Patent Application
20240115498
The object of the invention is a nanostructured lipid hydrogel consisting of a three-dimensional network of interconnected sheets and vesicles comprising at least one zwitterionic phospholipid and water. The present invention also relates to the preparation method and use thereof, particularly as an active ingredient release system.
The present invention could, therefore, be included in the field of gel formulations with potential biomedical applications.
Gels can be described as materials in which either an aqueous or organic solvent becomes trapped within a network of solute molecules. This composition allows gels to behave as soft solids and viscous liquids, depending on their composition. Their rheological behaviour is related to the molecular interactions that act on their microscopic structure. Chemical interactions in polymer gels, like covalent bonds, provide mechanical resistance to deformation. In contrast, physical interactions in supramolecular gels, like electrostatic forces, are weaker and allow the gels to deform and flow more easily. The ease of combining gelling agents to form gels with different properties gives rise to a rapid growth of gel byproducts. Understanding the parameters that control their formation and rheological behaviour is key to developing new applications for these materials.
In the prior art, a supramolecular gel made up of oleic acid and phospholipids has been described, as disclosed in document ES2754476 A1. This gel was formed from a dilute dispersion of lipids in water at pH between 5 and 8, which followed a freezing-heating process. Fluorescence confocal microscopy revealed that the gel structure consisted of aggregated lipid vesicles and lipid bilayers. The gels described in said document are stable at pH between 5 and 8, which limits their application to media with pH comprised between these values, and are not useful in strongly acidic or basic media.
In order to broaden the scope of application, the present invention proposes stable lipid gels that maintain their rheological properties in a broader range of pH between 2 and 13. In that sense, for example, it could be used for gastric applications where the pH ranges between 1 and 4.
The following prior art documents merit mention:
The two documents refer to a gel phase, making no reference to a material with a gel behaviour at the macroscopic level, as defined in the first paragraph of this section.
Different phases associated with the organisation of lipid membranes are described in the two mentioned articles. Among these phases are the so-called gel or crystalline solid phase and the liquid crystal phase (among others).
The organisation in gel or crystalline solid phase does not mean that the system behaves macroscopically like a gel, but rather that the hydrocarbon chains are packed at the molecular level, giving the system-forming membranes greater rigidity. Although the term used, that is, “gel”, is the same, the gel phase described in said documents has nothing to do with the lipid gel of the present invention which, due to its structure, presents macroscopic gel behaviour and rheological properties suitable for the application for which they are intended. In fact, the lipid mixtures mentioned in the two articles are diluted dispersions or referred to directly as liposomes, which means that they are liquid solutions.
The object of the present invention is a nanostructured lipid hydrogel consisting of a three-dimensional network of interconnected sheets and vesicles.
The term “lipid hydrogel” refers to a gel the major component of which is water, but which further comprises lipids. The lipids are grouped or arranged in structures in the form of sheets and vesicles within an aqueous matrix.
In the context of the present invention, the term “nanostructured” in reference to the hydrogel shall be understood as relating to materials with a gel-type rheological behaviour which are organised with at least one dimension below 100 nm. It has also been observed that they are organised on a micro scale. That is, they are organised both on a nano and micro scale.
The hydrogels of the present invention exhibit gel behaviour, since their elastic modulus value G′ is higher than the value of the viscous modulus G″ in a wide range of frequencies (preferably between 0.01 and 10 Hz).
Unlike other gels known in the prior art, the nanostructured hydrogel of the present invention can be formed with a low lipid content, where the intervention of polymers, surfactants, fatty acids or fatty alcohols is not required to favour dispersion.
The first aspect of the present invention relates to a nanostructured lipid hydrogel consisting of a three-dimensional network of interconnected sheets and vesicles, characterised in that it comprises:
Phospholipids are amphiphilic molecules that are made up of two chemically different parts: a hydrophilic polar head and one or two hydrophobic hydrocarbon chains. According to the nature of the polar head of phospholipids, these can be classified as cationic, anionic and zwitterionic phospholipids. Zwitterionic phospholipids have positive and negative formal charges on different atoms and can acquire a net charge depending on the pH of the solution in which they are found.
