Stimulus-responsive gelable liquid composition comprising vesicles

Abstract

The present invention provides stimulus-responsive gelable liquid composition comprising: vesicles comprising a sterol a cationic quaternary ammonium surfactant, and an active agent; and a stimulus-responsive polymer, wherein: the molar ratio between the sterol and the cationic quaternary ammonium surfactant is from 0.2:1 to 1:1; the sum of the concentration of the sterol and the cationic quaternary ammonium surfactant in the composition is from 1 to 40 mg / ml; and the stimulus-responsive polymer is a polymer selected from the group consisting of a block copolymer comprising at least: a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPO), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-ε-caprolactone) (PLCL), and polycaprolactone (PCL); chitosan and mixtures thereof; and wherein the vesicles comprise at least 50% w / w of the sterol and the cationic quaternary ammonium surfactant of the total weight of the vesicles.

Description

[0001] Stimulus-responsive gelable liquid composition comprising vesicles

[0002] This application claims the benefit of European Patent Application EP24383171.6 filed on 24 October 2024.

[0003] Technical Field

[0004] The present invention belongs to the field of liquid gelable compositions. In particular, the invention relates to stimulus-responsive gelable liquid compositions comprising vesicles and a stimulus-responsive polymer, its use as carriers, as well as a pharmaceutical composition comprising the liquid composition, and the pharmaceutical composition for use as a medicament.

[0005] Background Art

[0006] In the last two decades, new pharmacological modalities (highly hydrophobic compounds, oligonucleotides, antibody-drug conjugates, etc.) have emerged as potential alternative therapeutic tools to traditional orally bioavailable compounds. These new pharmacological modalities are directed to complex disease-relevant targets for which no therapy means were previously available. However, as the pharmaceutical landscape expands with a greater variety of these molecules, there is a parallel need for more creative drug delivery approaches, which are driven by the inherent challenges of the new modalities, such as poor stability, solubility and / or permeability under physiological conditions.

[0007] In response to the above challenges, micro- and nanocarriers, such as lipid-based nanovesicles, have gained increasing importance in drug delivery. Nanocarriers are submicron-sized vehicles with high versatility, allowing the administration of diverse therapeutic molecules ranging from small molecules to large and complex biomolecules. They increase drug bioavailability, protect and stabilize sensitive drugs, minimize side effects and provide means for active targeting to specific organs. On the other hand, although these nanostructures have demonstrated significant potential, nanocarriers by themselves are often insufficient to meet all the requirements for effective drug delivery, particularly for extended-release pharmaceutical compositions.

[0008] To overcome these limitations, the combination of nanocarriers with gels, such as hydrogels, offers a promising solution. Gels can act as a reservoir for drug-loaded nanocarriers, allowing for the gradual and controlled release of drugs over an extended period. This slow release reduces the frequency of drug administration and helps to maintain stable therapeutic drug levels for longer durations. The reduced frequency of drug administration afforded by gels can significantly improve patient adherence to treatment regimens, especially in chronic conditions where frequent dosing is burdensome, while providing a more sustained therapeutic effect.

[0009] According to the above, several strategies can be implemented for the delivery of active agents, such as the administration of pharmaceutical compositions as subcutaneous or intramuscular depots. A widely used approach involves the application of stimulus-responsive injectable compositions, which convert from liquid to gel in response to an external stimulus such as changes in pH, ionic composition, exposure to UV radiation, changes in temperature or precipitation upon contact with aqueous medium, thus being liquid during the injection and forming an in-situ solid or semi-solid depot once in the appropriate tissue. Among these depots, thermoreversible gels are attractive materials for biomedical applications, since their gelation can be triggered by the temperature increase when injected subcutaneously. As temperature or concentration increases, gelation occurs, which is attributed to the dehydration of the micellar cores resulting in an entropic change of state into a partly ordered cubic liquid crystalline gel state.

[0010] For instance, Zhang, J., et al., in “Deformable Liposomal Hydrogel for Dermal and Transdermal Delivery of Meloxicam”, International Journal of Nanomedicine 15 (2020) 9319-9335, assessed several encapsulating strategies for the delivery of Meloxicam (MX), a potent hydrophobic non-steroidal anti-inflammatory drug used to reduce inflammation and pain, since the oral dosage form can cause many adverse gastrointestinal effects. In this study, MX was encapsulated in phosphatidylcholine-based nanocarriers, (liposomes, transfersomes and flavosomes), incorporating other minor components such as cholesterol and / or cetylpyridinium chloride. These MX-loaded liposomal formulations were further incorporated into a poloxamer P407 solution to form a hydrogel. Then, the rheological properties and stability of the formulations were assessed, and an ex vivo permeation study through human cadaver skin by HPLC analysis and confocal laser scanning microscopy (CLSM) was carried out.

[0011] Despite its many advantageous properties, the formulation of drugs into stimulus-responsive gelable compositions presents some substantial challenges, such as the quantity and homogeneity of drug loading that can be achieved, especially for hydrophobic drugs in hydrogels, which are typically loaded in small amounts due to the predominance of water.

[0012] Overall, combining nanocarriers with stimulus-responsive gels provides an approach that enhances the efficacy of drug delivery systems by addressing the limitations of nanocarriers or gels alone. This combination offers a versatile platform for achieving both extended drug release and improved patient outcomes. However, preparing stable formulations comprising nanocarriers in stimulus-responsive gels still presents several challenges due to the complex interactions between the nanocarriers and the gel matrix. Such formulation challenges require extensive experimentation and cannot be easily deduced or predicted based on the properties of individual components, making the development of stable and functional nanocarrier-gel systems a complex task. Therefore, there remains a need for developing compositions that are both stable and capable of providing a sustained release of new pharmacological modalities over an extended period of time, overcoming the technical challenges in the preparation of new delivery systems as well as providing a wider range of drug delivery compositions. Overcoming these challenges would provide a much-needed therapeutic option for patients, potentially improving their adherence to treatment, reducing risk of relapse, and improving their quality of life.

[0013] Summary of Invention

[0014] The inventors have developed new stimulus-responsive gelable liquid compositions comprising vesicles which comprise a sterol and a quaternary ammonium surfactant in specific concentrations and ratios, and a stimulus-responsive polymer. These compositions are able to integrate and deliver compounds of interest, such as highly hydrophobic compounds, and provide an extended and sustained release thereof after administration and subsequent gelation.

[0015] The inventors surprisingly found that the vesicles according to the invention comprising a certain proportion between sterol and quaternary ammonium surfactants have an especially good stability within the liquid composition. Likewise, the concentration of the sterol and quaternary ammonium surfactants has proved to be highly relevant for the stability of the vesicles within the stimulus-responsive polymer. According to the macroscopical and microscopical study of the prepared formulations, selecting an appropriate sterol-to-surfactant ratio is essential to ensure that no precipitation or aggregation of the vesicles occurs. Similarly, the selected concentration of both the sterol and quaternary ammonium surfactant components determines the overall stability of the vesicles within the gel. It has also been shown by the inventors that the nature of the polymer influences the stability of the vesicles and that of the liquid composition, which, as evidenced by fluorescence resonance energy transfer (FRET) analysis, the vesicles according to the invention were stable within the gels for at least one month after preparation.

[0016] An in vitro release profile study demonstrated an 8-hour steady zero-order release of the vesicles, the zero-order release being a good kinetic profile for extended-release drug delivery applications. In some embodiments, the inventors have also observed that the release can be achieved over 7 days and even up to one month. The inventors also observed that the stimulus-responsive gelable liquid composition did not provide any initial burst release after administration. This is also partly achieved thanks to the stability of the vesicles, which have been shown to be stable after gelation at 37°C, preventing the disruption of the target drug release profile from the hydrogel matrix. This stability was confirmed even at different concentrations of the polymer. These liquid compositions of the invention additionally show good injectability, i.e. , flowing properties through a syringe having a needle with gauge from 22 to 25 (from 22 to 25-G needle) at 20-22 °C.

[0017] Accordingly, a first aspect of the invention relates to a stimulus-responsive gelable liquid composition comprising vesicles comprising a sterol and a quaternary ammonium surfactant; and a stimulus-responsive polymer, wherein the molar ratio between the sterol and the quaternary ammonium surfactant is from 0.2:1 to 1:1; the sum of the concentration of the sterol and the quaternary ammonium surfactant in the composition is from 1 to 40 mg / ml; and the stimulus-responsive polymer is a polymer selected from the group consisting of a block copolymer comprising at least: a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPG), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-£-caprolactone) (PLCL), and polycaprolactone (PCL); chitosan, and mixtures thereof.

[0018] The authors additionally found that combining a stimulus-responsive gel, to obtain in situ depots for extended-release, with the vesicles comprising sterol and quaternary ammonium surfactants in the ratios and concentrations of the invention, both contribute to the stabilization, transportation and delivery of challenging-to-formulate molecules, such as highly hydrophobic compounds and biomolecules.

[0019] In several experiments, the liquid composition according to the invention effectively stabilized hydrophobic and hydrophilic molecules, within the release medium, preventing its precipitation. This experiment demonstrates that the stimulus-responsive gelable liquid compositions effectively work as carriers of hydrophobic, as well as hydrophilic molecules, of interest. Thus, a second aspect of the invention relates to the use of the liquid composition, as defined above, as a carrier.

[0020] A third aspect of the invention relates to a pharmaceutical composition comprising the liquid composition, as defined above, wherein the vesicles comprise an active pharmaceutical ingredient, preferably a hydrophobic active pharmaceutical ingredient, and optionally pharmaceutically acceptable excipients or carriers.

[0021] A fourth aspect of the invention relates to the stimulus-responsive gelable liquid composition as defined above, or the pharmaceutical composition as defined above, for use as a medicament, wherein the use comprises subcutaneous or intramuscular administration of the composition.

[0022] Brief Description of Drawings

[0023] FIG. 1 shows shear stress (o) in Pascal (Pa) versus shear rate (y) in 1 / s for compositions described in Example 3. Black circles represent the compositions comprising 15% w / w of Plx407, dark grey triangles compositions comprising 17% w / w of Plx407, and light grey squares compositions comprising 20% w / w of Plx407. FIG. 1A corresponds to the shear stress of compositions comprising nanovesicles described in Example 3 at4°C. FIG. 1B corresponds to the shear stress of blank compositions described in Example 3 at 4°C.

[0024] FIG. 2 shows shear stress (o) in Pascal (Pa) versus shear rate (y) in 1 / s for compositions described in Example 3. Black circles represent the compositions comprising 15% w / w of Plx407, dark grey triangles compositions comprising 17% w / w of Plx407, and light grey squares compositions comprising 20% w / w of Plx407. FIG. 2A corresponds to the shear stress of compositions comprising nanovesicles described in Example 3 at 22°C. FIG. 2B corresponds to the shear stress of blank compositions described in Example 3 at 22°C.

[0025] FIG. 3 shows shear stress (o) in Pascal (Pa) versus shear rate (y) in 1 / s for compositions described in Example 3. Black circles represent the compositions comprising 15% w / w of Plx407, dark grey triangles compositions comprising 17% w / w of Plx407, and light grey squares compositions comprising 20% w / w of Plx407. FIG. 3A corresponds to the shear stress of compositions comprising nanovesicles described in Example 3 at 37°C. FIG. 3B corresponds to the shear stress of blank compositions described in Example 3 at 37°C.

[0026] FIG. 4 shows viscosity (q) in Pa*s versus shear rate (y) in 1 / s for compositions described in Example 3. Black circles represent the compositions comprising 15% w / w of Plx407, dark grey triangles the compositions comprising 17% w / w of Plx407, and light grey squares the compositions comprising 20% w / w of Plx407. FIG. 4A corresponds to the viscosity of compositions comprising nanovesicles described in Example 3 at 4°C. FIG. 4B corresponds to the viscosity of compositions comprising nanovesicles described in Example 3 at 4°C. FIG. 5 shows viscosity (q) in Pa*s versus shear rate (y) in 1 / s for compositions described in Example 3. Black circles represent the compositions comprising 15% w / w of Plx407, dark grey triangles the compositions comprising 17% w / w of Plx407, and light grey squares the compositions comprising 20% w / w of Plx407. FIG. 5A corresponds to the viscosity of compositions comprising nanovesicles described in Example 3 at 22°C. FIG.

[0027] 5B corresponds to the viscosity of compositions comprising nanovesicles described in Example 3 at 22°C.

