100 pL of BHI medium supplemented with hemin (10% v / v) and NAD (1% v / v) (sBHI) were added to 96-well polystyrene microtiter plates. The NLCs were then diluted in sBHI to a final concentration of 400 pg / mL, expressed as total lipid concentration. From this solution, labeled 1:2 dilutions were prepared in the 96-well microplate. The bacterial suspension was then adjusted to an optical density (OD) of 0.5 McFarland and diluted 1:100 in sBHI. Finally, 100 pL of the diluted bacterial solution was added to each well. The 96-well plate was incubated for 24 hours at 37°C and 5% CO2. After 24 h of incubation at 37°C and 5% CO2, the supernatant, containing the planktonic cells, was gently removed from the wells of the microplate. The wells were washed three times with 150 pL of ultrapure water.After washing, 200 pL of 1% CV were added, and the suspension was shaken for 20 minutes at room temperature. The plate was then rinsed three 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 minutes 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.

[0170] Bacterial cultures without the addition of the NLCs were used as a positive control for the assays. Only sBHI culture medium (microtiter wells containing uninoculated sBHI medium) was used as a negative control in all assays. The assay was performed in triplicate with three independent lots. The biological activity of the NLC-pO and NLC-p2 formulations was studied in three biofilm-forming strains of nontypeable H. influenzae: one strain from a culture collection, strain R2866 (NCBI taxonomic ID: 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).

[0171] As shown in Figure 11, the application of NLC-pO at a concentration of 25 pg / mL (in terms of total lipid) resulted in almost complete inhibition of biofilm formation in strains R2866 (Figure 11A) and P639 (Figure 11C). Furthermore, a concentration of 12.5 pg / mL was found to reduce biofilm formation by 50% compared to the untreated control. In strain P605, an NLC-pO concentration of 50 pg / mL achieved almost complete biofilm inhibition, while a concentration of 12.5 pg / mL also showed a 50% reduction compared to the control (Figure 11B).

[0172] These results demonstrate that the anti-biofilm effect of NLC-pO is not strain-specific, highlighting the versatility of these formulations as effective agents against non-typeable H. influenzae biofilms. Figure 11 shows that an NLC-p2 concentration of 25 pg / mL also produced almost complete inhibition of biofilm formation in strains R2866 (Figure 11D) and P639 (Figure 11F). As with NLC-pO, an NLC-p2 concentration of 12.5 pg / mL reduced biofilm formation by 50% compared to the untreated control. In strain P605, an NLC-p2 concentration of 50 pg / mL resulted in almost complete inhibition, while 12.5 pg / mL reduced biofilm formation by half compared to the control (Figure 11E).

[0173] This confirms that the anti-biofilm effect of NLC-p2 is also strain-independent, reaffirming the versatility of these formulations in combating non-typeable H. influenzae bacterial biofilms. Furthermore, this finding underscores the importance of formulation composition in antimicrobial activity, suggesting that variations in formulation, such as deriving NLC-p2 from NLC-pO, can maintain or even enhance treatment efficacy. This type of compositional flexibility highlights the ability to tailor formulations to optimize their effectiveness against a broad spectrum of bacterial strains.

[0174] Example 10. Evaluation of the effect of NLCs on the in vitro inhibition of planktonic growth in non-typeable H. influenzae

[0175] Nontypeable H. influenzae was cultured on a Petri dish with Mueller Hinton agar supplemented with factor X (hemin or hematin), factor V (nicotinamide adenine dinucleotide, or NAD) and yeast extract (HTM medium) at 37 eThe bacteria were heated to 5% CO2 for 12 hours. Subsequently, 100 µL of BHI medium supplemented with hemin (10% v / v) and NAD (1% v / v) (sBHI) were added to 96-well polystyrene microtiter plates. The NLCs were then diluted in sBHI to a final concentration of 400 pg / mL, expressed as total lipid concentration. From this solution, serial 1:2 dilutions were made in the 96-well microplate. The bacterial suspension was then adjusted to an optical density (OD) of 0.5 McFarland and diluted 1:100 in sBHI. Finally, 100 µL of the diluted bacterial solution was added to each well. The 96-well plate was incubated for 24 hours at 37 °C and 5% CO2. The minimum inhibitory concentration (MIC), defined as the lowest concentration of an antimicrobial agent required to inhibit the growth of planktonic cells, was determined by measuring the optical density (OD) at 600 nm.Bacterial cultures without the addition of NLCs were used as a positive control for the NLC assays. Only sBHI culture medium (microtiter wells containing uninoculated sBHI medium) was used as a negative control in all assays. The assay was performed in triplicate with three independent batches. The biological activity of the NLCs was studied in three nontypeable H. influenzae strains capable of biofilm formation (strain R2866, P605, and strain P639).

