Development of a novel family of nanocarriers derived from natural tetrameric acidic lipids

Abstract

Thus, the present invention provides a novel family of liposomal compositions (nanocarriers) comprising natural tetramer acid (TA) lipids and / or chemically functionalized novel tetramer acid (CFTA) lipids (hereinafter referred to as tetraacidosomes) for drug delivery applications.

Description

[Technical field]

[0001] Thus, the present invention provides a novel family of liposomal compositions (nanocarriers) comprising natural tetramer acid (TA) lipids and / or chemically functionalized novel tetramer acid (CFTA) lipids (hereinafter referred to as tetraacidosomes) for drug delivery applications. [Background technology]

[0002] Oral drug delivery is by far the most convenient and advantageous method of drug administration, since it is painless and does not require assistance or patient compliance. Other routes, such as intramuscular, intravenous, or pulmonary delivery routes, are more difficult to perform and / or may injure or be rejected by the patient. Currently, more than 60% of conventional small molecule (e.g., less than 800 Da) drug products available on the market are taken orally. However, this is very difficult for therapeutic peptides / proteins and nucleic acids due to their degradation in the gastrointestinal (GI) environment (stomach and intestines) and their low permeability through the intestinal barrier into the blood. In fact, this is a class of drug compounds with significant molecular size and unique therapeutic properties that are not efficiently taken up into the body, meaning that they have low bioavailability when administered orally. There are only a few scattered examples of routine oral application or clinical trials of peptide drugs (e.g., desmopressin, cyclosporine A).

[0003] The gastrointestinal (GI) environment (i.e., stomach and intestines) is surrounded by a protective barrier to contain the GI fluids necessary for digestion. The main components of this barrier are the mucus layer (a gel-like coating) and the lining of epithelial cells. The desired scenario during oral drug delivery is for the therapeutic components to be transported from inside the gastrointestinal tract, through the mucus and epithelial barrier, and into the blood circulation system. Low delivery efficiency or orally administered peptides / proteins and nucleic acids are mainly due to 1) degradation by acidic pH and proteolytic enzymes in the GI environment, and 2) low mucosal permeability.

[0004] This situation can be improved by using liposomal nanocarriers that encapsulate peptides / proteins and nucleic acids. Nanoencapsulation of peptides has attracted considerable attention as a way to protect peptides from the harsh GI environment and provide better adhesion and interaction with mucus. Mucus-inert nanocarriers favor the penetration of nanocarriers into mucus, increasing the time available for the drug to be released from the carrier and transported into the blood. However, the detailed delivery mechanism is still under debate.

[0005] Polymeric nanoparticles, nanoemulsions, and liposomes have all been investigated as nanocarriers to improve the bioavailability of peptides / proteins and nucleic acids. So far, they are not efficient enough to overcome the GI barrier. However, the performance of the current best systems is still insufficient for medical applications, and their price is too high. One example is insulin, where even the most promising studies have shown only 10-20% oral bioavailability of insulin in animals [1]. Such low bioavailability results in high production costs to deliver standard doses of drugs. Thus, orally treating diabetic patients remains a major scientific and technological challenge.

[0006] Liposomes are spherical colloids / vesicles consisting of an aqueous interior surrounded by at least one lipid bilayer. A novel class of liposomes named Archaeosomes Archaeosomes nanocarriers have recently been introduced as robust vehicles for oral peptide delivery [2,3] (Figure 1A). Archaeosomes consist of a monolayer of archaeal tetraether lipids (natural or synthetic) with conventional bilayer-forming lipids and a therapeutic peptide in an aqueous interior. Archaeal lipids consist of a macrocyclic core containing up to four cyclopentane rings per chain, and a polar head group at each end (Figure 1B). Compared to conventional liposomes, archaeosomes have shown long-term stability in the presence of stressors such as low pH, bile salts, or lipases [4]. Furthermore, in vivo studies have demonstrated improved oral bioavailability of several peptides, such as insulin [5], vancomycin, and the hepatitis B drug Myrcludex B [6]. Despite their remarkable stability against chemical and enzymatic degradation, there are two main obstacles that currently limit their use as nanocarriers in oral drug delivery. - The oral bioavailability of archaeosomes is higher than that of conventional liposomal formulations, but is not high enough for medical applications. The novel archaeolipid structures can be easily grafted with mucus-inert polymers such as polyethylene glycol, thus enhancing both GI protection and mucus penetration [7]. -The cost and availability of archaeal lipids may limit their commercial potential. Currently, they are extracted and purified from cultured archaeal biomass[8] or by synthesis from simpler components[9], costing more than $5,000 per gram. New sources of archaeal lipids could make them more affordable.