[0028] FIG. 6 shows viscosity (q) in Pa*s versus shear rate (y) in 1 / s for compositions described in Example 3. Black circles represent the compositions comprising 15% w / w of Plx407, dark grey triangles the compositions comprising 17% w / w of Plx407, and light grey squares the compositions comprising 20% w / w of Plx407. FIG. 6A corresponds to the viscosity of compositions comprising nanovesicles described in Example 3 at 37°C. FIG.

[0029] 6B corresponds to the viscosity of compositions comprising nanovesicles described in Example 3 at 37°C.

[0030] FIG. 7 shows the storage or loss modulus (G) in Pa versus temperature (T) in °C for compositions described in Example 3. Black circles represent the storage modulus (G’) and grey triangles the loss modulus (G”). A and B correspond to the storage / loss modulus of blank compositions comprising 15 and 17% w / w of Plx407, respectively, described in Example 3.

[0031] FIG. 8 shows the storage or loss modulus (G) in Pa versus temperature (T) in °C for compositions described in Example 3. Black circles represent the storage modulus (G’) and grey triangles the loss modulus (G”). A and B correspond to the storage / loss modulus of compositions comprising nanovesicles comprising 15 and 17% w / w of Plx407, respectively, described in Example 3.

[0032] FIG. 9 shows the absorbance (A) in arbitrary units (a.u) at different wavelengths (A) in nm of the DiD-NV and Dil / DiD-NV suspensions of Example 4. FIG. 9A and 9B show the absorbance spectra of DiD-NV and Dil / DiD-NV suspensions, respectively.

[0033] FIG. 10 shows the absorbance (A) in arbitrary units (a.u) at different wavelengths (A) in nm of the stimulus-responsive compositions comprising said nanovesicles at different concentrations of Plx407 of Example 4. FIG 10A and 10B show the absorbance spectra of DiD-NV and Dil / DiD-NV composition, respectively, comprising 17% w / w of Plx407. FIG.

[0034] 10C shows the absorbance spectra of Dil / DiD-NV composition comprising 15% w / w of Plx407. FIG. 10D shows the absorbance spectra of Dil / DiD-NV composition comprising 20% w / w of Plx407. FIG. 11 shows the emission (F) in arbitrary units (a.u) at different wavelengths (A) in nm of the DiD-NV (grey triangles) and Dil / DiD-NV (black circles) suspensions of Example 4.

[0035] FIG. 12 shows the emission (F) in arbitrary units (a.u) at different wavelengths (A) in nm of the Dil / DiD-NV suspensions of Example 4, measured at 2 days (FIG. 12A) and 30 days (FIG 12B) from preparation.

[0036] FIG. 13 shows the emission (F) in arbitrary units (a.u) at different wavelengths (A) in nm of the stimulus-responsive composition comprising Dil / DiD-NV nanovesicles at 15% w / w of Plx407 of Example 4, measured at 2 days (FIG. 13A) and 30 days (FIG 13B) from preparation.

[0037] FIG 14. shows the emission (F) in arbitrary units (a.u) at different wavelengths (A) in nm of the stimulus-responsive composition comprising Dil / DiD-NV nanovesicles at 17% w / w of Plx407 of Example 4, measured at 2 days (FIG. 14A) and 30 days (FIG 14B) from preparation.

[0038] FIG 15. shows the emission (F) in arbitrary units (a.u) at different wavelengths (A) in nm of the stimulus-responsive composition comprising Dil / DiD-NV nanovesicles at 20% w / w of Plx407 of Example 4, measured at 2 days (FIG. 15A) and 30 days (FIG 15B) from preparation.

[0039] FIG.16 shows the dissolution and release in vitro profile for compositions comprising DiD described in Example 5. FIG. 16A: release (%) of DiD for DiD-NV liquid composition comprising 17% w / w Plx-407 as a function of time (t) in hours (h). FIG. 16B: dissolution of gel (%) for DiD-NV liquid composition comprising 17% w / w Plx-407 as a function of time (t) in hours (h). FIG 16C: images of DiD-NV liquid composition comprising 17% w / w Plx-407 (left) and DiD free solution comprising 17% w / w Plx-407 (right).

[0040] FIG.17 shows the dissolution and release in vitro profile for compositions comprising lucifer yellow described in Example 5. FIG 17A: dissolution of gel (%) for lucifer yellow liquid composition comprising 17% w / w Plx-407 as a function of time (t) in hours (h). FIG 17B: release (%) of lucifer yellow for liquid composition comprising 17% w / w Plx-407 as a function of time (t) in hours (h).

[0041] FIG. 18 shows the cryo-TEM images of the chitosan-based stimulus-responsive gelable liquid composition according to Example 6. FIG. 19 shows the macroscopical images of the prepared formulations according to Example 7.

[0042] FIG. 20 shows cryo-TEM images of mixture M1 formulation according to Example 7

[0043] FIG. 21 shows cryo-TEM images of mixture M2 formulation according to Example 7

[0044] FIG. 22 shows cryo-TEM images of mixture M3 formulation according to Example 7

[0045] FIG. 23 shows cryo-TEM images of mixture M4 formulation according to Example 7

[0046] FIG. 24 shows cryo-TEM images for Examples 1.11 and 1.17. More specifically, FIG. 24A and B show images for Example 1.11 and FIG. 24C and D for Example 1.17.

[0047] FIG. 25 shows cryo-TEM images for Example 8

[0048] Detailed description of the invention

[0049] All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.

[0050] For the purposes of the invention, any ranges given include both the lower and the upper endpoints of the range.

[0051] The term “stimulus-responsive” refers to substance or composition whose properties or action can be modified by an external stimulus. In this application, when the term refers to a gelable liquid composition it is intended to mean that the gelation of the liquid composition is triggered by an external stimulus which induces the sol-to-gel transition. When the term is referred to a polymer, it is intended to mean that the dissolution of the polymer in a liquid composition confers to the liquid composition the stimulus-responsive properties.

[0052] The term “external stimulus” refers to a change in environmental conditions like temperature, pH, ionic composition, exposure to UV radiation, or the presence of body fluids. The changes in environmental conditions comprise the diffusion of the solvent in the body fluids (solvent may be substituted by body fluids), causing the precipitation of solid contents from the composition. When the external stimulus is a change in the temperature, the composition can be defined as a thermoreactive composition.

[0053] The term "thermoreversible” refers to substance or composition whose properties or action can be modified by heating and be restored by cooling, i.e., a thermoreactive composition having reversible properties. In this application, when the term refers to a gelable liquid composition it is intended to mean that the gelation of the liquid composition occurs by heating above a certain sol-to-gel transition temperature, and it can be reversed by cooling below the said temperature. When the term is referred to a polymer, it is intended to mean that the dissolution of the polymer in a liquid composition confers to the liquid composition the heat-reversible properties.

[0054] The term "gelable” refers to a composition which is capable of being converted into a gel. The term “gel” refers to colloids in which the liquid medium has become viscous enough to behave as a solid or a semi-solid. The term “colloid” refers to a mixture in which one substance comprising microscopically dispersed insoluble particles is suspended throughout another substance.

[0055] The term “in-situ depot” refers to a polymer solution or suspension that has solidified or semi-solidified upon subcutaneous or intramuscular injection.

[0056] The term “vesicles” refers to particles having spherical-shaped lipid bilayer structure surrounding an aqueous core.

[0057] The term “nanovesicles” refers to particles below 1 micrometre having spherical-shaped lipid bilayer structure surrounding an aqueous core.

[0058] The term “sterol” refers to a subgroup of steroids with a hydroxyl group at the 3-position. Suitable examples of sterols are cholesterol, ergosterol, p-sitosterol, stigmasterol and lanosterol.

[0059] The term “quaternary ammonium surfactants” refers to amphiphilic molecules with a positively charged quaternary ammonium polar head group comprising one or two apolar chains attached to it.

[0060] The term “cationic quaternary ammonium surfactant” refers to an amphiphilic compound containing a nitrogen atom that has four covalent bonds and carries a permanent positive charge (i.e., a quaternary ammonium), wherein the nitrogen is covalently bound to at least one long hydrophobic alkyl chain, particularly Cs-Cis. In the context of the present invention, the term “cationic quaternary ammonium surfactant” excludes quaternary ammonium surfactant that have a net zero formal charge.

[0061] The term “sol-to-gel transition” is the transformation of a colloidal solution or suspension into a gel through the growth of links between particles, or molecular species, resulting in the development of a three-dimensional solid network. The term “sol-to-gel transition temperature” is the temperature at which the transformation of a colloidal solution or suspension into a gel through the growth of links between particles, or molecular species, resulting in the development of a three-dimensional solid network, takes place.

[0062] The term “zero-order release” refers to a system or composition which releases a drug, molecule or conjugate of interest at a constant rate over a time period, independently of the concentration of the drug or molecule of interest.

[0063] The terms “long acting” and “extended release” are considered herein interchangeable and refer to a composition which releases an active ingredient or molecule of interest over a prolonged period after administration. The term “prolonged period” as used herein refers to a time longer than the time resulting from the absorption, metabolization and excretion of an active ingredient administered orally.

[0064] The term "effective amount” when used for therapeutic purposes, refers to the amount of a compound or composition that, when administered, is sufficient to provide a therapeutic benefit in relation to a disease condition and the symptoms caused by the said disease.

[0065] The term “sustained release” and “controlled release” are considered herein interchangeable and refer to a composition which releases an active ingredient at approximately a constant rate.

[0066] The term “bioavailability” refers to the proportion of an active ingredient which enters the bloodstream when administered to a subject.

[0067] The term “injectability” when used for a composition refers to the ability of the composition to be discharged through a needle of a syringe or a needle of an injection device. A 25-gauge (25G) needle has an inner diameter of about 0.26 mm. A lower value of needle gauge (e.g., 24G) corresponds to a larger inner diameter.

[0068] The term “biocompatible” refers to the condition of being compatible with living tissue or a living system by not being toxic or injurious and not causing immunological rejection. The term “molar ratio” refers to the relation of moles of the compounds indicated. For instance, a molar ratio (moles / moles) of 1:1 between the sterol and the thermoreversible polymer refers to 1 mol of sterol per 1 mole of thermoreversible polymer.

[0069] As used herein, "% by weight" or “% w / w” of a component refers to the weight amount of the single component relative to the total weight of the composition or, if specifically mentioned, of another component.

[0070] The term “hydrodynamic diameter” refers to the diameter of a perfect solid sphere that would exhibit the same hydrodynamic friction as the particle being measured. Therefore, the hydrodynamic diameter is an estimation of the absolute diameter of the particle.

[0071] The term “polydispersity index” refers to an estimation of a measure of the heterogeneity of a sample based on size. If the polydispersity index (Pdl) is referred to vesicles it refers to the heterogeneity of the size of the vesicles.

[0072] The term “apparent Z potential” refers to the potential difference between the stationary liquid layer affixed to the dispersed particle and the dispersion medium is known as the zeta potential.

[0073] The term “block copolymer” refers to a polymer prepared by combination of more than one type of monomers (i.e. , more than one block). The terms “di-block” and “tri-block” when referred to polymers indicate whether the polymer has been prepared by combination of two or three types of monomers, respectively. For instance, in the present application, a triblock polymer consisting of a hydrophobic polypropylene oxide block (PPO), flanked by two hydrophilic polyethylene oxide blocks (PEO), is expressed as PEO-PPO-PEO. The same nomenclature applies for other di-block (e.g., PLA-PEG) or other triblock polymers (e.g., PLA-PEG-PLA).

[0074] The term “carrier” refers to a system or composition that transports a molecule of interest and can change the way the molecule is released.

[0075] The term “hydrophobic compound” as used herein refers to a compound that is minimally soluble in water, while the term “highly hydrophobic compound” refers to a molecule that is insoluble in water. The term “hydrophobic active pharmaceutical ingredient” refers to an active pharmaceutical ingredient that is minimally soluble in water, typically a solubility lower than 0.1 mg / mL. The term “active agent” refers a substance that is added to a formulation to produce a specific chemical, physical, biological, or physiological effect. This term encompasses, among other categories of substances, therapeutic and diagnostic agents used for the treatment, diagnosis, or detection of a biological state or condition.

[0076] As mentioned above, the first aspect of the invention provides a stimulus-responsive gelable liquid composition comprising vesicles comprising a sterol and a quaternary ammonium surfactant; and a stimulus-responsive polymer, wherein the molar ratio between the sterol and the quaternary ammonium surfactant is from 0.2:1 to 1:1; the sum of the concentration of the sterol and the quaternary ammonium surfactant in the composition is from 1 to 40 mg / ml as measured by HPLC; and the stimulus-responsive polymer is a polymer selected from the group consisting of a block copolymer comprising at least: a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPG), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxy butyrate) (PHB), poly(l-lactide-co-E-caprolactone) (PLCL), and polycaprolactone (PCL); chitosan, and mixtures thereof.