[0176] In none of the three strains was a bactericidal effect of NLC-pO observed (Figure 12, AC). Nor was a bactericidal effect of NLC-p2 observed (Figure 12, DF). This supports the use of NLCs as antivirulence therapy. Antivirulence therapy is based on blocking the virulence factors that make bacteria pathogenic without affecting their cell viability. This type of therapy offers several advantages over conventional antibiotic therapy; among them is the fact that it only interferes with the expression or activity of virulence factors that, in most cases, are not essential for bacterial survival, thus not affecting the patient's microbiota and reducing bacterial selection pressure, a factor that contributes to the development of resistance.

[0177] Example 11. Evaluation of trimyristin and MCT NLCs (NLC-pO) in the in vitro disruption of preformed nontypeable H. influenzae biofilm

[0178] Nontypeable H. influenzae was cultured on a Petri dish with Mueller Hinton agar supplemented with factor X (hemin or hematin), factor V (nicotinamide adenine dinucleotide, or NAD) and yeast extract (HTM medium) at 37 eThe bacterial suspension was incubated at 37°C for 12 h and 5% CO2. The concentration was then adjusted to an optical density (OD) of 0.5 McFarland and diluted 1:100 in BHI medium supplemented with hemin (10% v / v) and NAD (1% v / v) (sBHI). 200 pL of this solution was transferred to 96-well polystyrene microtiter plates. After 24 h of incubation at 37°C and 5% CO2, the supernatant, containing the planktonic cells, was gently removed from the microplate wells. The trimyristin and MCT NLCs (NLC-pO) were diluted in 1X PBS to achieve final concentrations, expressed as total lipid concentrations, of 2360 µg / ml and 1300 µg / ml, respectively. Subsequently, each well was treated with 200 pL of each concentration of the trimyristin and MCT NLCs (NLC-p0). After 6 h of incubation at 37°C and 5% CO2, the liquid was removed and 200 pL of 1X PBS was added to each well.The plates were incubated at room temperature for 5 minutes and kept under low-speed shaking (200 rpm). The biofilm bacteria were then mechanically detached from the bottom of the well using a pipette tip for 45 seconds. 100-µL aliquots from each well were used to make 1 / 10 serial dilutions. These aliquots were then plated onto BHI agar plates.

[0179] After 24 h of incubation at 37°C and 5% CO2, the colony-forming units / mL (CFU / mL) were counted on the plates. The minimum biofilm eradication concentration (MBEC) was defined as the minimum nanoparticle concentration at which no growth was detected after dilution on plates (< 10 2 CFU / ml).

[0180] Bacterial cultures without the addition of trimyristin and MCT were used as a positive control for the trimyristin and MCT NLC assays (NLC-pO). Only sBHI culture medium (microtiter wells containing uninoculated sBHI medium) was used as a negative control in all assays. The assay was performed in triplicate with three independent batches. The biological activity of the trimyristin and MCT NLCs (NLC-pO) was studied in three nontypeable H. influenzae strains with biofilm-forming capacity (strain R2866, P605, and strain P639).

[0181] The trimyristin and MCT NLCs (NLC-pO) did not achieve complete eradication of preformed nontypeable H. influenzae biofilms. However, they demonstrated a 2-log reduction in bacterial concentration within these biofilms in strain R2866 (Figure 13A), strain P605 (Figure 13B), and strain P639 (Figure 13C), highlighting the ability of trimyristin and MCT NLCs (NLC-pO) to significantly reduce preformed nontypeable H. influenzae biofilms. Furthermore, this ability is not strain-influenced, underscoring the versatility of these formulations as agents against nontypeable H. influenzae bacterial biofilms. Example 12. Inhibition of in vitro bacterial biofilm formation in P. aeruginosa using NLCs