In the present invention, the term “surfactant” refers to synthetic compounds capable of lowering the surface tension of the water-air interface and exhibiting a critical micellar concentration, that is, they spontaneously form micelles in water. According to the present invention, the term “surfactant” does not include phospholipids.
In the present invention, the term “polymer” refers to a compound formed by the attachment, by means of covalent bonds, of at least five simple units called monomers (whether these are the same or different from each other).
The term “fatty acid” refers to a monocarboxylic acid with a linear hydrocarbon chain with a minimum of 4 carbons, and it can be saturated or unsaturated.
The term “fatty alcohol”, also called “long-chain alcohol”, refers to an aliphatic hydrocarbon which contains at least one primary hydroxyl group and has a linear hydrocarbon chain with a minimum of 5 carbons, and it can be saturated or unsaturated.
In a preferred embodiment, the hydrogel of the present invention consists of:
In a preferred embodiment of the method of the present invention, the lipid:water weight ratio is 5:95.
In a preferred embodiment, the zwitterionic phospholipid is selected from the list comprising: phosphatidylcholines and phosphatidylethanolamines. More preferably, the zwitterionic phospholipid is hydrogenated soy phosphatidylcholine (HSPC, also referred to as L-a-phosphatidylcholine, hydrogenated (Soy)).
In a preferred embodiment, the lipids comprise an anionic or cationic lipid in addition to zwitterionic phospholipid. In this case, the molar ratio between the zwitterionic phospholipid and the anionic or cationic lipid is preferably between 100:1 and 3:1, more preferably between 80:1 and 3:1, and even more preferably, the molar ratio is 20:1.
In a preferred embodiment, the cationic lipid is 1,2-dioleoyl-3-trimethylammonium propane (DOTAP).
In a preferred embodiment, the anionic lipid is 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS).
In a preferred embodiment, the hydrogel also comprises an active ingredient, such as, for example, a sphingolipid, cholesterol, an antioxidant, a drug (such as an antibiotic, an anti-inflammatory), a protein or combinations thereof.
According to the present invention, the term “active ingredient” refers to any component that potentially provides a pharmacological activity or other different effect in the diagnosis, cure, mitigation, treatment or prevention of a disease, or that beneficially affects the structure or function of a subject's body. The active ingredients that can be comprised in the hydrogel of the present invention can be lipophilic or hydrophilic compounds.
The weight ratio of the active ingredient in the hydrogel may vary between 0 and 20% by weight with respect to the total weight of the gel.
In a preferred embodiment, the hydrogel of the present invention consists of:
The structure and fluidity of the lipid hydrogels of the present invention respond reversibly to temperature and pH and are capable of transporting at least one hydrophilic substance within the skin and also to the follicles. Their particular organisation, with part of the water trapped in vesicles and these vesicles trapped or intercalated between extended sheets, makes them very suitable as systems for incorporating molecules of a different polar nature in different compartments. Their exclusively lipid composition ensures high biocompatibility and their rheological behaviour makes them easily applicable on the skin, eyes or mucous membranes.
As the inventors have demonstrated in the examples provided, the hydrogels described in the present invention are stable in a very broad pH range, between pH 2 and 13. To that end, for example, they can be used in gastric applications where the pH can range between 1 and 4.
Furthermore, the gels have proven to be stable over time, even without the need to add additives, maintaining their rheological behaviour, since their viscous modulus G″ and elastic modulus G′ values remain unchanged over time.
As mentioned above, the hydrogels of the present invention exhibit gel behaviour, since their elastic modulus G′ value is greater than the viscous modulus G″ value in a wide range of frequencies.
Another aspect of the invention relates to the preparation method for preparing a nanostructured lipid hydrogel as described in the first aspect of the invention, comprising the steps of:
The dispersion of the lipids in water is preferably carried out by mixing the corresponding lipids in an organic solvent at the specified concentrations and molar ratios and evaporation thereof in a rotary evaporator followed by desiccation and subsequent hydration by adding water in the range of specified concentrations under conditions of stirring and at room temperature.
The organic solvent used can be chloroform, methanol, ethanol, mixtures thereof, etc., depending on the lipids used. Preferably, the organic solvent is chloroform.