[0077] In a particular embodiment, the quaternary ammonium surfactant is a cationic quaternary ammonium surfactant.

[0078] In a particular embodiment, the stimulus-responsive polymer is a block copolymer comprising at least: a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPO), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-E-caprolactone) (PLCL), and polycaprolactone (PCL); and mixtures thereof.

[0079] In other particular embodiments, the molar ratio between the sterol and the quaternary ammonium surfactant is from 0.3:1 to 1:1, from 0.4:1 to 1:1, from 0.5:1 to 1:1, from 0.6:1 to 1:1, from 0.7:1 to 1:1, from 0.8:1 to 1:1, from 0.9:1 to 1:1, from 0.2:1 to 0.9:1, from 0.2:1 to 0.8:1, from 0.2:1 to 0.7:1, from 0.2:1 to 0.6:1, from 0.2:1 to 0.5:1, from 0.2:1 to 0.4:1, from 0.2:1 to 0.3:1, from 0.3:1 to 0.9:1, from 0.4:1 to 0.8:1, from 0.5:1 to 0.6:1, from 0.3:1 to 0.9:1, from 0.3:1 to 0.8:1, from 0.3:1 to 0.7:1, from 0.3:1 to 0.6:1, from 0.3:1 to 0.9:1, from 0.4:1 to 0.9:1, from 0.5:1 to 0.9:1, or from 0.6:1 to 0.9:1. The expression “the concentration of the sterol and the quaternary ammonium surfactant in the composition is from 1 to 40 mg / ml” is intended to mean that the sum of the concentration of the sterol and the quaternary ammonium surfactant is from 1 to 40 mg / ml, which is measured according to the HPLC method described in the examples. Each component was quantified by using its specific analytical method. In other particular embodiments, the concentration of the sterol and the quaternary ammonium surfactant in the composition is from 2 to 40 mg / ml, 2 to 35 mg / ml, from 2 to 30 mg / ml, from 2 to 25 mg / ml, from 2 to 20 mg / ml, from 2 to 15 mg / ml, from 2 to 10 mg / ml, from 2 to 6 mg / ml, from 3 to 40 mg / ml, from 5 to 40 mg / ml, from 10 to 40 mg / ml, from 15 to 40 mg / ml, from 20 to 40 mg / ml, from 25 to 40 mg / ml, from 30 to 40 mg / ml, from 3 to 35 mg / ml, from 5 to 30 mg / ml, from 10 to 25 mg / ml, from 15 to 20 mg / ml, from 3 to 30 mg / ml, from 5 to 25 mg / ml, from 5 to 20 mg / ml, from 5 to 30 mg / ml, or from 5 to 35 mg / ml, as measured by HPLC.

[0080] In a particular embodiment, the stimulus-responsive gelable composition is a thermoreversible gelable liquid composition, and the thermoreversible gelable liquid composition has a sol-to-gel transition temperature ranging from 25 to 42°C as measured by the oscillatory rheological analysis provided in the examples, preferably ranging from 25 to 40°C, more preferably ranging from 25 to 38°C, even more preferably ranging from 25 to 37°C. In a particular embodiment, the gelation of the liquid composition occurs progressively between the two temperatures of the given range. In a particular embodiment, the sol-to-gel transition temperature is from 30 to 37°C, as measured by the oscillatory rheological analysis provided in the examples. The selection of an appropriate range of sol-to-gel transition temperature allows to modulate the gelation behavior of the composition during and after the administration of the composition. Advantageously, the thermoreversible gelable liquid composition having a sol-to-gel transition temperature according to this embodiment allows to administrate topically, subcutaneously or intramuscularly the composition as a liquid, having good injectability, and subsequently gelling itself upon reaching the sol-to-gel transition temperature. The sol-to-gel transition temperature according to this embodiment may allow to administrate the liquid composition not only humans, but to other mammals, such as pigs, camels, horses, dogs, cats, among others, and cause the gelation of the composition. Likewise, the sol-to-gel transition temperature according to the invention may also trigger the gelation of the liquid composition upon administration to a human which has fever.

[0081] In a particular embodiment, the sterol is selected from the group consisting of cholesterol, ergosterol, p-sitosterol, stigmasterol, lanosterol, DC-Cholesterol and mixtures thereof. In a particular embodiment, the quaternary ammonium surfactant is selected from the group consisting of cetylpyridinium chloride (CPC), cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), benzalkonium chloride (BKC), myristalkonium chloride (MKC), benzethonium chloride or miripirium chloride, and mixtures thereof.

[0082] In a particular embodiment, the stimulus-responsive polymer is a di-block or a tri-block copolymer.

[0083] In a particular embodiment, the stimulus-responsive polymer is selected from the group consisting of PEO-PPO-PEO, PLGA-PEG-PLGA, PCL-PEG-PCL, PLCL-PEG-PLCL, PLA-PEG-PLA, PLA-PEG and mixtures thereof. These selected stimulus-responsive polymers have advantageously shown to provide stability for at least 30 days, when combined with the vesicles of the invention, and adequate gelation properties for administration in humans.

[0084] In a particular embodiment, the vesicles have a hydrodynamic diameter ranging from 20 to 300 nm, as measured by Dynamic Light Scattering (DLS) in the examples. In a particular embodiment, the vesicles are nanovesicles. Therefore, the inventors have found that nanoconjugates based on nanovesicles having the above hydrodynamic diameter are released in an adequate extended-release profile.

[0085] In a particular embodiment, the vesicles have a polydispersity index (Pdl) ranging from 0.01 to 0.45, as measured by Dynamic Light Scattering (DLS) in the examples. This Pdl ensures an adequate grade of homogeneity of size, reducing the variability in the performance of the liquid composition for the desired purpose.

[0086] In a particular embodiment, the vesicles have an apparent Z potential ranging from 30 to 150 mV, as measured by Electrophoretic Light Scattering (ELS), in the examples. The authors have found that Z-potential values greater than 30 mV, in absolute terms, provide sufficient repulsive forces for improved colloidal stability.

[0087] In a particular embodiment, the concentration of stimulus-responsive polymer is from 5% to 30% w / w of the total weight of the liquid composition. The administration by injection through a 25-G needle of compositions in this range was easier, and the flowability of the composition was higher, that for concentration of stimulus-responsive polymer above 30% w / w. In a particular embodiment, the concentration of stimulus-responsive polymer is from 5% to 25% w / w of the total weight of the liquid composition. The inventors surprisingly found that liquid compositions having a concentration of stimulus-responsive polymer in this range had even better gelation properties, and flowability / injectability, during the administration of the composition. Namely, the stimulus-responsive gelable liquid compositions having a concentration of stimulus-responsive polymer in the above range had low viscosity at injectable conditions (i.e. , 22 °C and high shear rates), what contributed to a better injectability, and behave as a solid or semi-solid at a temperature equal or above 25 °C, preferably equal or above 27°C, more preferably equal or above 30°C. For instance, the compositions according to the invention may in a particular embodiment have non-Newtonian fluid behavior and still be injectable, since the shear stress caused by the syringe lowers its viscosity.

[0088] In a particular embodiment, the concentration of stimulus-responsive polymer is from 5% to 20% w / w, from 5 to 17% w / w, from 10 to 17% w / w, from 10 to 15% w / w, from 5 to 15% w / w, from 15 to 20% w / w, or from 15 to 17% w / w, of the total weight of the liquid composition.

[0089] In a particular embodiment, the concentration % w / w of stimulus-responsive polymer in the stimulus-responsive gelable liquid composition is such that the composition is liquid at a temperature of injection. In a particular embodiment, the temperature of injection is room temperature (22°C). In a particular embodiment, the stimulus-responsive polymer is PEO-PPO-PEO and the concentration ranges from 10 to 17% w / w, preferably 15 to 17% w / w.

[0090] In a particular embodiment, the liquid compositions according to the invention are a solution or a colloidal dispersion.

[0091] In a particular embodiment, the liquid composition of the invention is an aqueous liquid composition. In a particular embodiment, the liquid composition comprises a buffer. In another particular embodiment, the liquid composition comprises a buffer selected from the group consisting of histidine, phosphate, and citrate buffer.

[0092] In a particular embodiment, the liquid compositions have a pH value ranging from 4 to 7, preferably from 5 to 7.

[0093] In a particular embodiment, the sterol is cholesterol. In a particular embodiment, the ammonium quaternary surfactant is CPC. In a particular embodiment, the stimulus-responsive gelable liquid composition comprising vesicles comprising a sterol and a quaternary ammonium surfactant; and a stimulus-responsive polymer, wherein the molar ratio between the sterol and the quaternary ammonium surfactant is from 0.2:1 to 1:1; the sum of the concentration of the sterol and the quaternary ammonium surfactant in the composition is from 1 to 40 mg / ml as measured by HPLC; the stimulus-responsive polymer is a polymer selected from the group consisting of a block copolymer comprising at least: a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPG), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-£-caprolactone) (PLCL), and polycaprolactone (PCL); chitosan and mixtures thereof; the stimulus-responsive gelable composition is a thermoreversible gelable liquid composition; and the thermoreversible gelable liquid composition has a sol-to-gel transition temperature ranging from 25 to 37°C.

[0094] In a particular embodiment, the stimulus-responsive gelable liquid composition comprising vesicles comprising a sterol and a quaternary ammonium surfactant; and a stimulus-responsive polymer, wherein the molar ratio between the sterol and the quaternary ammonium surfactant is from 0.2:1 to 0.8:1; the sum of the concentration of the sterol and the quaternary ammonium surfactant in the composition is from 2 to 30 mg / ml as measured by HPLC; the stimulus-responsive polymer is a polymer selected from the group consisting of a block copolymer comprising at least: a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPO), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-£-caprolactone) (PLCL), and polycaprolactone (PCL); chitosan and mixtures thereof; the stimulus-responsive gelable composition is a thermoreversible gelable liquid composition; and the thermoreversible gelable liquid composition has a sol-to-gel transition temperature ranging from 25 to 42°C.

[0095] In a particular embodiment, the stimulus-responsive gelable liquid composition comprising vesicles comprising a sterol and a quaternary ammonium surfactant; and a stimulus-responsive polymer, wherein the molar ratio between the sterol and the quaternary ammonium surfactant is from 0.3:1 to 1:1; the sum of the concentration of the sterol and the quaternary ammonium surfactant in the composition is from 5 to 40 mg / ml as measured by HPLC; the stimulus-responsive polymer is a polymer selected from the group consisting of a block copolymer comprising at least: a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPG), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-£-caprolactone) (PLCL), and polycaprolactone (PCL); chitosan and mixtures thereof; the stimulus-responsive gelable composition is a thermoreversible gelable liquid composition; and the thermoreversible gelable liquid composition has a sol-to-gel transition temperature ranging from 25 to 42°C.

[0096] In a particular embodiment, the stimulus-responsive gelable liquid composition comprising vesicles comprising a sterol and a quaternary ammonium surfactant; and a stimulus-responsive polymer, wherein the molar ratio between the sterol and the quaternary ammonium surfactant is from 0.2:1 to 1:1; the sum of the concentration of the sterol and the quaternary ammonium surfactant in the composition is from 2 to 25 mg / ml as measured by HPLC; the stimulus-responsive polymer is a polymer selected from the group consisting of a block copolymer comprising at least: a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPO), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-£-caprolactone) (PLCL), and polycaprolactone (PCL); chitosan and mixtures thereof; the stimulus-responsive gelable composition is a thermoreversible gelable liquid composition; and the thermoreversible gelable liquid composition has a sol-to-gel transition temperature ranging from 25 to 37°C, the sterol is cholesterol; and the quaternary ammonium surfactant is CPC.

[0097] In a particular embodiment, the stimulus-responsive gelable liquid composition comprising vesicles comprising a sterol and a quaternary ammonium surfactant; and a stimulus-responsive polymer, wherein the molar ratio between the sterol and the quaternary ammonium surfactant is from 0.2:1 to 1:1; the sum of the concentration of the sterol and the quaternary ammonium surfactant in the composition is from 2 to 40 mg / ml as measured by HPLC; the stimulus-responsive polymer is a block copolymer comprising at least: a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPO), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxy butyrate) (PHB), poly(l-lactide-co-£-caprolactone) (PLCL), and polycaprolactone (PCL); the stimulus-responsive gelable composition is a thermoreversible gelable liquid composition; and the thermoreversible gelable liquid composition has a sol-to-gel transition temperature ranging from 25 to 42°C.