[0007] Therefore, there remains a need to generate new nanocarriers that can be used to overcome GI barriers, without the above-mentioned side effects. Summary of the Invention

[0008] Natural TA lipids (also called ARN acids

[10] ) extracted from calcium naphthenate deposits

[11] are composed of four carboxylic acids with an "H-shaped" structure, 4 to 8 cyclopentane rings in the hydrocarbon backbone, and a molecular weight of 1227 to 1235 g.mol -1 The molecule can be thought of as consisting of two parts, one on the left and one on the right of the molecule, i.e. the polar head P 1 and P3 is located at the left end of the lipid, while the polar head group P 2 and P 4 means that it is at the right end of the lipid (Figure 2A). The most abundant TA lipid has six cyclopentane rings (formula (1c)) and a molar mass of 1232.01 g.mol -1 In the raw formula C 80 H 142 It has a C-O8 structure, the structure of which was first identified by Lutnaes et al. in 2006. It also loses symmetry due to an additional CH3. 81 (X=CH3, Y=H) analogues and C 82 The (X=Y=CH3) analogue was also found to be present in small proportions in the deposits

[13] (Figure 2B). 81 Analogs lose symmetry due to the additional CH3. Additional TA lipids (formulas (Ia), (Ib), (Id), and (Ie)) with different numbers of cyclopentanes (4, 5, 7, or 8) also exist, but in low proportions (<10% by weight). Natural TA lipids have the following general formula (Ia), (Ib), (Ic), (Id), or (Ie):

[0009] [ka] During the ceremony, P 1 =P 2 =P 3 =P 4 =COOH; X and Y are independently H or CH3.

[0010] The present invention provides novel lipid compounds, chemically functionalized TA (CFTA) lipids containing four terminal COOH that allow the introduction of many functional groups (polyethylene glycol (PEG), fluorescent probes, targeting ligands, cationic moieties). The source of these complex molecules (oil industrial waste) with enormous potential pharmaceutical applications ensures a low price and facilitates future commercialization. The novel chemically functionalized tetrameric acids were generated by a highly efficient chemical process. PEGylation of the tetrameric acids was achieved using a peptide coupling reaction to obtain the corresponding PEGylated tetrameric acids. More specifically, a method based on a selective esterification reaction was developed to distinguish between the four carboxylic acids of the lipids and the number of PEG chains introduced onto the lipids. Not only that, they controlled which part of the molecule the chain was attached to (specifically, asymmetric functionalization of two carboxylic acid groups on one side of the lipid). Fluorescent probes were grafted onto lipids to allow monitoring of liposomes by fluorometry. Cationic tetraacids were synthesized via esterification reactions with choline chloride for nucleic acid delivery. These formulations were found to exhibit stability over time and are suitable for encapsulation of both hydrophilic and hydrophobic substrates (including probes).

[0011] Thus, an object of the present invention is a novel lipid compound having the following general formula (II):

[0012] [ka] (In the formula, R A , R B , R C , and R D each independently represents a linear or branched aliphatic or alicyclic saturated hydrocarbon group containing 2 to 8 carbon atoms, and preferably R A , R B , R C , and R D is independent,

[0013] [ka] represents

[0014] [ka] P 1 , P 2 , P 3 , and P 4 or the point of attachment to the main molecule; P 1 , P 2 , P 3 , and P 4 are the same or different and each represent one of the following substituents: -CH2OH; -COOH; -COOR 1 , R 1 represents an aliphatic linear or branched, saturated or unsaturated alkyl chain having 1 to 22 carbon atoms, in particular having 4 carbon atoms (butyl chain); -A 1 -CH2-CH2-N + (CH3)3, X2 - X2 represents a halogen or a sulfonate; A 1 represents an ester (OC(O)) bond; -A 2 -(PEG x1 -A 3 ) n -R 2 , n is 0 or 1, and PEG x1 is a polyethylene glycol having a molecular weight of X1, where X1 is 5000 daltons or less; 2 and A 3 may be the same or different and represent an ester (C(O)O), an amide (C(O)NH), or a triazole; R 2 represents a methoxy group, a targeting agent (peptide, antibody, vitamin) or a probe (Nile Red, fluorescein, etc.); X and Y are independently H or CH3.

[0015] In these novel lipid compounds of the above general formula (II), P 1 , P 2 , P 3 , and P 4 are not all COOH at the same time.

[0016] According to a particular embodiment of the present invention, in the novel lipid compounds of general formula (II) above, P1, P2, P3 and P4 are identical or different and each represent one of the following substituents: -A 1 -CH2-CH2-N + (CH3)3, X2 - X2 represents a halogen or a sulfonate; A 1 represents an ester (OC(O)) bond; -A 2 -(PEGxi-A 3 ) n -R 2 , n is 0 or 1, and PEG x1 is a polyethylene glycol having a molecular weight of X1, where X1 is 5000 daltons or less; 2 and A 3 may be the same or different and represent an ester (C(O)O), an amide (C(O)NH), or a triazole; R 2 represents a methoxy group, a targeting agent, or a probe. For example, the targeting agent is selected from the group consisting of a peptide, an antibody, and a vitamin, and the probe is preferably a fluorescent probe selected from the group consisting of Nile Red and fluorescein.

[0017] According to another particular embodiment of the present invention, in the novel lipid compounds of general formula (II) above, P 1 , P 2 , P 3 , and P 4 are the same or different and each represent one of the following substituents: -A 1 -CH2-CH2-N + (CH3)3, X2 - X2 represents a halogen or a sulfonate; A 1represents an ester (OC(O)) bond; -A 2 -(PEG x1 -A 3 ) n -R 2 , n is 0 or 1, and PEG x1 is a polyethylene glycol having a molecular weight of X1, where X1 is 5000 daltons or less; 2 and A 3 may be the same or different and represent an ester (C(O)O), an amide (C(O)NH), or a triazole; R 2 represents a methoxy group.