[0182] The ability of NLCs (NLC-pO) to prevent biofilm formation was evaluated using the crystal violet (CV) assay. P. aeruginosa was cultured in a Falcon tube with LB liquid medium at 37°C. eC for 12 hours. Subsequently, 100 pL of a medium designed to simulate the sputum conditions of a patient with cystic fibrosis (SCFM medium) were added to polystyrene microtiter plates with a special lid. This lid has 96 prongs that fit snugly into the wells of the microtiter plates, allowing for future biofilm formation both at the bottom of the wells and on the prongs of the lid. SCFM medium is a laboratory-developed medium that mimics the biochemical environment of the lungs of patients with cystic fibrosis, replicating the nutritional composition of sputum and including essential components such as mucus glycoproteins, salts, and amino acids. This provides a more realistic platform for investigating biofilm formation. The SCFM used in this study follows the composition proposed by Palmer et al., with the exception that glucose was replaced with deionized water (Palmer, KL).etal. J Bacterio!, 2007, 189).

[0183] Next, the NLCs were diluted in SCFM to a final concentration of 1320 pg / mL in terms of total lipid. From this solution, labeled 1:2 dilutions were prepared in the 96-well microplate. Subsequently, the concentration of the bacterial suspension was adjusted to an optical density (OD) of 0.2 and diluted 1:2 in SCFM. Finally, 100 pL of the diluted bacterial solution was added to each well. The spiked lid was immersed in the 96-well plate and incubated for 24 hours at 37 °C.

[0184] After incubation, the supernatant, containing the planktonic cells, was removed, and the wells were washed three times with 150 pL of ultrapure water. Following the washes, 200 pL of 1% crystal violet (CV) was added, and the suspension was shaken for 20 minutes at room temperature. The plate was then rinsed three times with 200 pL of ultrapure water in each well, and the crystal violet was dissolved by adding 200 pL of 30% acetic acid. The plate was shaken for 20 minutes at room temperature. Separately, the spiked lid was washed twice by immersing it in a 96-well polystyrene plate containing 150 pL of ultrapure water per well. The lid was then immersed in a plate containing 150 pL of 1% CV for 15 minutes and allowed to air dry for 30 minutes. To remove excess crystal violet, the lid was immersed twice in 96-well polystyrene plates with 150 pL of ultrapure water per well.Finally, the lid was immersed in a 96-well polystyrene plate with 150pL of 30% acetic acid per well for 15 minutes without stirring.

[0185] The minimum biofilm inhibitory concentration (MBIC), defined as the lowest concentration of an antimicrobial agent needed to inhibit biofilm formation, was determined from the sum of the optical density at 570 nm (OD570) of the plate and the lid.

[0186] Untreated bacterial cultures were used as a positive control, while uninoculated SCFM medium (uninoculated wells) was used as a negative control. All assays were performed in triplicate with three independent batches. The biological activity of the NLC-p0 formulation was evaluated against the P. aeruginosa PAO1 strain (NCBI Taxon ID: 208964), known for its biofilm-forming ability.

[0187] As shown in Figure 14, an NLC-p0 concentration of 41.25 pg / mL (in terms of total lipid) resulted in approximately 50% inhibition of biofilm formation compared to the untreated control. This inhibitory effect was maintained at all higher concentrations tested. These results demonstrate that the anti-biofilm effect of NLC-p0 is not exclusive to non-typeable H. influenzae, highlighting the versatility of these formulations as agents against P. aeruginosa bacterial biofilms.

[0188] Example 13. Evaluation of the effect of NLCs on the in vitro inhibition of planktonic growth in P. aeruginosa

[0189] P. aeruginosa was cultured in a Falcon tube with LB liquid medium at 37 eThe samples were incubated at room temperature for 12 hours. Subsequently, 100 pL of a medium designed to simulate the sputum conditions of a patient with cystic fibrosis (SCFM medium) were added to polystyrene microtiter plates with a special lid. This lid has 96 prongs that fit snugly into the wells of the microtiter plates, allowing for future biofilm formation both at the bottom of the wells and on the prongs of the lid. The NLCs were then diluted in SCFM to a final concentration of 1320 pg / mL in terms of total lipid. From this solution, serial 1:2 dilutions were made in the 96-well microplate. The bacterial suspension was then adjusted to an optical density (OD) of 0.2 and diluted 1:2 in SCFM. Finally, 100 pL of the diluted bacterial solution was added to each well. The spiked lid was immersed in the 96-well plate and incubated for 24 hours at 37 °C.