[0098] In a particular embodiment, the composition further comprises a secondary polymer selected from the group consisting of hyaluronic acid, heparin, hydroxypropyl methyl cellulose, collagen, ethylene vinyl acetate, poly(N-isopropylacrylamide), and polyacrylic acid.

[0099] In a particular embodiment, the cationic quaternary ammonium surfactant is selected from the group consisting of cetylpyridinium chloride (CPC), cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), benzalkonium chloride (BKC), myristalkonium chloride (MKC), benzethonium chloride or miripirium chloride, and mixtures thereof.

[0100] In other particular embodiments, the molar ratio between the sterol and the cationic quaternary ammonium surfactant is from 0.3:1 to 1:1, from 0.4:1 to 1:1, from 0.5:1 to 1:1, from 0.6:1 to 1:1, from 0.7:1 to 1:1, from 0.8:1 to 1:1, from 0.9:1 to 1:1, from 0.2:1 to 0.9:1, from 0.2:1 to 0.8:1, from 0.2:1 to 0.7:1, from 0.2:1 to 0.6:1, from 0.2:1 to 0.5:1, from 0.2:1 to 0.4:1, from 0.2:1 to 0.3:1, from 0.3:1 to 0.9:1, from 0.4:1 to 0.8:1, from 0.5:1 to 0.6:1, from 0.3:1 to 0.9:1, from 0.3:1 to 0.8:1, from 0.3:1 to 0.7:1, from 0.3:1 to 0.6:1, from 0.3:1 to 0.9:1, from 0.4:1 to 0.9:1, from 0.5:1 to 0.9:1, or from 0.6:1 to 0.9:1.

[0101] The expression “the concentration of the sterol and the cationic quaternary ammonium surfactant in the composition is from 1 to 40 mg / ml” is intended to mean that the sum of the concentration of the sterol and the cationic quaternary ammonium surfactant is from 1 to 40 mg / ml, which is measured according to the HPLC method described in the examples. Each component was quantified by using its specific analytical method. In other particular embodiments, the concentration of the sterol and the cationic quaternary ammonium surfactant in the composition is from 1 to 35 mg / ml, 2 to 35 mg / ml, from 2 to 30 mg / ml, from 2 to 25 mg / ml, from 2 to 20 mg / ml, from 2 to 15 mg / ml, from 2 to 10 mg / ml, from 2 to 6 mg / ml, from 3 to 40 mg / ml, from 5 to 40 mg / ml, from 10 to 40 mg / ml, from 15 to 40 mg / ml, from 20 to 40 mg / ml, from 25 to 40 mg / ml, from 30 to 40 mg / ml, from 3 to 35 mg / ml, from 5 to 30 mg / ml, from 10 to 25 mg / ml, from 15 to 20 mg / ml, from 3 to 30 mg / ml, from 5 to 25 mg / ml, from 5 to 20 mg / ml, from 5 to 30 mg / ml, or from 5 to 35 mg / ml, as measured by HPLC.

[0102] In a particular embodiment, the stimulus-responsive gelable liquid composition comprises: vesicles comprising a sterol, a cationic quaternary ammonium surfactant, and an active agent; and a stimulus-responsive polymer, wherein: the molar ratio between the sterol and the cationic quaternary ammonium surfactant is from 0.2:1 to 1:1; the sum of the concentration of the sterol and the cationic quaternary ammonium surfactant in the composition is from 1 to 40 mg / ml; and the stimulus-responsive polymer is selected from the group consisting of a block copolymer comprising at least:

[0103] a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and

[0104] b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPG), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-E-caprolactone) (PLCL), and polycaprolactone (PCL); chitosan and mixtures thereof; and

[0105] wherein the vesicles comprise at least 50% w / w of the sterol and the cationic quaternary ammonium surfactant of the total weight of the vesicles.

[0106] In a particular embodiment, the vesicles comprise at least 50% w / w of the sterol and the cationic quaternary ammonium surfactant of the total weight of the vesicles, as measured by HPLC. In a particular embodiment, the vesicles comprise at least 60% w / w of the sterol and the cationic quaternary ammonium surfactant of the total weight of the vesicles, as measured by HPLC. In a particular embodiment, the vesicles comprise at least 70% w / w of the sterol and the cationic quaternary ammonium surfactant of the total weight of the vesicles, as measured by HPLC.

[0107] As those skilled in the art would appreciate, the expression “wherein the vesicles comprise at least 50% w / w of the sterol and the cationic quaternary ammonium surfactant of the total weight of the vesicles, as measured by HPLC” is intended to mean that the vesicles comprise at least 50% w / w of the sterol and the cationic quaternary ammonium surfactant of the total weight of the membrane components of the vesicles. That is to say, from those components which form part of the membrane of the vesicles, the sterol and the cationic quaternary ammonium surfactant represent at least 50% w / w.

[0108] In a particular embodiment, the stimulus-responsive gelable liquid composition comprises: vesicles comprising a sterol, a cationic quaternary ammonium surfactant, an active agent; and a stimulus-responsive polymer, wherein:

[0109] the molar ratio between the sterol and the cationic quaternary ammonium surfactant is from 0.3:1 to 1:1; the sum of the concentration of the sterol and the cationic quaternary ammonium surfactant in the composition is from 2 to 40 mg / ml; and the stimulus-responsive polymer is a block copolymer comprising at least:

[0110] a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPO), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-E-caprolactone) (PLCL), and polycaprolactone (PCL); and

[0111] wherein the vesicles comprise at least 50% w / w of the sterol and the cationic quaternary ammonium surfactant of the total weight of the vesicles.

[0112] In a particular embodiment, the stimulus-responsive gelable liquid composition comprises: vesicles comprising a sterol, a cationic quaternary ammonium surfactant, an active agent; and a stimulus-responsive polymer, wherein:

[0113] the molar ratio between the sterol and the cationic quaternary ammonium surfactant is from 0.3:1 to 1:1; the sum of the concentration of the sterol and the cationic quaternary ammonium surfactant in the composition is from 2 to 40 mg / ml; and the stimulus-responsive polymer is a block copolymer comprising at least:

[0114] a) one block of a polymer selected from the group consisting of polyethylene oxide (PEG) and polyethylene glycol (PEG); and

[0115] b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPO), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-E-caprolactone) (PLCL), and polycaprolactone (PCL); and

[0116] wherein the vesicles comprise at least 60% w / w of the sterol and the cationic quaternary ammonium surfactant of the total weight of the vesicles.

[0117] In a particular embodiment, the stimulus-responsive gelable liquid composition comprises: vesicles comprising a sterol, a cationic quaternary ammonium surfactant, an active agent; and a stimulus-responsive polymer, wherein:

[0118] the molar ratio between the sterol and the cationic quaternary ammonium surfactant is from 0.3:1 to 1:1; the sum of the concentration of the sterol and the cationic quaternary ammonium surfactant in the composition is from 3 to 40 mg / ml; and the stimulus-responsive polymer is a block copolymer comprising at least:

[0119] a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and

[0120] b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPO), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-E-caprolactone) (PLCL), and polycaprolactone (PCL); and

[0121] wherein the vesicles comprise at least 50% w / w of the sterol and the cationic quaternary ammonium surfactant of the total weight of the vesicles. In a particular embodiment, the stimulus-responsive gelable liquid composition comprises: vesicles comprising a sterol, a cationic quaternary ammonium surfactant, an active agent; and a stimulus-responsive polymer, wherein:

[0122] the molar ratio between the sterol and the cationic quaternary ammonium surfactant is from 0.3:1 to 1:1; the sum of the concentration of the sterol and the cationic quaternary ammonium surfactant in the composition is from 3 to 40 mg / ml; and the stimulus-responsive polymer is a block copolymer comprising at least:

[0123] a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and

[0124] b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPG), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-e-caprolactone) (PLCL), and polycaprolactone (PCL); and

[0125] wherein the vesicles comprise at least 60% w / w of the sterol and the cationic quaternary ammonium surfactant of the total weight of the vesicles.

[0126] In a particular embodiment, the stimulus-responsive gelable liquid composition comprising vesicles comprises a sterol, an active pharmaceutical ingredient, and a quaternary ammonium surfactant; and a stimulus-responsive polymer, wherein the molar ratio between the sterol and the quaternary ammonium surfactant is from 0.2:1 to 1:1; the sum of the concentration of the sterol and the quaternary ammonium surfactant in the composition is from 2 to 40 mg / ml; the stimulus-responsive polymer is a polymer selected from the group consisting of a block copolymer comprising at least: a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPO), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-£-caprolactone) (PLCL), and polycaprolactone (PCL); chitosan and mixtures thereof; and the cationic quaternary ammonium surfactant is selected from the group consisting of cetylpyridinium chloride (CPC), cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), benzalkonium chloride (BKC), benzethonium chloride, miripirium chloride, and mixtures thereof.

[0127] In a particular embodiment, the stimulus-responsive gelable liquid composition comprising vesicles comprises a sterol, an active pharmaceutical ingredient, and a quaternary ammonium surfactant; and a stimulus-responsive polymer, wherein the molar ratio between the sterol and the quaternary ammonium surfactant is from 0.2:1 to 1:1; the sum of the concentration of the sterol and the quaternary ammonium surfactant in the composition is from 2 to 40 mg / ml; the stimulus-responsive polymer is a polymer selected from the group consisting of a block copolymer comprising at least: a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPG), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-£-caprolactone) (PLCL), and polycaprolactone (PCL); chitosan and mixtures thereof; and the cationic quaternary ammonium surfactant is selected from the group consisting of cetylpyridinium chloride (CPC), cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), benzalkonium chloride (BKC), benzethonium chloride, miripirium chloride, and mixtures thereof; and the vesicles comprise at least 50% w / w of the sterol and the cationic quaternary ammonium surfactant of the total weight of the vesicles.

[0128] The use of the liquid composition, as defined above, as a carrier.

[0129] In a particular embodiment, the use of the liquid composition as a carrier comprises the delivery of at least one molecule of interest to a target site by administration of the composition.

[0130] In a particular embodiment, the use of the liquid composition as a carrier comprises the extended and sustained release of the carrier and / or at least one molecule of interest.

[0131] In a particular embodiment, the use of the liquid composition comprises the delivery of at least one molecule of interest, wherein the molecule of interest is a hydrophobic compound or highly hydrophobic compound.

[0132] In a particular embodiment, the vesicles comprise an active agent. In a particular embodiment, the vesicles comprise a hydrophobic active agent. In a particular embodiment, the active agent is an active pharmaceutical ingredient. In a particular embodiment, the vesicles comprise an active pharmaceutical ingredient. In a particular embodiment, the vesicles comprise a hydrophobic active pharmaceutical ingredient.

[0133] Suitable examples of hydrophobic active pharmaceutical ingredients are class II and IV molecules according to the Biopharmaceutics Classification System (BCS) classification, such as ivermectin, meloxicam, flunixin, thiabendazole and other. In a particular embodiment, the vesicles comprise a hydrophilic active agent. In a particular embodiment, the vesicles comprise a hydrophilic active pharmaceutical ingredient. In a particular embodiment, the vesicles comprise peptides and / or proteins. Suitable examples of hydrophilic active pharmaceutical ingredients include class I and III molecules according to the Biopharmaceutics Classification System (BCS), such as metformin hydrochloride, amoxicillin, atenolol and cimetidine. Furthermore, several peptide-based hydrophilic active pharmaceutical ingredients include insulin, glucagon, oxytocin, vasopressin, calcitonin, leuprolide, buserelin, exenatide, tirzepatide, and teriparatide.

[0134] In a particular embodiment, the vesicles are nanovesicles. In a particular embodiment, the vesicles are unilamellar vesicles. In a particular embodiment, the vesicles are unilamellar nanovesicles. In a particular embodiment, the vesicles are unilamellar vesicles comprising at least 50% w / w of the sterol and the cationic quaternary ammonium surfactant of the total weight of the vesicles, as measured by HPLC.

[0135] A pharmaceutical composition comprising the liquid composition as defined above, wherein the vesicles comprise an active pharmaceutical ingredient, preferably a hydrophobic active pharmaceutical ingredient, and optionally pharmaceutically acceptable excipients or carriers.

[0136] In a particular embodiment, the pharmaceutical composition comprises the liquid composition as defined above, and pharmaceutically acceptable excipients or carriers; and wherein the active agent is an active pharmaceutical ingredient, particularly a hydrophobic active pharmaceutical ingredient.