[0018] According to certain embodiments of the present invention, the novel lipid compounds have the following general formula (IIa), (IIb), (IIc), (IId) or (IIe):

[0019] [ka] During the ceremony, X, Y, P 1 , P 2 , P 3 , and P 4 is as stated above.

[0020] The present invention also provides a novel family of stable liposomal compositions (nanocarriers) with low polydispersity index (e.g., less than 0.3, preferably less than 0.1) and diameters ranging from about 30-40 nm to about less than 250 nm, preferably less than 200 nm, hereafter referred to as tetraacidosomes. They are liposomes prepared by advanced microfluidic techniques, using, for example, one or more tetrameric acid (TA) lipids extracted from oil industry wastes (calcium naphthenate deposits) and / or one or more chemically functionalized natural molecules (novel CFTA lipids), in combination with conventional phospholipids (e.g., EggpC) and / or cholesterol for drug delivery applications, providing fine-tuning of the protective and transport properties of tetraacidosomes, as well as cell activity targeting and biopharmaceutical characterization. Mainly These innovative liposomal compositions (nanocarriers) designed for oral peptide / protein and nucleic acid drug delivery allow to efficiently deliver therapeutic agents into the body due to their resistance to the gastrointestinal environment (GI) induced by the tetrameric acid structure, as well as the unique possibility to control the interaction with the mucus and epithelial barriers through tetrameric acid functionalization. Indeed, their H-shaped structure strengthens the vesicles by limiting the flip-flop movement. The presence of four negatively charged carboxylate groups improves the permeability of the intestinal wall. Finally, the presence of four terminal COOH allows the introduction of many functional groups (polyethylene glycol (PEG), fluorescent probes, targeting ligands) required to stabilize these nanocarriers and / or to improve oral bioavailability and mucus / intestinal permeability and / or to perform biopharmaceutical studies.

[0021] Another object of the present invention is therefore a tetraacidosome comprising an inventive lipid compound as defined above, namely a lipid compound having the following general formula (II):

[0022] [ka] During the ceremony, R A , R B , R C , and R D each independently represents a linear or branched, aliphatic or alicyclic, saturated hydrocarbon group containing 2 to 8 carbon atoms, preferably R A , R B , R C , and R D is independent,

[0023] [ka] represents

[0024] [ka] P 1 , P 2 , P 3 , and P 4 represents the point of attachment to either the P 1 , P 2 , P 3 , and P 4 are the same or different and each represent one of the following substituents: -CH2OH; -COOH; -COOR 1 , R 1 is an aliphatic alkyl group having 1 to 22 carbon atoms, particularly 4 carbon atoms represents a linear or branched, saturated or unsaturated alkyl chain (butyl chain); -A1-CH2-CH2-N + (CH3)3, X2 - X2 represents a halogen or a sulfonate; A 1 represents an ester (OC(O)) bond; -A 2 -(PEG x1 -A 3 ) n -R 2 , n is 0 or 1, and PEG x1 is a polyethylene glycol having a molecular weight of X1, where X1 is 5000 daltons or less; 2 and A 3 may be the same or different and represent an ester (C(O)O), an amide (C(O)NH), or a triazole; R 2 represents a methoxy group, a targeting agent (peptide, antibody, vitamin), or a probe (Nile Red, fluorescein, etc.); X and Y are independently H or CH3.

[0025] According to a particular embodiment of the invention, the tetraacidosomes of the invention comprise a lipid compound having the following general formula (II): P1, P2, P3 and P4 are identical or different and each represent one of the following substituents: -A 1 -CH2-CH2-N +(CH3)3, X2 - X2 represents a halogen or a sulfonate; A 1 represents an ester (OC(O)) bond, -A 2 -(PEG x1 -A 3 ) n -R 2 , n is 0 or 1, and PEG x1 is a polyethylene glycol having a molecular weight of X1, where X1 is 5000 daltons or less; 2 and A 3 may be the same or different and represent an ester (C(O)O), an amide (C(O)NH), or a triazole; R 2 represents a methoxy group, a targeting agent, or a probe. For example, the targeting agent is selected from the group consisting of a peptide, an antibody, and a vitamin, and the probe is preferably a fluorescent probe selected from the group consisting of Nile Red and fluorescein.

[0026] According to another particular embodiment of the invention, the tetraacidosome of the invention comprises a lipid compound having the following general formula (II): 1 , P 2 , P 3 , and P 4 are the same or different and each represents one of the following substituents: -A 1 -CH2-CH2-N + (CH3)3, X2 - X2 represents a halogen or a sulfonate; A 1 represents an ester (OC(O)) bond; -A 2 -(PEG x1 -A 3 ) n -R 2 , n is 0 or 1, and PEG x1 is a polyethylene glycol having a molecular weight of X1, where X1 is 5000 daltons or less; 2 and A 3 may be the same or different and represent an ester (C(O)O), an amide (C(O)NH), or a triazole; R2 represents a methoxy group.

[0027] According to the present invention, the term "tetraacidosome" refers to a liposomal carrier consisting of an archeosome containing the lipid compounds of the present invention and / or natural tetrameric acid (TA) lipids. In other words, the tetraacidosome is derived from an archeosome in which all or part of the tetraether lipids are replaced by the lipid compounds of the present invention and / or natural tetrameric acid (TA) lipids.