[0190] 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 the addition of NLC were used as a positive control for the NLC assays. Only SCFM culture medium (microtiter wells containing uninoculated SCFM medium) was used as a negative control in all assays. The assay was performed in triplicate with three independent batches. The biological activity of the NLC-p0 formulation was evaluated against the P. aeruginosa PAO1 strain (NCBI Taxon ID: 208964), known for its biofilm-forming ability.

[0191] As shown in Figure 15, the NLC-p0 did not produce a bactericidal effect. This supports the use of NLC as an antivirulence therapy against P. aeruginosa.

[0192] Example 14. Inhibition of in vitro bacterial biofilm formation in C. violaceum using NLCs

[0193] The ability of NLCs (NLC-p0) to prevent biofilm formation in vitro was evaluated using the crystal violet (CV) assay. Chromobacterium violaceum was cultured in a Falcon tube with LB liquid medium at 30°C. eThe bacteria were incubated at C for 12 hours. Subsequently, 100 pL of LB were added to polystyrene microtiter plates with a special lid. This lid has 96 prongs that fit snugly into the wells of the microtiter plates, allowing for the future formation of biofilms both at the bottom of the wells and on the prongs of the lid. The NLCs were then diluted in LB to a final concentration of 1320 pg / mL in terms of total lipid. From this solution, serial 1:2 dilutions were made in the 96-well microplate. The concentration of the bacterial suspension was then adjusted to an optical density (OD) of 0.2 and diluted 1:2 in LB. Finally, 100 pL of the diluted bacterial solution was added to each well. The lid with spikes was submerged in the 96-well plate and incubated for 24 hours at 30 °C.

[0194] After incubation, the supernatant, which contained the planktonic cells, was removed, and the wells were washed three times with 150 pL of ultrapure water. After washing, 200 pL of 1% crystal violet (CV) was added, and the suspension was shaken for 20 minutes at room temperature. The plate was then rinsed three times with 200 pL of ultrapure water in each well, and the crystal violet was dissolved by adding 200 pL of 30% acetic acid. The plate was shaken for 20 minutes at room temperature.

[0195] On the other hand, the spiked lid was washed twice by immersing it in a 96-well plate with 150 pL of ultrapure water per well. Subsequently, the lid was immersed in a plate with 150 pL of 1% crystal violet (CV) for 15 minutes and allowed to air dry for 30 minutes. To remove excess crystal violet, the lid was washed twice in 96-well plates with 150 pL of ultrapure water per well. Finally, the lid was immersed in a 96-well plate with 150 pL of 30% acetic acid per well for 15 minutes without agitation.

[0196] The minimum biofilm inhibitory concentration (MBIC), defined as the lowest concentration of an antimicrobial agent needed to inhibit biofilm formation, was determined from the sum of the optical density at 570 nm (OD570) of the plate and the lid.

[0197] Untreated bacterial cultures were used as a positive control, while untreated LB medium (uninoculated wells) was used as a negative control. All assays were performed in duplicate with two independent batches. The biological activity of the NLC-p0 formulation was evaluated in the C. violaceum strain ATCC 12472 (NCBI Taxon ID: 243365), known for its biofilm-forming capacity. As shown in Figure 16, an NLC-p0 concentration of 41.25 pg / mL (in terms of total lipid) produced approximately 50% inhibition of biofilm formation compared to the untreated control. This inhibitory effect was maintained at all higher concentrations tested. These results demonstrate that the anti-biofilm effect of NLC-pO is not exclusive to non-typeable H. influenzae and P. aeruginosa, highlighting the versatility of these formulations as agents against bacterial biofilms of other species, such as C. violaceum.