[0137] The liquid composition as defined above, for use as a medicament, wherein the use comprises subcutaneous or intramuscular administration of the composition. This aspect can also be formulated as use of the liquid composition as defined above for the preparation of a medicament for the treatment of a disease, wherein the treatment comprises subcutaneous or intramuscular administration of the composition. Alternatively, this aspect can also be formulated as a method for the treatment of a disease, the method comprising administering subcutaneously or intramuscularly an amount of the liquid composition as defined above to a subject in need thereof.

[0138] The pharmaceutical composition as defined above, for use as a medicament, wherein the use comprises subcutaneous or intramuscular administration of the composition. This aspect can also be formulated as use of the pharmaceutical composition as defined above for the preparation of a medicament for the treatment of a disease, wherein the treatment comprises subcutaneous or intramuscular administration of the composition. Alternatively, this aspect can also be formulated as a method for the treatment of a disease, the method comprising administering subcutaneously or intramuscularly an amount of the pharmaceutical composition as defined above to a subject in need thereof. The stimulus-responsive gelable liquid composition as defined above can be prepared by a method which comprises the steps of:

[0139] a) loading an organic solution comprising a sterol and a quaternary ammonium surfactant, optionally a molecule of interest into a high-pressure vessel at atmospheric pressure to form a mixture;

[0140] b) volumetrically expanding the mixture by adding compressed gas into the high- pressure vessel until a first pressure, wherein the first pressure is at least 50 bar, preferably at least 70 bar, more preferably at least 85 bar;

[0141] c) depressurizing the mixture out of the high-pressure vessel and over a buffer solution at a pH ranging from 5 to 7, by using compressed nitrogen at a second pressure, wherein the second pressure is higher than the first pressure, to obtain a vesicles suspension;

[0142] d) optionally diafiltrating the vesicles suspension;

[0143] e) adding a stimulus-responsive polymer or stimulus-responsive polymer solution to the vesicles suspension and stirring to form a stimulus-responsive gelable liquid composition, wherein preferably the stimulus-responsive polymer or stimulus- responsive polymer solution is added up to an amount ranging from 5 to 30% w / w of the total weight of the stimulus-responsive gelable liquid composition.

[0144] In a particular embodiment, the sterol, the quaternary ammonium surfactant, the molecule of interest and / or the stimulus-responsive polymer are as defined above.

[0145] In a particular embodiment, the organic solution comprises acetone, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), acetonitrile, dimethylformamide (DMF), dichloromethane (DCM), methanol, ethanol, ethyl acetate, toluene or mixtures thereof.

[0146] In a particular embodiment, the buffer is selected from the group consisting of histidine, phosphate or citrate buffer.

[0147] In a particular embodiment, the gas is selected from the group consisting of CO2, ethane, propane, hydrochlorofluorocarbons, hydrofluorocarbons, and mixtures thereof.

[0148] In a particular embodiment, the organic solution and / or buffer comprises an additive selected from the group consisting of polysorbates, sodium bis(2-ethylhexyl) sulfosuccinate (AOT), polyethylenglycols (PEGs), sterol conjugates, lecithin, ascorbil palmitate, tocopherol, phospholipids, medium or large-chain triglycerides, fatty acids, silicate compounds, organosilane compounds, carbocyanine dyes, succinyl ester compounds, alkylpolyglucosides. In a particular embodiment, the second pressure is at least 90 bar, preferably 100 bar.

[0149] In a particular embodiment, the first temperature is below 15°C, preferably below 10°C, more preferably below 5°C. In a particular embodiment the first and second temperatures are equal.

[0150] In a particular embodiment, the sterol in the organic solution is an amount ranging from 3 mg / mL to 35 mg / mL. In a particular embodiment, the quaternary ammonium surfactant in the organic solution is in an amount ranging from 4 mg / mL to 39 mg / mL.

[0151] In a particular embodiment, the diafiltration is carried out using a 100 kDa cut-off hollow fiber. In a particular embodiment, the diafiltration is carried out for concentrate the vesicle suspension, for exchanging the solvent and / or to remove non-integrated payloads.

[0152] The invention also relates to the stimulus-responsive gelable liquid composition obtainable by the method defined above.

[0153] Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps.

[0154] Furthermore, the word “comprise” encompasses the case of “consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular embodiments described herein.

[0155] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0156] Clause 1. A stimulus-responsive gelable liquid composition comprising:

[0157] - vesicles comprising a sterol and a quaternary ammonium surfactant; and

[0158] - a stimulus-responsive polymer,

[0159] wherein:

[0160] the molar ratio between the sterol and the quaternary ammonium surfactant is from 0.2:1 to 1:1;

[0161] the sum of the concentration of the sterol and the quaternary ammonium surfactant in the composition is from 2 to 40 mg / ml as measured by HPLC;

[0162] and the stimulus-responsive polymer is a polymer selected from the group consisting of hyaluronic acid, chitosan, heparin, a block copolymer comprising at least: a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); and

[0163] b) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPG), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-E-caprolactone) (PLCL), and polycaprolactone (PCL);

[0164] and mixtures thereof.

[0165] Clause 2. The liquid composition according to clause 1, wherein the stimulus-responsive gelable composition is a thermoreversible gelable liquid composition, and the thermoreversible gelable liquid composition has a sol-to-gel transition temperature ranging from 25 to 42°C.

[0166] Clause 3. The liquid composition according to any of the clauses 1-2, wherein the sterol is selected from the group consisting of cholesterol, ergosterol, p-sitosterol, stigmasterol, lanosterol, DC-cholesterol and mixtures thereof.

[0167] Clause 4. The liquid composition according to any of the clauses 1-3, wherein the quaternary ammonium surfactant is selected from the group consisting of cetylpyridinium chloride (CPC), cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), benzalkonium chloride (BKC), benzethonium chloride or miripirium chloride and mixtures thereof.

[0168] Clause 5. The liquid composition according to any of the clauses 1-4, wherein the stimulus-responsive polymer is a di-block or a tri-block copolymer selected from the group consisting of PEO-PPO-PEO, PLGA-PEG-PLGA, PCL-PEG-PCL, PLA-PEG-PLA, PLA-PEG and mixtures thereof.

[0169] Clause 6. The liquid composition according to any of the clauses 1-5, wherein the vesicles have a hydrodynamic diameter ranging from 20 to 300 nm, as measured by Dynamic Light Scattering (DLS).

[0170] Clause 7. The liquid composition according to any of the clauses 1-5, wherein the vesicles have a polydispersity index ranging from 0.01 to 0.45, as measured by Dynamic Light Scattering (DLS).

[0171] Clause 8. The liquid composition according to any of the clauses 1-7, wherein the vesicles have an apparent Z potential ranging from 30 to 150 mV, as measured by Electrophoretic Light Scattering (ELS). Clause 9. The liquid composition according to any of the clauses 1-8, wherein the concentration of stimulus-responsive polymer is from 5% to 30% w / w of the total weight of the liquid composition.

[0172] Clause 10. The liquid composition according to any of the clauses 1-9, wherein the sterol is cholesterol.

[0173] Clause 11. The liquid composition according to any of the clauses 1-10, wherein the ammonium quaternary surfactant is CPC.

[0174] Clause 12. The liquid composition according to any of the clauses 1-11, wherein the vesicles comprise at least 50% w / w of the sterol and the quaternary ammonium surfactant of the total weight of the vesicles, as measured by HPLC.

[0175] Clause 13. Use of the liquid composition according to any of the clauses 1-12, as a carrier.

[0176] Clause 14. A pharmaceutical composition comprising the liquid composition according to any of the clauses 1-12, wherein the vesicles comprise an active pharmaceutical ingredient, preferably a hydrophobic active pharmaceutical ingredient, and optionally pharmaceutically acceptable excipients or carriers.

[0177] Clause 15. The pharmaceutical composition according to clause 14, for use as a medicament, wherein the use comprises subcutaneous or intramuscular administration of the composition.

[0178] Examples

[0179] Materials

[0180] 5-Cholesten-3p-ol (Cholesterol (Choi) BioChemica, purity > 95%) was obtained from PanReac AppliChem (Barcelona, Spain). The cetylpyridinium chloride (CPC, purity > 98%) was purchased from Sigma-Aldrich (Madrid, Spain). Ethanol (EtOH, High-Performance Liquid Chromatography -HPLC- grade) was obtained from PanReac AppliChem (Barcelona, Spain). Carbon dioxide (CO2, purity > 99.9%) and nitrogen (N2, purity > 99.9%) were obtained from Nippon Gases (Barcelona, Spain). Purified water was obtained from Grifols (Barcelona, Spain). Acetic Acid, Glacial (Optima™) was purchased from Fisher Scientific (Madrid, Spain). Poloxamer 407 (Plx407) and Chitosan were acquired from Merck (Darmstadt, Germany). Heparin and Hyaluronic Acid were acquired from MedChem Express (New Jersey, United States). Human insulin (Purity > 99%) was purchased from MedChemExpress (New Jersey, USA) and PLCL-PEG-PLCL was obtained from PolySciTech (Akina; West Lafayette, USA).

[0181] Example 1. Preparation of the nanovesicles

[0182] Nanovesicles according to the first aspect of the invention comprising cholesterol (Choi) and cetylpyridinium chloride (CPC) were prepared by DELOS (Depressurization of an Expanded Liquid Organic Solution) technology as previously described in Cabrera, I., et al., “Multifunctional Nanovesicle-Bioactive Conjugates Prepared by a One-Step Scalable Method Using CO2-Expanded Solvents”, Nanoletters 13 (2013) 3766-3774. An organic solution containing Choi (from 0.4 to 32.8 mg / mL), CPC (from 0.5 to 39 mg / mL), and EtOH was loaded into a 25 mL high-pressure vessel at atmospheric pressure. Next, the solution was volumetrically expanded with compressed CO2, at a working temperature of 40°C and until a working pressure of 85 bar. Next, depressurization was conducted over histidine buffer at pH 7 using a flow of compressed N2 (100 bar) as a plunger to push down the CCh-expanded solution from the vessel. In this way, nanovesicles suspensions were obtained (Examples 1.1-1.11, 1.13 and 1.16) containing 10% (v / v) of EtOH. The obtained nanovesicles were characterized as explained in Example 3.

[0183] Fluorescent-labeled nanovesicles comprising 1,T-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil) and / or 1,T-dioctadecyl-3,3,3',3'-tetramethyl-indodicarbocyanine perchlorate (DiD) dyes were prepared by dissolving these compounds in an EtOH solution containing 27.5 mg / mL of Choi and 24.5 mg / mL of CPC, until reaching a concentration of 45 pM DiD for the nanovesicles comprising DiD (DiD-NV) or a total concentration of 90 pM (45 pM DiD and 45 pM Dil) for the nanovesicles comprising Dil and DiD (Dil / DiD-NV). The solutions were loaded into a high-pressure vessel and the same conditions as for nanovesicles of the above paragraphs were applied.

[0184] In addition, fluorescent-labeled nanovesicles comprising dilithium 6-amino-2-(hydrazinecarbonyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-5,8-disulfonate (lucifer yellow) as an example of a hydrophilic small molecule were produced by dissolving the lucifer yellow in the histidine buffer at pH 7. Next, the despressurization was conducted over the buffer containing the lucifer yellow to reach a final concentration of lucifer yellow of 0.4 mg / mL.

[0185] All the obtained suspensions were transferred to different containers, hermetically sealed, and stored until use at 5 ± 3 °C. The nanovesicles suspensions were diafiltrated by tangential flow filtration to eliminate the EtOH and, in the case of loaded DiD-NV, Dil / DiD-NV and lucifer yellow, the non-integrated payloads. An AKTA flux Tangential Flow Filtration (TFF) System with modified polyethersulfone (mPES) hollow fiber module of 100 kDa cut-off was employed.

[0186] Physicochemical characterization of the suspension comprising the nanovesicles after TFF reveals minimal impact on the concentration of Choi and CPC, and other parameters such as hydrodynamic diameter, Pdl and apparent Z potential, i.e., they have approximately the same values as before the TFF.

[0187] Formulations with high concentration of membrane components (i.e., 40-50 mg / mL) require an extra concentration step by tangential flow filtration (TFF) to achieve the target concentration prior to the preparation of the stimulus-responsive gelable liquid compositions according to the invention. Formulations with 40-50 mg / mL membrane components were thus achieved by concentration through TFF in a AKTA flux TFF system using a 100 kDa molecular weight cut-off hollow fiber column. A portion of the aqueous media of the nanovesicles formulations is eliminated through the column pores while the nanovesicles are retained and concentrated (Examples 1.12, 1.14 and 1.15).