The adjustment of the pH of the lipid dispersion between 2 and 13 is performed by adding an acidic or basic solution, for example, with a hydrochloric acid or sodium hydroxide solution.
The pH value to which the dispersion must be adjusted so that at least one lipid is charged is something that the skilled person can easily determine based on the lipid or lipids present in the dispersion. If there is a positive lipid, it is preferably adjusted to neutral or basic pH; if there is a negative lipid, it is adjusted to neutral or acidic, so that they hold their charge. If there are only zwitterionic lipids, the pH can be adjusted to both acidic and basic pH for the lipid to become ionised.
Lastly, a third aspect of the invention relates to the use of a hydrogel described in the first aspect of the invention as an active ingredient release system or as a protective agent for the skin and mucous membranes, more preferably as a gastric protective agent.
The hydrogel can be for cutaneous, mucosal or ocular, as well as oral, application.
Throughout the description and the claims, the word “comprises” and its variants do not intend to exclude other technical features, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention may be partially deduced from both the description and the embodiment of the invention. The following examples and figures are provided by way of illustration and are not intended to limit the present invention.
The three gels have been formed at a 5% lipid concentration following the protocol described in Example 1 of the present invention. A rigid gel behaviour is observed with the naked eye since the gels do not move due to the effect of gravity inside the Eppendorf tubes.
Next, the invention will be illustrated by means of assays carried out by the inventors that demonstrate the effectiveness of the product of the invention.
Hydrogenated soy phosphatidylcholine (HSPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were purchased from Lipoid GmbH (Ludwigshafen, Germany). The chloroform was purchased from Merck (Darmstadt, Germany). 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) was purchased from Lipoid GmbH.
First, an aqueous dispersion of HSPC and DOTAP was formed (the molar ratio between said lipids was set at 3:1, 20:1 and 80:1), for which purpose the lipids were dissolved in chloroform and then the solvent was evaporated by means of a rotary evaporator to form a lipid film, and water was added until a total lipid concentration of 5% by weight was obtained. Next, the pH was adjusted to 7 by adding a 0.1% by weight NaOH solution. To form the gels, the aqueous lipid dispersion was first stored at −20° C. in a closed vial for two hours to completely freeze the sample. Then, the samples were heated at 70° C. for 10 minutes until all the dispersion was clear. The gels were left to cool to room temperature and stored 24h in the refrigerator (5° C.) before any measurement. Gelling and the absence of water separation were confirmed by inverting the vials.
The hydrogels with an HSPC/DOPS composition were prepared following the same methodology as in example 1.1., but using DOPS instead of DOTAP.
This hydrogel was prepared like in the previous cases, but using only HSPC as the lipid and adjusting the pH to 2.5 in one case and to 11.5 in another (two gels of the same composition were prepared at different pH).
This technique allows us to observe the microstructure of the gel and how it is organised in the aqueous medium. The formation of structures with different morphologies and sizes, as well as the interconnection between said structures in the gel matrix, can be detected.
Fluorescence confocal microscopy tests were performed with the following hydrogels prepared in Example 1: HSPC/DOTAP at a ratio of 80:1, 20:1 and 3:1; and HSPC/DOPS at a ratio of 20:1.
In order to label the lipid part of the gels, the fluorescent probe 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) from Avanti Polar Lipids Inc. (Alabaster, AL, USA) was used. The final concentration of the probe was 0.005% by weight in all the samples to have sufficient fluorescent intensity without interfering with the gel structure.
A Zeiss LSM 880 confocal microscope equipped with a 561 nm DPSS laser was used for Rhodamine B excitation. Images were obtained using a 10×0.4NA objective and a 63×1.4NA glycerol immersion objective. Images were processed using ImageJ 1.52d software (J. Schindelin, et al. Nat. Methods. 9 (2012) 676).
The organisation of the lipids forming the microstructure of the HSPC/DOTAP and HSPC/DOTAP gel can be seen in
In
Rheology allows the macroscopic properties of the gel to be described. In the rheological study described below, the response of the material to an oscillatory stress is determined, determining the variables elastic modulus (G″) and viscous modulus (G″). In materials that exhibit gel behaviour, elasticity, measured with G″, will be greater than the dissipated energy or viscous behaviour, determined by G″ for a given range of frequencies.