[0028] According to a particular embodiment of the invention, said tetraacidosomes have an average size of less than 250 nm, preferably an average size of less than 200 nm, and / or a polydispersity index (PDI) of less than 0.3.

[0029] According to a particular embodiment of the invention, said tetraacidosome further comprises an encapsulated molecule of interest.

[0030] The use of TA and / or CFTA lipids in nanocarriers for peptide / protein and nucleic acid delivery has not been investigated before. The unique structure of the molecule allows multiple opportunities for chemical functionalization, providing CFTA lipids and allowing fine tuning of the protective and transport properties of tetraacaeosomes. This novel approach overcomes the limitations associated with archaeosomes in several ways. -The characteristic "H" structure, not found in conventional archaeal lipids, makes the lipid bilayer in the tetraacidosome more rigid by reducing transbilayer flip-flop motion, which provides better protection for therapeutic peptides against the harsh GI environment

[14] . - The presence of four carboxylic acid groups, which are lacking in conventional archaeal lipids, allows the incorporation of a wide variety of functional groups by synthetic routes based on peptide coupling reactions and Huisgen "click" cycloaddition. In this way, one to four dyes (e.g., Nile Red or Fluorescein) introduced at one or two termini of TA lipids provide original biological lipid probes designed for biopharmaceutical characterization of tetraacidosomes. In particular, TA lipids with one or two similar dyes (e.g., Nile Red or Fluorescein) on each side of the lipid backbone provide fluorescent tri- and tetra-Nile Red TAs and tri- and tetra-fluorescein TAs that constitute efficient donor (fluorescein-based TAs) and acceptor (Nile Red-based TAs) dye pairs for fluorescence resonance energy transfer (FRET) studies

[15] . Moreover, the controlled introduction of one or two identical dyes (e.g., Nile Red in Fluorescein) on one side of the TA lipids provides fluorescent mono- and di-labeled TA lipids designed for Fluorescence Recovery After Photobleaching (FRAP) experiments. This opens the study of nanoparticle stability in the gastrointestinal tract and intestinal permeability by FRET, and the interaction between nanocarriers and mucus by FRAP. The presence of four identical probes is also suitable for biopharmaceutical research. -Furthermore, asymmetric incorporation of polyethylene glycol (PEG) into the TA structure leads to PEGylated TA lipids (two PEG chains on one side of the lipid) that can give PEG-coated nanoparticles for improving oral bioavailability and mucus / intestinal permeability.

[16] The PEG chains can be functionalized at their termini with targeting ligands (peptides, antibodies, vitamins, etc.) to exhibit active targeting through cellular recognition and uptake by cell membrane receptors. -Furthermore, the four carboxylic acids can be reduced to hydroxyl functions to provide tetraol lipids as more hydroxylated analogues of the tetraetherdiols found in the archaeosomes. -Finally, the introduction of cationic moieties based on choline units onto the TA lipids by esterification reaction led to cationic versions of the tetraacidosomes useful for nucleic acid delivery. The presence of carboxyl groups on -TA also renders the tetraacidosomes negatively charged at neutral pH, which can increase the permeability of the intestinal wall, as recently shown for negatively charged silica nanoparticles

[17] , which is favorable for drug delivery. -Calcium naphthenate deposits are used as the TA lipid source. They typically contain 20-40% by weight of the molecule and can be easily extracted to obtain 80-90% purity

[18] . This abundance significantly reduces the time of lipid preparation compared to traditional lipid sources.

[0031] Therefore, another object of the present invention is the use of the above-mentioned tetraacidosomes as carriers, in particular as carriers for the gastrointestinal environment (GI).

[0032] Another object of the present invention is a tetraacidosome as described above for its use as a medicament.

[0033] Another object of the present invention is a method for the synthesis of said novel lipid compounds, comprising the step of reacting a TA lipid with a reducing agent, an alcohol, an amine, or an ester. [Brief description of the drawings]

[0034] [Figure 1] (A) Archaeosomes (tetraether lipids circled), (B) detailed structure of tetraether lipids present in Sulfolobus acidocaldarius. [Diagram 2] (A) Simplified representation of the most abundant TA lipids with P representing the polar head group (for TA lipids P1=P2=P3=P4=COOH), (B) suggested structure of C81 or C82 TA lipid. The two dotted lines represent two methyl groups that may be present to account for one or two carbons in addition to the 80 first carbons. [Diagram 3]FIG. 1 depicts the structure of the partially esterified TA lipid (P1:P2:P3:P4=COOH OR COOR1) of Example 2. Carbons C17 and C17''' (circles). [Figure 4] (A) The tetraacidosome of the present invention having natural TA lipid (TA lipid surrounded by a circle), (B) the detailed structure of the main TA lipid present in large amounts in petroleum calcium naphthenate deposits (=industrial waste). [Diagram 5] 1 shows the stability of the tetraacidosomes of the invention with respect to size. [Figure 6] FIG. 1 shows the stability of the tetraacidosomes of the invention in terms of the PolyDispersity Index (PDI). [Figure 7] PEGylation of TA lipids: represents an asymmetric introduction to prevent the chains from possibly filling the hydrophilic core of the tetraacidosomes. [Figure 8] 1 shows the structure of peptide-loaded tetraacidosomes composed of TA (with different polar head groups), fluorescently labeled TA (Nile Red TA), and PEGylated TA lipids. EXAMPLES

[0035] Materials and Methods General: 1 H NMR spectra were recorded on a Bruker Avance III 400 spectrometer operating at 400.13 MHz equipped with a BBFO probe with a Z-gradient coil and a GREAT 1 / 10 gradient unit. All experiments were performed at 25 °C.