[0188] Example 2. Preparation of the stimulus-responsive qelable liquid compositions

[0189] To prepare the stimulus-responsive gelable liquid compositions comprising the nanovesicles of Example 1, a thermoreversible polymer, PEO-PPO-PEO (Poloxamer 407, “Plx407”), was added to the suspensions of the nanovesicles to obtain a final concentration of 15, 17 and 20% w / w of the polymer. The samples were left overnight at 5 ± 3 °C for hydrating the polymer, and the day after magnetic stirring was applied in a bath at the same temperature until no changes in appearance were observed. The macroscopical appearance of the stimulus-responsive gelable liquid compositions obtained for each polymer (thus, obtaining thermoreversible gelable liquid composition) was studied in search of signs of aggregation or precipitation of the nanovesicles. No precipitation or aggregation was observed for any of the Examples 1.1 to 1.14. The examples 1.15 and 1.16 show a cloudy appearance, which was found to be related to a destabilization phenomenon in the liquid compositions, as explained in Example 3, wherein the microscopical appearance was investigated.

[0190] The gelation performance of the resulting thermoreversible gelable liquid compositions was assessed at 4, 22 and 37°C by inversion of a vial containing them. The compositions were liquid at 4 °C and 22°C (the composition flows from the base of the vial to the cap of the vial when inverted) and had good injectability through a 25-G needle, and at 37 °C all compositions behave similarly, becoming solid and remaining at the base of the vial despite gravitational force.

[0191] The same procedure was followed for the Dil / DiD-NV (molar ratio of ChokCPC 1:1, and Chol+CPC concentration of 5 mg / mL) by adding 15, 17 and 20% w / w of Plx4017, and their stability and integrity evaluated as discussed in Example 4. The DiD-NV were also gelled with 17% w / w Plx407 as a control. Blank compositions of Plx407 without nanovesicles (“blank compositions”) in histidine buffer at pH 7 were also prepared following the same procedure. The stability and the integrity of the resulting compositions is discussed in Example 4. The lucifer yellow nanovesicles were also gelled using 17% w / w Plx407 and their stability and integrity of the resulting compositions is discussed in Example 4. Two compositions were also prepared having a molar ratio of ChokCPC 1:1, and Chol+CPC concentration of 5 mg / mL and gelled with 25% w / w of PLGA-PEG-PLGA or PCL-PEG-PCL. The stability of these two compositions was analyzed by Cryo-TEM as explained in Example 3.

[0192] With the aim of assessing the effect of the nature of the thermoreversible polymer, the same procedure was followed with different polymers (PEO-PPO-PEO, PLGA-PEG-PLGA, PCL-PEG-PCL, PLA-PEG-PLA, PLA-PEG, acrylamide / sodium acryloyldimethyl taurate copolymer, carboxymethylcellulose, and cross-linked polyacrylic acid) at a concentration of 17% w / w of the polymer, molar ratio ChokCPC 1:1, and Chol+CPC concentration of 5 mg / mL. All obtained formulations were stored at 5 ± 3 °C before analysing. The macroscopical appearance of the liquid compositions obtained for each polymer was studied in search of signs of aggregation, precipitation of the nanovesicles, or other destabilization phenomena and the results are shown below:

[0193] Polymer Stability

[0194] PEO-PPO-PEO No aggregation or precipitation PLGA-PEG-PLGA No aggregation or precipitation PCL-PEG-PCL No aggregation or precipitation PLA-PEG-PLA No aggregation or precipitation PLA-PEG No aggregation or precipitation Acrylamide / Sodium acryloyldimethyl

[0195] Cloudy appearance / Precipitation taurate copolymer

[0196] Carboxymethylcellulose Cloudy appearance / Precipitation Cross-linked polyacrylic acid Cloudy appearance / Precipitation

[0197]

[0198] Example 3. Nanovesicles and thermoreversible qelable liquid composition characterization

[0199] Particle hydrodynamic diameter, size polydispersity index, and apparent zeta potential assessment

[0200] The particles’ size as intensity-weighted hydrodynamic diameter, the size polydispersity index (Pdl), and the apparent zeta potential (Z-potential) of the different nanovesicles dispersions of Examples 1.1 to 1.10 were determined at 25 °C using a Zetasizer Ultra (Malvern Instruments, Worcestershire, UK) with non-invasive backscatter optics, equipped with helium-neon laser at 633 nm. The hydrodynamic diameter and size Pdl were determined by Dynamic Light Scattering (DLS), derived from a cumulants analysis of the measured correlation curve. When measuring DLS of the DiD-NV or Dil / DiD-NV, a fluorescent filter was applied; otherwise, no filter was utilized. The Electrophoretic Light Scattering (ELS) was employed for the quantification of the apparent Z-potential. The samples (1 mL) were analyzed without any previous modification or dilution. All reported results are averaging over three consecutive measurements of the same samples.

[0201] Examples 1.1 to 1.10 were prepared according to Example 1 and the nanovesicles were characterized (hydrodynamic diameter, Pdl and apparent Z potential) as described above:

[0202] Hydrodynamic Apparent Z -potential Example Pdl

[0203] diameter (nm) (mV)

[0204] 1.1 86.3 ± 1.5 0.3327 ± 0.0018 101 ± 4

[0205] 1.2 64.33 ± 0.15 0.191 ± 0.015 102 ± 3

[0206] 1.3 49.3 ± 1.2 0.104 ± 0.012 65 ± 3

[0207] 1.4 59 ± 3 0.163 ± 0.003 83 ± 5

[0208] 1.5 46.5 ± 1.6 0.0639 ± 0.0021 53 ± 3

[0209] 1.6 52.1 ± 1.0 0.130 ± 0.013 82 ± 3

[0210] 1.7 67.4 ± 1.2 0.1948 ± 0.010 102 ± 6

[0211] 1.8 44.6 ± 1.9 0.080 ± 0.012 71 ± 4

[0212] 1.9 91 ± 3 0.433 ± 0.010 107 ± 7

[0213]

[0214] 1.10 53.0 ± 1.3 0.131 ± 0.005 76 ± 8

[0215] The Dil / DiD-NV had a mean hydrodynamic diameter of 100.7 ± 0.6 nm, a Pdl of 0.3485 ± 0.021 , and an apparent Z-Potential of 76 ± 7 mV, while the DiD-NV had a mean hydrodynamic diameter of 114.4 ± 2.8 nm, a Pdl of 0.432 ± 0.026, and an apparent Z-Potential of 52 ± 7 mV.

[0216] Quantification of the nanovesicles membrane components by HPLC

[0217] The concentration of Choi and CPC components from the nanovesicles samples were determined using an Acquity Arc LIHPLC from Waters Corporation (Milford, MA, USA) coupled to a PDA 2998 detector. Each component was quantified by using its specific analytical method. Choi and CPC calibration curves were elaborated by separately preparing 5 different standards from 25 pg / mL to 250 pg / mL dissolved in methanol. The nanovesicles were analyzed in triplicate by dissolving them in methanol. All standards and nanovesicles samples were eventually filtered with a 0.20 pm hydrophobic polytetrafluoroethylene (PTFE) filter from Merck (Darmstadt, Germany) into an HPLC vial.

[0218] Choi was quantified by using an XBridge BEH C18 column (2.5 pm 3.0 x 75 mm, Waters, Milford, MA, USA) in an isocratic mode of 90% of mobile phase A (volume ratio 5:95 water: methanol) and 10% of mobile phase B (0.1% formic acid in 2-propanol) at a flow rate of 1.0 mL / min for 4 min. The Choi detection wavelength was 206 nm. CPC was quantified by using an InfinityLab Poroshell 120 EC-C18 column (4 pm 4.6 x 100 mm, Agilent) in a gradient mode starting at 12% of mobile phase A (50 mM ammonium acetate at pH 3.6) and 88% of mobile phase B (1% glacial acetic acid in methanol) for 3 min, then at 5% of mobile phase A and 95% of mobile phase B for 12 min, and eventually going back to the initial 12% of mobile phase A and 88% of mobile phase B for 5 min, at a flow rate of 1.0 mL / min for a total of 20 min. The CPC detection wavelength was 260 nm.

[0219] The concentration of Choi and CPC, and the ChokCPC molar ratio was determined for Examples 1.1 to 1.16 according to the above HPLC procedures.

[0220] Concentration

[0221] ChokCPC molar

[0222] Example Chol+CPC

[0223] ratio

[0224] (mg / mL)

[0225] 1.1 6 1

[0226] 1.2 6 0.5

[0227] 1.3 1 0.5

[0228] 1.4 3.5 0.67

[0229] 1.5 1 1

[0230] 1.6 3.5 0.67

[0231] 1.7 6 0.5

[0232] 1.8 1 1

[0233] 1.9 6 1

[0234] 1.10 1 0.5

[0235] 1.11 2 0.2

[0236] 1.12 40 0.2

[0237] 1.13 2 1

[0238] 1.14 40 1

[0239] 1.15 50 1

[0240]

[0241] 1.16 2 1.5 Microscopical appearance

[0242] Optical microscopy was used to assess the homogeneity of the thermoreversible gelable liquid compositions prepared. A drop of the liquid compositions sample was placed on a glass slide and covered with a coverslip. Then, the microscopic examination was carried out at room temperature by a Morphologi G3S optical microscope (Malvern Instruments, Worcestershire, UK) and images were taken at 20x magnifications to get a proper observation of the liquid composition.

[0243] The microscopic images of the Examples 1.1 to 1.14 did not show a significative presence of particles, thus concluding that the thermoreversible gelable liquid compositions were stable. In contrast, the thermoreversible gelable liquid composition Example 1.15, comprising 50 mg / mL of Choi and CPC and ChokCPC ratio of 1, showed a significant number of small particles, which suggests a certain destabilization and precipitation of the system. Other compositions comprising a molar ratio of ChokCPC of 1 were stable and thus, it indicates that the destabilization is caused by the excessive concentration of these two components in the nanovesicles. The microscopic image of the Example 1.16 composition having a 1.5:1 ChokCPC molar ratio revealed some crystals of micrometric size. Examples 1.11 and 1.13 having the same concentration of membrane components (2 mg / mL) were stable and no crystal formation occurred, thus suggesting that crystal formation was caused by the used ratio in this formulation.

[0244] Morphological characterization by Cryo-TEM

[0245] The morphology of the distinct types of produced DELOS-NV was assessed by cryogenic transmission electron microscopy (Cryo-TEM) using a JEOL JEM-2011 transmission electron microscope (JEOL Ltd., Tokyo, Japan) operating at 200 kV. In the case of the nanovesicles solutions, a small drop of the sample was directly placed on a copper grid coated with a perforated polymer film. Conversely, in the case of gelable liquid compositions, they were diluted 1:5 in water to adjust the viscosity for an adequate fixation and preservation of the composition sample in the grid. Afterward, the excess of the sample was removed by blotting with filter paper. Immediately after film preparation, the grid was plunged into liquid ethane held at a temperature just above its freezing point (-179.15 °C). The vitrified sample was then transferred to the microscope for analysis. To prevent sample perturbation and the formation of ice crystals, the specimens were kept cold (-196.15 °C) during both the transfer and viewing procedures. Images were recorded on a Gatan RIO 16 camera using Digital Micrograph 3.50.3584.0 (Gatan Inc., Pleasanton, CA, USA). Cryo-TEM images showed well-formed spherical, unilamellar, and homogeneous nanovesicles in all the tested formulations. Those compositions having a molar ratio of ChokCPC 1:1, and Chol+CPC concentration of 5 mg / mL to which 25% w / w of PLGA-PEG-PLGA or PCL-PEG-PCL were added, showed adequate stability of the nanovesicles inside the composition, which could be perfectly observed, showing no signs of precipitation, either at 22 (liquid) or 37°C (gelled).

[0246] An additional ratio between the membrane components of the nanovesicles (Example 1.17, 0.1:1 ChokCPC molar ratio) was prepared and gelled with 17% w / w Plx407. This new ratio was compared with Example 1.11 to determine the lowest ratio compatible with Plx407, according to the table below, by just changing the ratio ChokCPC to 0.1.

[0247] Nanovesicles Chol+CPC Plx407 Example ChokCPC concentration concentration molar ratio (mg / mL) (% w / w) Example 1:11 0.2 2 17

[0248]

[0249] Example 1:17 0.1 2 17

[0250] Cryo-TEM images in Figures 24A-24D show the most representative microscopic results obtained using the JEOL JEM-2011 transmission electron microscope for Examples 1.11 and 1.17. The results indicate that Plx407 hydrogels containing nanovesicles prepared with 2 mg / mL membrane components and a 0.2:1 molar ratio of ChokCPC (Figures 24A and 24B) are stable, as clearly evidenced by the presence of unilamellar vesicles. In contrast, for hydrogels containing nanovesicles prepared with 2 mg / mL membrane components and a 0.1:1 molar ratio of ChokCPC (Figures 24C and 24D), no vesicles were observed, suggesting structural instability.