Tests were carried out with the following hydrogels prepared in Example 1: HSPC/DOTAP at a ratio of 80:1, 20:1 and 3:1; and HSPC/DOPS at a ratio of 20:1.
3.1. Test method
The rheological behaviour of the gels was studied by performing oscillatory tests with an AR-G2 controlled stress rheometer (TA Instruments) equipped with a Peltier temperature control system. The measurements were carried out by the Nanostructured Liquids Unit (U12) of CIBER-BBN, in ICTS NANBIOSIS. A cone-plate geometry of 40 mm in diameter, a cone angle of 4° and a gap of 105 μm were used. The samples were evaluated at 25° C. and drying of the samples was prevented by using a solvent trap. Before each oscillatory measurement, gels were pre-cut at 10 s−1 for 10 s to obtain homogeneous conditions. Then, the sample was subjected to sinusoidal shear deformation while the stress response was measured at each point. Data was analysed using TRIOS software (TA Instruments, U.S.A.). The viscoelastic behaviour of the sample was described by the complex modulus G*=where
the shear deformation. With the phase angle δ between the applied and the resulting oscillation response, the software determined the elastic modulus G′ and the viscous modulus G″ of the vector G* as follows:
G′=G*cos δ
G″=G*sin δ
The elastic modulus G′ is related to stored energy, while the viscous modulus G″ represents dissipated energy.
In these tests, the elastic modulus (G′) and the viscous modulus (G″) of the sample are measured by applying a fixed deformation in a range of frequencies. It is thereby possible to see how the material behaves depending on the time that the applied deformation lasts.
It can be seen in
Table 1 shows the differences in G′ and G″ values from
The same gel behaviour can be observed in
Regarding stability, these gels have been found to be stable over time.
X-ray scattering is an analytical technique that measures the intensities of scattered X-rays in a sample based on the scattering angle. From Bragg's law, it is understood that with the decrease of the scattering angle, increasingly larger structural characteristics are analysed.
A small angle X-ray scattering signal (SAXS) is observed when the material contains structural characteristics on the nanometer scale, generally, in the range of between 1 and 100. On the other hand, wide angle X-ray scattering (WAXS) analyses the structures in the material on a much smaller length scale, as interatomic and smaller distances.
In the experiments for the present invention, these techniques allow detection of the nanostructure of the lipid bilayer of the sheets and vesicles forming the gel (SAXS) as well as the lateral packing between the lipids (WAXS).
Tests were carried out with the following hydrogels prepared in Example 1: HSPC/DOTAP at ratios of 80:1, 20:1 and 3:1 and HSPC/DOPS at ratios of 80:1, 20:1 and 3:1.
X-ray scattering measurements were performed with synchrotron radiation at the BL11-NCD-SWEET beamline of ALBA (Cerdanyola del Valles, Barcelona, Spain). Samples were loaded into a 1.5 mm glass capillary and placed in a Linkam chamber, which is used to control the temperature at 25° C. The samples were aligned with the X-ray beam with an energy of 12.4 keV. Small angle (SAXS) measurements were acquired using a 1M Pilatus detector placed 2.71 m from the sample and wide angle (WAXS) measurements were acquired using a Rayonix LX255-HS detector placed at 0.134 m. The spectra were collected with an acquisition time of 5 s.
In order to study the organisation of the lipid structure at the molecular level, an X-ray scattering technique using synchrotron radiation was used.
It can be seen how the SAXS pattern (
The WAXS pattern makes it possible to study the lateral packing of the molecules making up the lipid bilayers. In
The ability of gels to be stable at different pH, even extreme acidic and basic pH, allows a very wide application range. For example, they can be used in gastric applications where the pH can range between 1 and 4. The 20:1 HSPC/DOTAP gel was subjected to an incubation simulating gastric digestion. In this test, a saline solution at pH 2 was prepared with HCl and NaCl and then the gel was added and left to stir back and forth at 37° C. for 2 hours. It was observed (
| Number | Date | Country | Kind |
|---|---|---|---|
| P202031135 | Nov 2020 | ES | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/ES2021/070815 | 11/11/2021 | WO |