[0036] The noesygpprld Broca pulse program was used for 1D NMR with presaturation using the following parameters: TD of 64, relaxation delay d1 = 2 s and 16 scans. The spectral width was set to 18 ppm. Fourier transformation of the acquired FID was performed without apodization in most cases.

[0037] 13C NMR spectra were recorded at 100.61 MHz. Several sequences such as jmod, dept135, or zgpg30 were used, usually with 64 to 1024 scans depending on the concentration of the sample. The TD was 64k and a relaxation delay of 2 seconds was used for a spectral width of 220 ppm. Fourier transformation was performed after exponential apodization using a LB of 0.6 Hz.

[0038] For mass spectrometry, SI+, ESI and MALDI ionization was used.

[0039] All reagents were obtained from known suppliers such as Merck, Fischer, and Alfa Aesar and were used without purification.

[0040] General procedure for formulation: Procedure A: 4 mg.mL -1 Stock solutions of EggpC, cholesterol, and lipids (depending on solubility) were prepared in MeOH (HPLC grade) and CHCl3 (HPLC grade). To ensure that all lipids were fully dissolved, the solutions were placed under ultrasound for 5 minutes. Formulations were then prepared at 1 mL depending on the selected weight percent. The NanoAssembler® operating conditions were selected by the software.

[0041] Working Example: Capacity: 3.3mL FRR:3:11 TFR: 8 mL.min -1 Left syringe: Aqueous phase - 3 mL syringe (dispensing: 2.47 mL) Right syringe: Organic phase - 1 mL syringe (dispensing: 0.83 mL) Star waste volume: 0.350mL Final waste volume: 0.050 mL

[0042] The aqueous phase is Dulbecco's Phosphate Buffered Saline (DPBS). The organic phase was extracted with a rotavapor: 2 x 1000 μL of liposome solution was placed in a 10 mL flask, itself placed in a 40°C bath. The pressure was set at 300 mbar for 10 min, then reduced by 20 mbar every 20 s until it reached 100 mbar, and maintained at 100 mbar for a further 5 min. The formulation was withdrawn with a syringe, sterilized with an Acrodisc, and then adjusted to a mass of 2 g with DPBS. The theoretical final concentration was 1 mg.L. -1 Each formulation was Analysis was performed by DLS using Malvern ZS90:3, corresponding to 10 runs of 20 seconds each with an initial equilibration of 90 seconds. The temperature was set at 25°C.

[0043] Procedure B: 4 mg.mL -1 Stock solutions of EggpC, cholesterol and lipids were prepared in EtOH. The solutions were placed under ultrasound for 5 minutes to ensure that all lipids were fully dissolved. Formulations were then prepared at 1 mL according to the selected mole %. The NanoAssembler® operating conditions were selected in the software.

[0044] Working Example: Capacity: 2.4mL FRR:3:11 TFR: 12 mL min -1 Left syringe: Aqueous phase - 3 mL syringe (dispensing: 1.8 mL) Right syringe: Organic phase - 1 mL syringe (dispensing: 0.6 mL) Star waste volume: 0.350mL Final waste volume: 0.050 mL

[0045] The aqueous phase is Dulbecco's Phosphate Buffered Saline (DPBS). The organic phase (EtOH) was removed by dialysis: 1000 μL of liposome solution was placed in a 1 mL hydration dialysis tube (Spectrum Laboratories G235035, cutoff = 100 kDa) and then the filled tube was placed in 250 mL of MilliQ water with magnetic stirring at 350 rpm. The MilliQ water bath was changed after 1 h. 500 μL of liposome solution in the dialysis tube was analyzed by DLS using a Zetasizer Malvern ZS90:3, measured by samples corresponding to 10 runs of 20 s with an initial equilibration of 90 s. The temperature was set to 25 °C.

[0046] Example 1: Synthesis of Tetraol Lipids (P 1 =P 2 =P 3 =P 4 =CH2OH)

[0047] [ka]

[0048] The reducing agent used was LiAlH4 (19.5 mg, 5.13.10 -4 TA lipid (95.8 mg, 7.78.10 mol, 6 equiv.) was dissolved in 8 mL of anhydrous THF. -5 mol, 1 equiv.) was added dropwise under inert atmosphere. The resulting mixture was then stirred under reflux for 5 h. Excess LiAlH4 was destroyed by addition of 6 mL of diethyl ether followed by 3 mL of water. The aqueous phase was extracted with diethyl ether and then the combined organic phases were washed with 5% sulfuric acid in water and water. After drying over MgSO4, the solvent was removed under reduced pressure to give a yellow oil (90.2 mg, 99% yield). 1H NMR in CDCl3: δ=2.3-1.5 (m), 1.4-1.0 (m), 0.91 (d, J=6.5 Hz), 0.89-0.83 (m), in CDCI3 13C NMR:62.4, 61.2, 46.5, 45.7, 45.1, 44.9, 44.8, 39.9, 39.5, 39.4, 39.1, 38.3, 37.9, 37.3, 37.2, 36.6, 36.1, 35.7, 34.7, 34.1, 33.7, 33.3, 33.2, 31.9, 31.7, 31.5, 31.2, 30.3, 29.7, 29.6, 29.3, 29.2, 25.9, 25.7, 24.4, 24.1, 22.7, 19.9, 19.6, 17.8, 14.1