[0198] Example 15. Evaluation of the effect of NLCs on the in vitro inhibition of planktonic growth in C. violaceum

[0199] C. violaceum was cultured in a Falcon tube with LB liquid medium at 30 eThe bacteria were incubated at C for 12 hours. Subsequently, 100 pL of LB were added to polystyrene microtiter plates with a special lid. This lid has 96 prongs that fit snugly into the wells of the microtiter plates, allowing for the future formation of biofilms both at the bottom of the wells and on the prongs of the lid. The NLCs were then diluted in LB to a final concentration of 1320 pg / mL in terms of total lipid. From this solution, serial 1:2 dilutions were made in the 96-well microplate. The concentration of the bacterial suspension was then adjusted to an optical density (OD) of 0.2 and diluted 1:2 in LB. Finally, 100 pL of the diluted bacterial solution was added to each well. The lid with spikes was submerged in the 96-well plate and incubated for 24 hours at 30 °C.

[0200] 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 the addition of NLC were used as a positive control for the NLC assays. Only LB culture medium (microtiter wells containing uninoculated LB medium) was used as a negative control in all assays. The assay was performed in duplicate with two independent batches. The biological activity of the NLC-pO formulation was evaluated against the C. violaceum strain ATCC 12472 (NCBI Taxon ID: 243365), known for its biofilm-forming ability. As shown in Figure 17, no bactericidal effect was observed with the NLC-pO. This supports the use of NLC as an antivirulence therapy against C. violaceum.

[0201] Example 16. Inhibition of violacein production in C. violaceum by NLCs

[0202] C. violaceum was cultured in a Falcon tube with LB liquid medium at 30 eThe bacterial suspension was incubated at 30°C for 12 hours. The concentration was then adjusted to an optical density (OD) of 0.2. The bacterial suspension was subsequently incubated with different concentrations of NLC-pO in sterile tubes at 30°C, maintaining shaking at 120 rpm for 24 hours. After incubation, the cultures were centrifuged at 13,000 rpm for 15 minutes to pellet the bacteria. To solubilize the violacein, the supernatant was discarded, and 250 pL of DMSO was added to the pellet. The tubes were vigorously shaken to extract the violacein pigment. After extraction, bacterial cell debris was removed by centrifugation at 13,000 rpm for 10 minutes. The absorbance of soluble violacein (supernatant) was measured at 570 nm (OD570), considering the production of violacein in the untreated C. violaceum culture (control) as 100%.Bacterial cultures without the addition of NLC were used as a positive control in the assays. Only LB culture medium (microtiter wells containing uninoculated LB medium) was used as a negative control in all assays. The assay was performed in duplicate with two independent batches. The biological activity of the NLC-pO formulation was evaluated in strain ATCC 12472 (NCBI Taxon ID: 243365), known for its biofilm-forming ability.

[0203] C. violaceum was chosen as a model organism to study quorum sensing because of its ability to synthesize the pigment violacein, whose production level is directly related to quorum sensing mediated by N-acylhomosenin lactones (AHL) signaling molecules.

[0204] Quorum sensing is a bacterial communication mechanism that allows bacterial cells to detect and respond to population density by releasing and sensing signaling molecules called autoinducers, such as AHLs. When the concentration of these molecules reaches a critical threshold, specific genes are activated that regulate collective behaviors, such as biofilm formation and virulence production. Furthermore, quorum sensing is responsible for the production of various virulence factors, as well as biofilm synthesis.

[0205] The use of C. violaceum as a model organism allows the study of the effect of quorum sensing inhibitors mediated by AHL molecules through the production of violacein, which allows the observation of variations in the production of this pigment and, therefore, the inference of the activity of the compounds in the interference of quorum sensing.

[0206] It is crucial to highlight that the AHL system is not exclusive to C. violaceum; it is also found in other bacteria, including P. aeruginosa. This provides an easily quantifiable system for evaluating the activity of compounds that affect quorum sensing mediated by AHL molecules in different bacteria, such as P. aeruginosa.

[0207] As shown in Figure 18, NLC-pO inhibited violacein production, achieving 50% inhibition at a lipid concentration of 660 pg / mL. These findings suggest that the composition of NLC-pO plays a significant role in disrupting the AHL signaling system in bacteria, indicating that their mechanism of action in combating biofilm formation may be related to interference with AHL synthesis or perception, altering the ability of bacteria, such as P. aeruginosa, to communicate and, consequently, their ability to form biofilms.