[0251] Rheological characterization

[0252] The rheological measurements were performed using a Kinexus Pro+® (Netzsch, Germany). The geometry employed was a stainless-steel parallel plate (diameter 40 mm) at a 1 mm gap between plates. Thermoreversible gelable liquid compositions (ChokCPC 1:1, and Chol+CPC concentration of 5 mg / mL prepared by adding 15, 17 and 20% w / w of Plx4017) or blank compositions (without nanovesicles) were poured on the rheometer lower plate at a given temperature, trimmed, covered with a thermal cover, and rested 10 min before each analysis. Three replicates of each composition were produced and characterized with the rheometer.

[0253] Shear Viscosity Measurements: Flow measurements were performed at 4 °C (storage temperature), 22 °C (room temperature), and 37 °C (body temperature). Each measurement was performed with a three-step method. First a shear rate ramp from 0.1 to 500 s-1 followed by 60 seconds of a steady shear rate at 500 s-1 and a shear rate ramp from 500 to 0.1 s-1. Viscosity and shear stress were measured at each point and plotted.

[0254] Oscillatory Measurements: The shear oscillatory measurements were conducted at 10 Pa and 1 Hz, as optimized with preliminary amplitude and frequency sweeps, respectively. A temperature ramp from 4 °C to 42 °C at a rate of 1 °C / min was performed and the storage -or elastic- modulus (G’), the loss -or viscous- modulus (G”), and the phase angle (5) across the temperature ramp were investigated. The gelation temperature (sol-to-gel transition temperature) was determined as the temperature at which both modulus (G’ and G”) crossover.

[0255] At 4 °C, all liquid compositions exhibited a Newtonian fluid behavior, where the viscosity remained constant regardless of the applied shear force. In contrast, at 37 °C, all the compositions exhibit pseudo-plastic behavior with shear-thinning characteristics, showing yield stress, a minimum force required to start to flow, and decreasing viscosity with increasing shear rates. At 22 °C, the compositions containing 15% and 17% w / w of Plx407 remain Newtonian fluids, while the composition having 20% w / w of Plx407, although not behaving as Newtonian fluid, had a shear thinning behavior which, at similar shear rates to what is expected under injection conditions can be injected.

[0256] The shear stress analysis results (FIG.1 , 2 and 3) suggest that in addition to the concentration of Plx407 employed in the liquid composition, the presence of the nanovesicles has also an impact on the thermoreversible gelable liquid compositions flow behavior. When behaving as Newtonian fluids, the flow curves of the liquid compositions with or without nanovesicles overlapped. However, when behaving as pseudo-plastic fluids, the presence of nanovesicles elevated the shear stress compared to blank compositions. All hydrogels exhibited low thixotropy at 37 °C. This is evidenced by their small or non-existent hysteresis loop area, indicating that their inner structure recovers quickly after shear-thinning. This property could facilitate prompt depot formation after injection, preventing spread.

[0257] The viscosity results (FIG. 4, 5 and 6) showed that thermoreversible gelable liquid compositions comprising nanovesicles exhibit higher viscosities than blank compositions. Further, lower polymer concentrations result in lower viscosities. The 15% w / w Plx407 liquid compositions had viscosities below 0.05 Pa s, making them easily injectable through a 25 G syringe, which is a preferred option for subcutaneous administration. Although the 17% w / w Plx407 liquid compositions had higher viscosities, a test using a 25 G syringe demonstrated satisfactory injectability, as well as it happened with the 20% w / w composition which, under injectability condition could be injected.

[0258] The storage modulus relates to the material's ability to store energy elastically, and the loss modulus relates to the material's ability to dissipate energy through heat and describes the viscose part of the sample. As shown in FIG. 8, the ramp temperatures showed a gradual decrease in the sol-to-gel transition temperature (Tsol-gel) in response to the Plx407 concentration increase. Blank compositions and the thermoreversible gelable liquid compositions according to the invention comprising 15% and 17% w / w of Plx407 present Tsol-gel values above 25 °C (FIG 7 and 8), making them appropriate for injection at room temperature (about 22 °C). The addition of nanovesicles to blank compositions causes a slight decrease in the Tsol-gel.

[0259] Example 4. Stability evaluation by FRET efficiency

[0260] Absorption and fluorescence spectroscopy measurements of fluorescent-labelled nanovesicles

[0261] Absorption spectra were recorded using a Varian Cary 5000 UV-Vis-NIR spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) while for fluorescence emission and excitation spectra collection, a Varian Cary Eclipse equipment (Agilent Technologies, Santa Clara, CA, USA) was used.

[0262] Absorption spectra were measured at appropriate dilutions in histidine pH 7 buffer to have absorption values between 0.1 and 0.3. A 1 cm path quartz cuvette was employed.

[0263] FRET emission spectra were collected at 500-800 nm after excitation at 490 nm for Dil / DiD-NV and for DiD-NV as controls. In addition, the emission spectra (Aex = 590 and Aem = 600 - 800 nm) and the excitation spectra (Aem = 710 and Aex = 400 - 700) were also collected for Dil / DiD-NV and DiD-NV, as an extra control. In all the cases, samples were diluted 10 times in water and placed in a 1 cm quartz cuvette.

[0264] The FRET efficiency was calculated with the following equation:

[0265] Intensity DID

[0266] Efficiency FRET (EFBET,° / b) =

[0267]

[0268] Intensity Dtl+Intensity DID

[0269] Measurements were performed in triplicate. The FRET phenomenon occurs between a pair of light-sensitive molecules, where the emission spectrum of one molecule (the donor) overlaps with the excitation spectrum of the other (the acceptor). When these pairs of molecules are closer than 10 nm of each other, there is an energy transfer from the donor chromophore, initially in its electronically excited state, to the acceptor chromophore. This technique is known as the spectroscopic ruler because the efficiency of the energy transfer is inversely proportional to the sixth power of the distance between the donor and acceptor, see Algar, W.R. et al., “FRET as a biomolecular research tool - understanding its potential while avoiding pitfalls”, Nat.

[0270] Methods 16 (2019) 815-829. For this reason, it can be employed as a research tool to determine nanovesicle stability and integrity: If nanovesicles break down or dissociate, a decrease in FRET efficiency should be detected.

[0271] As a control, a batch of nanovesicles containing DiD (DiD-NV) at the same concentration as it is present in the Dil / DiD-NV formulation was also produced. Afterward, 15%, 17% and 20% w / w of the thermoreversible polymer Plx407 was added to the Dil / DiD-NV and their stability and integrity evaluated through the fluorescence emission of the dye pair,. The DiD-NV were also prepared with 17% w / w of the same polymer as a control after gelation.

[0272] As shown in FIG. 9 and FIG. 10, the absorbance spectra of the pair of fluorophores incorporated into the Dil / DiD-NV composition did not present significant variations between different formulations and concentrations of the thermoreversible polymer, indicating stability of the Dil / DiD-NV and residence of the fluorophores in the membranes despite the formulation into the composition (FIG. 9B, 10B, 10C and 10D). The absorbance spectra of the control with only DiD-NV shows a peak at 650 nm and no peak at 551 nm, as expected (FIG. 9A). Predominance of monomers of DiD (instead of Flaggregates and J-aggregates with differentiated absorption peaks) non-dependent on the formulation was also demonstrated with the 17% w / w loaded polymer with DiD-NV composition. (FIG. 10A)

[0273] As shown in FIG. 11A, the excitation of the Dil at Aex = 490 nm resulted in two emission bands at ~569 nm and at ~673 nm, corresponding to the Dil and DiD emission, respectively, thus indicating the FRET phenomenon. As anticipated, excitation of the control DiD-NV at Aex = 490 nm did not result in DiD emission due to the absence of FRET (FIG. 11B).

[0274] Next, the FRET efficiency was investigated for the nanovesicles suspension (FIG. 12) and the different concentrations of Plx407 compositions comprising Dil / DiD-NV (FIG 13, 14, and 15). The efficiency of this energy transfer was similar for each formulation at 2 days and 30 days after incorporation of the Plx407 polymer (FIG. 13, 14 and 15).

[0275] Overall, the experimental results confirmed the stability of the nanovesicles gelled with different concentrations of thermoreversible polymer according to the invention.

[0276] Example 5. In vitro injectability, thixotropy, dissolution test and release test

[0277] The injectability of the DiD-NV liquid composition comprising 17% w / w Plx-407 was evaluated with a simple injection through a 1 mL syringe coupled to a 25 G needle. The injection was performed directly into dPBS, at pH 7.4, and pre-warmed to 37 °C and permitted evaluation of the flowability through the syringe and the thixotropic behavior of the hydrogel.

[0278] The composition dissolution profile and the in vitro release profile of DiD-NV comprising 17% w / w Plx-407 were obtained simultaneously, by adapting the method of Lee S.Y. et al. “A supramolecular host-guest interaction-mediated injectable hydrogel system with enhanced stability and sustained protein release”, Acta Biomater. 131 (2021) 286-301. Vials with an inner diameter of 15 mm and 60 mm height, containing 1.5 mL of DiD-NV comprising 17% w / w Plx-407 were placed in an incubator with orbital agitation at 37 °C and 30 rpm. 45 minutes later, after a complete liquid-to-gel transition, 4.5 mL of Dulbecco’s Phosphate Buffer Saline (dPBS) (pH 7.4) pre-warmed to 37 °C were carefully layered over the gelled composition’s surface. Every hour, the entire release medium was removed by decantation, and the weight of the vial and remaining composition recorded. The composition weight lost (corresponding to the dissolved composition) was calculated at each time point by subtracting the remaining weight of the composition from the initial weight. Next, the percentage of dissolved composition was calculated by dividing the composition weight lost at the different time points by the initial composition weight. The dissolution test was performed in triplicates. The same procedure was followed for the composition comprising lucifer yellow nanovesicles and 17% w / w of Plx407. The dPBS was prepared with 140 mM Sodium chloride (NaCI, purity > 99.5%), 2.7 mM potassium chloride (KCI, purity > 99.0%), 8.1 mM sodium hydrogen phosphate (Na2HPO4, purity > 99.0%) and 1.5 mM sodium phosphate monobasic dihydrate (NaH2PO42H2O, purity > 99.0%).

[0279] In vitro release behavior was assessed using a membrane-less diffusion method, according to the procedure described in the composition dissolution test. The release profile of the DiD-NV was determined by UV-Vis spectroscopy in a Varian Cary 5000 UV-Vis-NIR spectrophotometer (Agilent Technologies, Santa Clara, CA, USA), as described in Example 4, by employing calibration curves.

[0280] As a control, a similar release test was performed with the DiD freely dispersed in the blank composition. Due to its hydrophobic nature, the DiD (45 pM) was solubilized in a volume ratio of 1:2:97 EtOH:benzyl alcohokwater. Next, the solution was added Plx407 to 17% w / w and gelled, and the in vitro release was performed as with the DiD-NV composition.

[0281] DiD is an amphiphilic carbocyanine that integrates hydrophobically within the membranes of the nanovesicles according to the invention and presents negligible leakage. This molecule was chosen for its easiness to be quantified by spectroscopic techniques. In addition, it cannot be dissolved in water and requires formulation into nanovesicles to be stabilized in aqueous media, as it would occur with a hydrophobic drug. For the DiD-NV compositions with 17% w / w Plx407 (same hydrogel employed as a control in FRET analysis), a sustained release over 8 h was achieved at 37 °C using dPBS pH 7.4 as the release medium (FIG. 16A). The release of the DiD-NV was steady and constant, without any initial burst release.

[0282] In parallel to the release test, a dissolution test of the Plx407 hydrogel was also performed, which also demonstrated a zero-order kinetics dissolution (FIG. 16B). The weight loss of the hydrogel over time was constant and it aligns with the dissolution kinetics, providing evidence that the release kinetics mechanism of the nanovesicles is led by the hydrogel dissolution and not by diffusion of the DiD-NV through the hydrogel channels, a commonly undesired release mechanism in hydrogels. As a control, a release test without nanovesicles was carried out, containing exclusively the DiD solubilized in the formulation (1:2:97 EtOH:benzyl alcohokwater + 17% w / w Plx407).