[0049] The complete reduction of the four carboxylic acids to primary alcohol functions was achieved in semi-quantitative yields. 1D NMR spectra showed that the signals of the carboxylic acid carbons were no longer present, and two signals corresponding to the hydroxyl functions were visible in the 1D 13 The presence of the 1D protons at 61.23 ppm and 62.43 ppm on the C spectrum proved that tetraol was obtained. 1 Synthesis was also confirmed by high-resolution mass spectrometry, as a peak at m / z 1175 was observed in the H spectrum, specifically a doublet at 0.91 ppm, which was 0.98 ppm for the initial TA lipid.

[0050] Example 2: Partially esterified Ta lipid (containing at least one P 1 :P 2 :P 3 :P 4 =COOR 1 Synthesis of

[0051] [ka]

[0052] Selective esterification of TA lipids was carried out through the use of an acidic resin. TA lipids were dissolved in a mixture of a large excess of octane and butyl formate, and the transesterification reaction was carried out at the interface of a strongly acidic ion exchange resin, Dowex 50W-X2 (50-100 mesh). The explanation for the selectivity was based on the higher solubility of tetracarboxylic acid (TA) lipids in butyl formate compared to octane. Due to the acidic aqueous properties of the resin, the transesterification took place at the interface between the resin and butyl formate. The resulting partially esterified TA lipids, which are much more soluble in octane, were then less reactive and interacted less with the resin. Several experiments were carried out by varying the reaction time or the solvent ratio. The results are shown in Table 1 below, along with the calculated total esterification rate and the right and left esterification rates as a function of the reaction time or the solvent ratio.

[0053] Three Signals 1 The H NMR spectrum allowed the calculation of both the overall esterification rate and the esterification rate on the right side of the lipid. The doublet seen at 0.94 ppm and the doublet at 0.98 ppm correspond to carbon C 17’ and C 17’’’ The triplet corresponds to the CH3 of the butyl ester (Figure 3). When the polar head is a carboxylic acid, a doublet is found at 0.98 ppm, whereas when it is a butyl ester, a doublet is found at 0.94 ppm. However, a triplet corresponding to the terminal CH3 of the butyl ester was found at 0.95 ppm, overlapping with the doublet at 0.94 ppm. Line fitting of the region from 1.01 to 0.92 ppm allowed the three signals to be integrated separately.

[0054] There is a mathematical relationship between the integrals of these three signals. 17’ and C 17’’’(Figure 3) always corresponds to a CH3, so the sum of the integrals of the two doublets is always equal to 6. As for the triplet, the integral can range from 0 to 12 protons, since a lipid can have 0 to 4 butyl ester functionalities, i.e., 0 to 4 terminal CH3 of butyl esters. Using the cross product, we determine the integral of the triplet and multiply it by the theoretical maximum of 12, i.e., overall esterification rate = (integral triplet0.95ppm / 12) * It is possible to compare the esterification rate of the right side of the molecule with that of the right side of the molecule (integral doublet0.94ppm / 6) * is given by 100.

[0055] [Table 1]

[0056] Entries 1-5 in Table 1 gave information on the reaction time required to convert the carboxylic acid to a butyl ester with a solvent ratio of 90 / 10. Within 24 hours, the conversion was limited since it only reached 13%. It was still possible to obtain a satisfactory conversion rate since it reached 61% after 3 days (entry 5). Next, the effect of the solvent ratio was important. The esterification rate was increased 4.5 times in the same reaction time by increasing the proportion of butyl formate (entry 4 and entry 6). After a longer reaction time, with enough butyl formate, the carboxylic acid could be almost completely converted to a butyl ester (entry 7). In addition, the esterification was selective for all conditions tested (entries 1-7). In fact, the esterification rate of the right side of the molecule increased slowly compared to the overall esterification. The conditions of this reaction are therefore optimized to obtain TA lipids in which the number of ester functional groups was controlled and the left side of the lipid was more substituted than the right side. In fact, the longer the reaction time, the more tetraesters were obtained. This means that after a certain time, the difference in solubility in octane and butyl formate was not enough to prevent the reaction from continuing. Also, it was not clear why the left part of the molecule seemed more susceptible to this reaction. This may be due to the geometry of the lipid's approach to the resin, which may be more favorable for one side of the molecule.

[0057] In conclusion, a 50 / 50 solvent ratio and a reaction time of 24 h (entry 6) were the most suitable conditions for the asymmetric esterification of TA lipids, since the esterification rate on the right side was lower (14%) compared to the left side (100%). Furthermore, removal of tetraester compounds (fraction 1) by column chromatography allowed the isolation of partially esterified TA lipids (fraction 2) with a total esterification rate of 51%, an esterification rate of 5% on the right side, and an esterification rate of 97% on the left side (see below).