[0208] Example 17. Inhibition of bacterial biofilm formation in P. aeruginosa by NLCs using an ex vivo porcine lung model

[0209] Biofilm formation inhibition assays were performed using an ex vivo porcine lung model, following a modified protocol (Harrison, F. et al. Infect Immum, 2014, 82(8), 3312-3323). First, cubes of approximately 5 mm were extracted 3 From the ventral surface of the left caudal lobe of the lungs, using a sterile scalpel, carefully avoiding the large bronchioles and veins to ensure uniformity between tissue samples. Prior to dissection, the ventral pleural surface was briefly sealed with a heated palette knife to remove surface contaminants and facilitate cutting. During dissection, the tissue was washed three times with cell culture medium, consisting of a 1:1 mixture of Roswell Park Memorial Institute (RPMI 1640) and Dulbecco's Modified Eagle Medium (DMEM), and then a fourth time in SCFM.

[0210] In a sterile 24-well plate, 400 µl of SCFM supplemented with 0.8% agarose was added to each well to create a soft surface for the tissue. Each tissue cube was placed in a well and covered with 250 µl of SCFM and 250 µl of NLC-p0 suspension.

[0211] To inoculate lung tissue, the bacterial strains P. aeruginosa LESB58 and PA14 were cultured overnight on LB agar plates at 37 eC. Using a 30-gauge needle attached to a 1 ml disposable syringe, each tissue cube was inoculated with one colony from each plate. The cubes were then incubated at 37°C on an orbital shaker for 24 hours. After incubation, the cubes were rinsed with 1 ml of 1X PBS to remove loosely adhering cells. Biofilm growth was assessed by homogenizing each cube in 1000 µl of 1X PBS using tubes with metal beads in a homogenizer, thoroughly diluting the homogenate, and seeding aliquots. The plates were incubated for 24 hours at 37°C. e C and then the colony-forming units / mL (CFU / mL) were counted.

[0212] Bacterial cultures without the addition of NLC were used as a positive control for the NLC assays. SCFM culture medium (microtiter wells containing uninoculated SCFM medium) was used as a negative control in all assays. The assay was performed in triplicate with three independent batches. The biological activity of NLC-p0 was studied in two biofilm-forming strains of P. aeruginosa: strain LESB58 (NCBI Taxon ID: 557722) and strain PA14 (NCBI Taxon ID: 652611).

[0213] The ex vivo porcine lung model used in this study allows for the evaluation of the biofilm inhibition efficacy of NLC-p0 against P. aeruginosa in an environment that simulates a lung chronically infected with cystic fibrosis. This model overcomes several key disadvantages associated with in vitro and live animal models in lung infections, providing a structurally and chemically relevant environment.

[0214] As shown in Figure 19, a significant decrease of 1 CFU / ml and 2 CFU / ml of viable biofilm bacteria was recorded with the P. aeruginosa strains LESB58 and PA14, respectively.

[0215] In the context of cystic fibrosis, biofilms formed by P. aeruginosa play a central role in the persistence and chronicity of pulmonary infections, contributing to airway obstruction, inflammation, and increased morbidity. The lungs of patients with cystic fibrosis are characterized by thick, viscous mucus and a hypoxic environment, creating ideal conditions for biofilm formation and bacterial persistence. The observed efficacy of NLC-pO in this model suggests that they can inhibit biofilms even under these conditions. Furthermore, this strain-specific response highlights the importance of considering bacterial heterogeneity when developing treatments for chronic infections. P. aeruginosa strains such as PA14 and LESB58 are more virulent and prone to biofilm formation in patients with cystic fibrosis.

[0216] Example 18. In vitro evaluation of biocompatibility using a hepatocellular carcinoma (HepG2) cell line

[0217] Biocompatibility assessment of nanoparticles (NPs) is essential to ensure their suitability for pharmaceutical applications. The potential hepatotoxicity of NPs was evaluated in hepatocellular carcinoma cells (HepG2 cell line) using a colhomethical assay. Specifically, the MTT assay, based on the reagent 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), was used. The MTT assay works by measuring the metabolic activity of live cells. MTT is a yellow tetrazolium salt that, when introduced into cell culture, is enzymatically reduced by mitochondrial dehydrogenases in viable and metabolically active cells. This reduction results in the formation of formazan, an insoluble purple product, which can be quantified spectrophotometrically as an indirect measure of cell viability and metabolic activity. A higher production of formazan indicates a higher number of viable cells.