[0283] The release test of the free DiD from Plx407 could not be properly quantified because sedimentation of the DiD in the dPBS occurred during the release test due to the low solubility of this hydrophobic molecule in the aqueous release media (FIG. 16C), despite the presence of the organic solvents employed for its formulation. This result also highlights the potential of the nanovesicles as nanocarriers for stabilizing highly hydrophobic compounds in physiological media.

[0284] Similar results to those obtained for the DiD-NV compositions with 17% w / w Plx407 were obtained for the dissolution and in vitro release tests with the compositions comprising lucifer yellow nanovesicles and 17% w / w of Plx407 (FIG. 17), thus suggesting that the stimulus-responsive gelable compositions according to the invention may serve to deliver hydrophobic as well as hydrophilic molecules of interest. These in vitro release and dissolution tests demonstrate the potential of formulating the nanovesicles with the thermoreversible polymers according to the invention to achieve an extended release with zero-order kinetics. Zero-order kinetics is the desired release kinetics for drug delivery because it allows maintenance of drug concentrations within the therapeutic window for an extended period, thus, reducing dosing frequency and adverse effects. The dissolution test demonstrates that the nanovesicles release is driven by hydrogel dissolution rather than diffusion of the nanovesicles through the polymeric matrix. This lack of diffusion of the nanovesicles is additionally adequate for avoiding an initial burst release.

[0285] Example 6. Stimulus-responsive hydrogels with Chitosan

[0286] Preparation of stimulus-responsive hydrogels based on mixtures of chitosan and loaded with DELOS nanovesicles.

[0287] DELOS nanovesicles composed of cholesterol and CPC at a molar ratio 1:1 and with a final concentration of membrane components of 3 mg / mL have been prepared and loaded into a chitosan aqueous solution.

[0288] The nanovesicles were produced by DELOS process. First, the organic solution containing the vesicle-forming components (i.e. , Choi and CPC) solubilized in ethanol are loaded at atmospheric pressure in the vessel; Then, compressed CO2 is injected into the vessel to form a CO2-expanded solution at working temperature Tw = 40 °C, and working pressure Pw = 85 bar; Finally, the CO2-expanded solution is depressurized into purified water. Nitrogen (N2) at a working pressure of Pw = 100 bar is injected into the vessel to eject the CCh-expanded solution, keeping a constant pressure inside the vessel during depressurization.

[0289] To prepare the hydrogels enriched with the nanovesicles, a stock solution of chitosan was prepared in 1% acetic acid (2.0%, w / v). The solution was mixed in a 1:1 ratio with the DELOS-NVs to obtain a formulation having 1.5 mg / ml of nanovesicles and 1%w / v of chitosan.

[0290] Macroscopical and microscopical images of the formulations

[0291] The structure and integrity of the nanovesicles within the hydrogels has been confirmed by cryogenic transmission electron microscopy (Cryo-TEM). The images were acquired using a JEOL JEM-2011 transmission electron microscope (JEOL Ltd., Tokyo, Japan) operating at 200 kV. Vitrified samples were prepared using a controlled environment vitrification system (CEVS) Leica EMCPC (Leica Microsystems, Germany). FIG. 18 demonstrates the integrity of the nanovesicles within the chitosan-based hydrogel.

[0292] Example 7. Stimulus-responsive hydrogels based on mixtures of polymers and loaded with DELOS nanovesicles.

[0293] DELOS nanovesicles composed of cholesterol and CPC at a molar ratio 1:1 and with a final concentration of membrane components of 3 mg / mL have been prepared and loaded into different mixtures of polymers.

[0294] Nanovesicles HA Heparin PLX 407 Sample

[0295] (mg / mL) (% w / v) (% w / v) (% w / v) M1 2 0.66 0 20 M2 2 0 0.20 20 M3 2 1 0.20 20

[0296]

[0297] M4 2 1 0 20

[0298] The nanovesicles were produced by DELOS process. First, the organic solution containing the vesicle-forming components (i.e. , Choi and CPC) solubilized in ethanol are loaded at atmospheric pressure in the vessel. Then, compressed CO2 is injected into the vessel to form a CO2-expanded solution at working temperature Tw = 40 °C, and working pressure Pw = 85 bar; Finally, the CO2-expanded solution is depressurized into purified water. Nitrogen (N2) at a working pressure of Pw = 100 bar is injected into the vessel to eject the CCh-expanded solution, keeping a constant pressure inside the vessel during depressurization.

[0299] To prepare the hydrogels enriched with the nanovesicles, Poloxamer 407 was dissolved into the nanovesicles suspension to achieve a concentration of 30% w / v.

[0300] Next, hyaluronic acid (2.0%, w / v) and heparin (0.4%, w / v) solutions were prepared in purified water.

[0301] Known volumes of the solution of nanovesicles and 30% Poloxamer 407 and the stock solutions of hyaluronic acid and heparin were mixed to obtain the formulations listed in the table above.

[0302] Macroscopical images and Cryo-TEM of the formulations

[0303] The macroscopical appearance of the obtained hydrogels was studied in search of signs of precipitation or destabilization phenomena. Images of the macroscopical appearance are obtained on a black background.

[0304] Moreover, the structure and integrity of the nanovesicles within the hydrogels was confirmed by cryogenic transmission electron microscopy (Cryo-TEM). The images were acquired using a JEOL JEM-2011 transmission electron microscope (JEOL Ltd., Tokyo, Japan) operating at 200 kV. Vitrified samples were prepared using a controlled environment vitrification system (CEVS) Leica EMCPC (Leica Microsystems, Germany).

[0305] FIG. 19 shows the macroscopical appearance of the different hydrogels obtained in the table above at room temperature, behaving as liquids, and at 37 °C, behaving as solidlike. FIG. 20 - 23 demonstrates the integrity of the nanovesicles of the Examples M1-M4, respectively, within the mixtures of polymers. In all cases small unilamellar vesicles are detected.

[0306] Example 8. Preparation of thermoreversible hydrogels incorporated with insulin-loaded nanovesicles.

[0307] DELOS nanovesicles composed of cholesterol and CPC at a molar ratio 1:1 and with a final concentration of membrane components of 30 mg / mL have been prepared and loaded into a PLCL-PEG-PLCL aqueous solution, with an insuline concentration of 3 mg / ml.

[0308] Briefly, the nanovesicles were produced by the DELOS process. First, the organic solution containing the vesicle-forming components (i.e. , Choi and CPC) solubilized in ethanol were loaded at atmospheric pressure in the vessel. Then, compressed CO2 was injected into the vessel to form a CO2-expanded solution at working temperature Tw = 40 °C, and working pressure Pw = 85 bar. Finally, the CO2-expanded solution was depressurized into 5 mM histidine buffer containing 10% of insulin (over the total nanovesicles suspension). Nitrogen (N2) at a working pressure of Pw = 100 bar was injected into the vessel to eject the CO2-expanded solution, keeping a constant pressure inside the vessel during depressurization.

[0309] Aiming to increase the insulin concentration in the formulation, the obtained nanovesicle suspension was further concentrated 10-fold through tangential flow filtration (TFF), reaching the final concentration of 3 mg / ml of insulin.

[0310] Next, the formulations were mixed with an aqueous solution of PLCL-PEG-PLCL. That is, the nanovesicle-enriched hydrogels were prepared by adding the concentrated insulin-loaded nanovesicle suspension to the corresponding amount of PLCL-PEG-PLCL (25% w / v). The formulation was magnetically stirred at room temperature until the polymer was fully dissolved.

[0311] Cryo-TEM of the formulations

[0312] The structure and integrity of the nanovesicles within the hydrogels was confirmed by cryogenic transmission electron microscopy (Cryo-TEM). To adjust the viscosity to the technique, the samples were diluted 1:10. The images were acquired using a JEOL JEM-2011 transmission electron microscope (JEOL Ltd., Tokyo, Japan) operating at 200 kV. Vitrified samples were prepared using a controlled environment vitrification system (CEVS) Leica EMCPC (Leica Microsystems, Germany).

[0313] The cryo-TEM images of FIG. 25 show that nanovesicles remained stable within the hydrogel.

[0314] Citation List

[0315] Non-Patent Literature

[0316] - Zhang, J., et al., in “Deformable Liposomal Hydrogel for Dermal and

[0317] Transdermal Delivery of Meloxicam”, International Journal of Nanomedicine 15 (2020) 9319-9335

[0318] - Cabrera, I., et al., “Multifunctional Nanovesicle-Bioactive Conjugates Prepared by a One-Step Scalable Method Using CO2-Expanded Solvents”, Nanoletters 13 (2013) 3766-3774.

[0319] - Algar, W.R. et al., “FRET as a biomolecular research tool - understanding its potential while avoiding pitfalls”, Nat. Methods 16 (2019) 815-829

[0320] - Lee S.Y. et al. “A supramolecular host-guest interaction-mediated injectable hydrogel system with enhanced stability and sustained protein release”, Acta Biomater. 131 (2021) 286-301

Claims

Claims1. A stimulus-responsive gelable liquid composition comprising:- vesicles comprising a sterol a cationic quaternary ammonium surfactant, and an active agent; and- a stimulus-responsive polymer,wherein:the molar ratio between the sterol and the cationic quaternary ammonium surfactant is from 0.2:1 to 1:1;the sum of the concentration of the sterol and the cationic quaternary ammonium surfactant in the composition is from 1 to 40 mg / ml;and the stimulus-responsive polymer is a polymer selected from the group consisting of a block copolymer comprising at least:a) one block of a polymer selected from the group consisting of polyethylene oxide (PEO) and polyethylene glycol (PEG); andb) one block of a polymer selected from the group consisting of polypropylene glycol (PPG), polypropylene oxide (PPG), polycarbonate (PC), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate) (PHB), poly(l-lactide-co-£- caprolactone) (PLCL), and polycaprolactone (PCL);chitosan and mixtures thereof; andwherein the vesicles comprise at least 50% w / w of the sterol and the cationic quaternary ammonium surfactant of the total weight of the vesicles.

2. The liquid composition according to claim 1 , wherein the sum of the concentration of the sterol and the cationic quaternary ammonium surfactant in the composition is from 2 to 40 mg / ml.

3. The liquid composition according to any of the claims 1-2, wherein the sterol is selected from the group consisting of cholesterol, ergosterol, p-sitosterol, stigmasterol, lanosterol, DC-cholesterol and mixtures thereof.

4. The liquid composition according to any of the claims 1-3, wherein the cationic quaternary ammonium surfactant is selected from the group consisting of cetylpyridinium chloride (CPC), cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), benzalkonium chloride (BKC), myristalkonium chloride (MKC), benzethonium chloride or miripirium chloride and mixtures thereof.

5. The liquid composition according to any of the claims 1-4, wherein the stimulus- responsive polymer is a di-block or a tri-block copolymer selected from the group consisting of PEO-PPO-PEO, PLGA-PEG-PLGA, PLCL-PEG-PLCL, PCL-PEG-PCL, PLA-PEG-PLA, PLA-PEG and mixtures thereof.

6. The liquid composition according to any of the claims 1-5, wherein the vesicles have a hydrodynamic diameter ranging from 20 to 300 nm, as measured by Dynamic Light Scattering (DLS).

7. The liquid composition according to any of the claims 1-6, wherein the vesicles have a polydispersity index ranging from 0.01 to 0.45, as measured by Dynamic Light Scattering (DLS).

8. The liquid composition according to any of the claims 1-7, wherein the vesicles have an apparent Z potential ranging from 30 to 150 mV, as measured by Electrophoretic Light Scattering (ELS).

9. The liquid composition according to any of the claims 1-8, wherein the concentration of stimulus-responsive polymer is from 5% to 30% w / w of the total weight of the liquid composition.

10. The liquid composition according to any of the claims 1-9, wherein the sterol is cholesterol.

11. The liquid composition according to any of the claims 1-10, wherein the ammonium cationic quaternary surfactant is CPC.

12. The liquid composition according to any of the claims 1-11, further comprising a secondary polymer selected from the group consisting of hyaluronic acid, heparin, hydroxypropyl methyl cellulose, collagen, ethylene vinyl acetate, poly(N- isopropylacrylamide), and polyacrylic acid.

13. The liquid composition according to any of the claims 1-12, the molar ratio between the sterol and the cationic quaternary ammonium surfactant is from 0.3:1 to 1:

114. A pharmaceutical composition comprising the liquid composition according to any of the claims 1-13, and pharmaceutically acceptable excipients or carriers; and wherein the active agent is an active pharmaceutical ingredient.

15. The liquid composition according to any of the claims 1-13, or the pharmaceutical composition according to claim 14, for use as a medicament, wherein the use comprises subcutaneous or intramuscular administration of the composition.

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