[0058] A representative reaction for partial esterification of TA lipids is shown below. TA lipid (107 mg, 0.086 mmol, 1 equiv.) was dissolved in 20 mL of a 50 / 50 mixture of octane and butyl formate. Dowex 50W-X2 (50-100 mesh) (500 mg) was added and the mixture was stirred at 100 °C for 24 h. The resin was removed by filtration. The solvent was removed under reduced pressure to give a yellow oil (114 mg). Two fractions were isolated after flash chromatography (CH2Cl2MeOH: 100 / 0 to 90 / 10). Fraction 1 (10 mg) was identified as tetrabutyl esters (P1 = P2 = P3 = P4 = CO2Bu) and fraction 2 (83 mg, 72%) consisted of partially esterified TA lipid (total esterification rate: 51%; 5% esterification rate on the right and 97% esterification rate on the left). Esterification rate) in CDCI3 1 H NMR: Fraction 1, same as tetraester: 4.08 (8H, 2t, J = 6.7 Hz), 2.37-2.05 (10H, m), 1.95 (4H, m), 1.76 (10H, m), 1.61 (14H, m), 1.5-1.0 (80H, m), 0.95 (12H, t, J = 7.2 Hz), 0.94 (6H, d, J = 7.2 Hz), 0.86 (20H, m); Fraction 2: 4.08(2H,2t,J=6.7Hz), 2.4-1.9(14H,m), 1.9-1.5(20H,m), 1.4-1.0 (80H,m), 0.98(6H,d,J=7.0Hz), 0.95(6H,t,J=7.1Hz), 0.9-0.7(20H,m).

[0059] Example 3: TA lipid (containing at least one P 1 :P 2 :P 3 :P 4 =A 2 -(PEGx1-A 3 )nR 2 or COOR 1 ) Here, the goal was to introduce polyethylene glycol chains into lipids to endow tetraacidosomes with mucus-inert properties. The PEGylation strategy was based on a peptide coupling reaction between the carboxylic acids of the lipids and the amines of PEG. TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate) and DIPEA (N,N-diisopropylethylamine) were chosen because of their known efficiency in coupling reactions with other lipids

[19] . The use of uronium salts such as TBTU allowed the formation of HOBt (1-hydroxybenzotriazole hydrate) by itself, while DIPEA ensured that the carboxylic acids were deprotonated. Also, to avoid the formation of secondary products, the TA lipids were stirred with TBTU and DIPEA for at least 20 min before the addition of the amines, so that the carboxylate functional groups were fully activated as esters of HOBt.

[0060] N3-PEG 500 Tetrazide PEGylation using -NH2 (N3PEG 500 )Synthesis of 4-TA lipid The first coupling reaction was carried out with N3-PEG 500 -NH2 and the first TA lipid, followed by tetrazide PEGylation (NPEG 500 ) to give the 4-TA lipid (Scheme 3), which functions in the copper-catalyzed cycloaddition described in the next part related to the functionalization of PEG chains (Scheme 5).

[0061] [ka]

[0062] TBTU (75 mg, 0.23 mmol, 4.8 equiv), DIPEA (4.8 equiv), and TA lipid (60 mg, 0.049 mmol, 1 equiv) were dissolved in 9 ml of anhydrous CHCl under an inert atmosphere. DIPEA (30 mg, 0.23 mmol, 4.8 equiv) was added to the mixture. After 20 min, N3-PEG dissolved in 3 mL of anhydrous CHCl was added. 500-NH2 (102 mg, 0.23 mmol, 4.8 equiv.) was added and the mixture was refluxed for 48 h. DIPEA was then neutralized by adding a few drops of 4% HCl solution. The organic phase was washed with distilled water (3 × 2 mL). The organic phase was dried over MgSO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography (CH2Cl2 / MeOH: 100 / 0 to 90 / 0) to give the tetrazide PEGylation (N3PEG 500 ) The 4-TA lipid was obtained as a yellow oil in excellent yield (92%). 1 H NMR: δ=3.8-3.2(160H,m,HPEG), 2.1(6H,m), 1.9(6H,m), 1.8-1.59(16H,m), 1.4-0.9(78H,m), 0.85(6H,d,J=6.2Hz), 0.78(16H,m). 1 The H NMR spectrum confirms the structure as the integration is accurate and the shielding resulting from the formation of amides is observed, being more significant for protons located close to these amides.

[0063] MeO-PEG 2000 Representative PEGylation of partially esterified TA lipids using -NH2 Additionally, partially esterified TA lipids (46%) with known esterification rates were synthesized using MeO-PEG 2000 Used in coupling reactions with -NH2.