[0218] For the study, 20,000 cells per well were seeded in 96-well tissue culture plates and incubated for 24 hours at 37 °C in a 5% CO2 atmosphere. After incubation, the culture medium was removed, and increasing concentrations of NLC (NLC-p0) were added. After 24 hours of incubation with NLC, 10 µl of MTT stock solution (5 mg / ml in 1X PBS) were added to each well, including untreated controls. The plates were incubated for 2 hours at 37 °C in a 5% CO2 atmosphere. The culture medium was then removed, and 100 µl of DMSO was added to each well. The mixture was stirred at room temperature for 10 minutes and the OD570 was recorded using a CLARIOstar microplate reader (BMG Labtech) to determine cell viability, using untreated cells as a 100% viability reference.As shown in Figure 20, NLC-pO showed minimal cytotoxicity at most of the tested concentrations, except at the highest concentration (2.5 mg / ml), at which cell viability decreased by 50%. This concentration is 12.5 times higher than the concentration that showed complete in vitro inhibition of biofilm formation in NTHi, and 1.5 times higher than the concentration that showed antibiofilm activity in the ex vivo lung infection model, indicating a favorable balance between safety and efficacy.

[0219] Example 19. Evaluation of in vivo biocompatibility in a zebrafish (Danio rerio) embryo model

[0220] Zebrafish are considered a good in vivo model for toxicological and biocompatibility studies, as they exhibit 65% to 100% concordance with mammalian models. To evaluate the toxicity of the NLCs (NLC-pO), the OECD harmonized protocol for acute toxicity in fish embryos (FET) was used. This test is performed on embryos from 120 h post-fertilization to 5 days post-fertilization by immersion exposure. Five concentrations of NLC, expressed as total NLC concentration, were tested in triplicate: 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, and 1.5 mg / mL, equivalent to total lipid concentrations of 0.4 mg / mL, 0.54 mg / mL, 0.67 mg / mL, 0.81 mg / mL, and 1.01 mg / mL, respectively, using a total of 24–36 embryos for each concentration. Mortality was measured every 24 h for a total of 96 h.

[0221] At the end of the study, the lethal concentration 50 (LC50) was determined 50) by Probit regression, while the minimum observed effect concentration (LOEC) and the minimum observed effect concentration (NOEC) were calculated using Fisher's exact test. Thus, with respect to total lipid concentration, the LC 50 The concentration of NLC-pO was 0.71 mg / mL, while the LOEC was 0.54 mg / mL and the NOEC was 0.4 mg / mL. These results (Figure 21) demonstrate the low toxicity of the developed NLC-pO formulations, suggesting remarkable biocompatibility.

Claims

CLAIMS 1. Nanostructured lipid transporters comprising: (a) a lipid core comprising at least one solid lipid and at least one liquid lipid, characterized in that the solid lipid is a triglyceride with a fatty acid chain of between 12 and 16 carbons, and (b) a coating of at least two surfactants, characterized in that one of the surfactants is phosphatidylcholine; characterized in that said nanostructured lipid transporters do not comprise any active agent or principle.

2. The nanostructured lipid transporters according to claim 1, wherein the triglyceride with a fatty acid chain between 12 and 16 carbons is trilaurin, trimyristin and / or tripalmitin.

3. The nanostructured lipid transporters according to claim 1 or 2, wherein the liquid lipid is selected from the list consisting of: medium chain triglyceride (MCT), glycerol monolinoleate, mineral oil and propylene glycol monolaurate, preferably medium chain triglyceride.

4. The nanostructured lipid transporters according to claim 3, wherein the medium-chain triglyceride is a mixture of saturated fatty acid triglycerides, preferably caprylic acid and capric acid.

5. Nanostructured lipid transporters according to any one of claims 1 to 4, wherein the surfactant is phosphatidylcholine, polysorbate 20, polysorbate 80, poloxamer 188, poloxamer 407, PEG 40 stearate and / or any combination thereof, preferably phosphatidylcholine, PEG 40 stearate and poloxamer 188.

6. Nanostructured lipid transporters according to any one of claims 1 to 5, wherein said nanostructured lipid transporters have a size of less than 200nm, preferably between 150 and 190nm.

7. A composition comprising nanostructured lipid transporters according to any one of claims 1 to 6.

8. The composition according to claim 7, wherein the composition is a pharmaceutical composition.

9. The composition according to claim 8, further comprising a pharmaceutically acceptable vehicle and / or excipient.

10. A method for obtaining nanostructured lipid transporters according to any one of claims 1 to 6 comprising: (i) preparing an organic phase by dissolving at least one solid lipid, characterized in that the solid lipid is a triglyceride with a fatty acid chain of between 12 and 16 carbons, at least one liquid lipid, and at least one surfactant (1) characterized in that it is at least phosphatidylcholine, in at least one solvent, preferably ethanol and / or isopropanol; (i) preparing an aqueous phase by dissolving at least one other surfactant (2) in an aqueous solvent, preferably water, (iii) independently heating the phases obtained in i) and i) to the same or 5 temperature e C above the melting temperature of the solid lipid, iv) inject the organic phase onto the aqueous phase, obtained in (iii), by continuous stirring at a speed between 600 and 1000 rpm, leave stirring at the same temperature or 5 eC above the melting temperature of the solid lipid and for at least 10 minutes, preferably between 10 and 20 minutes, (v) maintain continuous stirring at a speed between 600 and 1000 rpm until a temperature between 20 and 25 is reached e C and 1 atmosphere of pressure, and (vi) purifying the dispersion obtained in step (v), preferably by means of size exclusion chromatography columns or tangential filtration membranes; characterized in that said nanostructured lipid carriers obtained do not comprise any active agent or principle.

11. Method according to claim 10, wherein the solid lipid is trilaurin, trimyristin and / or thpalmitin.

12. Method according to claim 10 or 11, wherein the concentration of solid lipid in the organic phase of i) is between 15 and 40 mM, preferably between 26.1 mM and 31.9 mM.

13. Method according to any one of claims 10 to 12, wherein the liquid lipid is selected from the list consisting of: medium chain triglyceride (MCT), glycerol monolinoleate, mineral oil and propylene glycol monolaurate, preferably medium chain triglyceride.

14. Method according to claim 13, wherein the medium-chain triglyceride is a mixture of saturated fatty acid triglycerides, preferably caprylic acid and capric acid.

15. Method according to claim 13 or 14, wherein the concentration of liquid lipid in the organic phase of i) is between 15 and 35 mM, preferably between 22.3 and 27.3 mM.

16. Method according to any one of claims 10 to 15, wherein the surfactant is phosphatidylcholine, polysorbate 20, polysorbate 80, poloxamer 188, poloxamer 407 and / or PEG 40 stearate, preferably phosphatidylcholine, PEG 40 stearate and poloxamer 188.

17. Method according to claim 16, wherein the concentration of PEG 40 stearate in the aqueous phase of iii) is between 0.15% and 0.250%, preferably 0.2% w / v; and the concentration of poloxamer 188 is between 0.01% and 0.05%, preferably 0.02% w / v.

18. Method according to any one of claims 10 to 17, wherein the organic phase:aqueous phase ratio is 1:

9.

19. Method according to any one of claims 10 to 18, wherein the solvent in step (i) is isopropanol.

20. Nanostructured lipid transporters obtained by the method according to any one of claims 10 to 19.

21. Non-therapeutic use of the nanostructured lipid transporters according to any one of claims 1 to 6, the composition according to claim 7 or the nanostructured lipid transporters according to claim 20, for inhibiting the formation of bacterial biofilms on implants or surfaces, preferably where the bacteria is a gram-negative bacterium, more preferably H. influenzae, C. violaceum and P. aeruginosa.

22. Use according to claim 21, wherein the concentration of nanostructured lipid transporters is between 2 and 200 pg / mL, preferably between 5 and 50 Hg / mL.

23. Nanostructured lipid transporters according to any one of claims 1 to 6, the composition according to any one of claims 7 to 9 or the nanostructured lipid transporters according to claim 20, for use as a medicament.

24. Nanostructured lipid transporters according to any one of claims 1 to 6, the composition according to any one of claims 7 to 9 or the nanostructured lipid transporters according to claim 20, for use in the treatment or prevention of diseases caused by biofilm-forming bacterial pathogens.

25. Nanostructured lipid transporters, the composition or nanostructured lipid transporters for use according to claim 24, wherein the biofilm-forming bacterial pathogens are gram-negative bacteria, preferably Haemophilus influenzae, Pseudomonas aeruginosa and / or Chromobacterium violaceum.

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