[0064] [ka]

[0065] Partially esterified TA lipid (see fraction 2 in Example 2) (10 mg, 0.008 mmol, 1 eq.), TBTU (6.2 mg, 0.019 mmol, 2.4 eq.: 1.2 eq. for each equivalent of carboxylic acid) were dissolved in 2 mL of anhydrous CHCl under an inert atmosphere. DIPEA (6.7 mg, 0.019 mmol, 2.4 eq.: 1.2 eq. for each equivalent of carboxylic acid) was added to the mixture. After 20 min, MeO-PEG dissolved in 5 mL of anhydrous CHCl was added. 2000-NH2 (38 mg, 0.019 mmol, 2.4 equiv.: 1.2 equiv. for each equivalent of carboxylic acid) was added and the mixture was refluxed for 48 h. DIPEA was then neutralized by adding a few drops of 4% HCl solution. The organic phase was washed with distilled water (3 × 2 mL). The organic phase was dried over MgSO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography (CH2Cl2 / MeOH: 100 / 0 to 90 / 0) to give a yellow oil (42 mg). 1 H NMR: δ = 3.8-3.2 (HPEG), 2.4-1.9 (m), 1.9-1.4 (m), 1.4-0.95, 0.95-0.70 (m). The NMR spectra demonstrated that the remaining carboxylic acids of the TA lipids were semi-quantitatively PEGylated. The first goal of controlling where the PEG chains were grafted onto the TA lipids could then be achieved in excellent yields by prior selective protection of the appropriate carboxylic acids (Figure 7).

[0066] A subsequent coupling reaction with the (unprotected) initial TA lipid was performed to confirm the need for prior selective esterification of the TA lipid to obtain a di-PEGylated lipid with the PEG chain on the same side of the molecule. 2000 TBTU (2 eq.), DIPEA (2 eq.) and TA lipid (1 eq.) were added in anhydrous CH2Cl2 before adding -NH2 (2 eq.). We obtained di-PEGylated TA lipid in semi-quantitative yield, 1 Integration of the H NMR spectrum showed that the PEG chains were statistically attached to the right or left moiety of the lipid, therefore, a way to control where the PEG chains are attached is to selectively preprotect the carboxylic acids, as previously described.

[0067] Example 4: PEGylated TA lipid (P 1 :P 2 :P 3 :P 4 =A 2 -(PEGX1-A 3 ) n -R 2 or COOR 1 ) The introduction of specific functional groups (targeting agents, probes) at the end of the PEG chain can be achieved by esterification reactions, This can be achieved by peptide coupling reactions, or by click chemistry reactions (e.g., using copper-catalyzed Huisgen cycloaddition). 500 ) Introduction of probes at the four ends of 4-TA lipids was carried out for the characterization of biopharmaceuticals. Copper-catalyzed Huisgen cycloaddition is currently common, for example, to easily add probes or specific molecules to lipids. However, it is fundamental to find the right conditions, i.e., the right solvent and catalyst, since the yield can easily decrease. Classically, copper sulfate with ascorbic acid as an additive is used in a polar solvent such as water or alcohol.

[0068] Considering the aliphatic structure of the TA lipids, it seemed appropriate to try the reaction under other conditions.

[0069] [ka]

[0070] Tetrazide PEGylation (N3PEG 500 )4-TA lipid (50 mg, 61.9 mmol, 1 equiv.) and 9-diethylamino-2-(prop-2-ynyloxy)-5H-benzo[α]phenoxazin-5-one (Nile Red probe

[20] : 23 mg, 61.9 mmol, 4 equiv.) were dissolved in 9 mL of a 2 / 1 ratio mixture of two organic solvents: anhydrous toluene and anhydrous methanol (MeOH) (Scheme 5). The catalyst copper iodide (CuI) (13.6 mg, 40% mol), with a direct oxidation degree of 1, was added, followed by DIPEA, and the reaction mixture was stirred at 60 °C overnight. The mixture was cooled, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (CHCl / MeOH: 100 / 0 to 90 / 0) to give the tetra-Nile Red functionalized (Nile Red-PEG-2 tetrahydrofuran). 500 ) A dark purple oil (57 mg, 78%) was obtained corresponding to the 4-TA lipid. 1H NMR: δ = 8.2 (8H,m), 7.97 (s,4H), 7.59 (4H,m), 7.24 (4H,m), 6.65 (4H,m), 6.44 (4H,m), 6.29 (4H,m), 4.6 (8H,m), 3.9-3.3 (145H,HPEG), 2.3-1.8 (34H,m), 1.73 (6H,m), 1.63 (8H,m), 1.55-0.96 (100,m), 0.91 (6H,m), 0.83 (12H,m). The formation of triazole is 1 In the 1 H NMR, this was evidenced by the presence of a singlet at 7.9 ppm corresponding to the four protons and the loss of a signal at 2.58 ppm for the alkyne.

[0071] Example 5: Functionalization of ta lipids with cationic moieties or butanol Choline (TA) Functionalization of lipids (P 1 :P 2 :P 3 :P 4 =A1-CH2-CH2-N + (CH3)3, X - (In the formula, X=Cl) Esterification of TA lipids was achieved in semi-quantitative yields using MSA as a catalyst in a two-step reaction.

[0072] [ka]

[0073] First, choline chloride (27 mg, 0.19 mmol, 4.8 equiv.) was placed in a Dean-Stark flask under inert atmosphere. Then, MSA (20 mg, 0.21 mmol, 5.0 equiv.) was added and the mixture was stirred for 30 min. During this first step, the counterion of choline became mesylate while HCl was released.

[0074] The second step was the esterification with the hydroxyl function of choline under acidic conditions at a temperature of 130° C. TA lipid 1 (50 mg, 0.040 mmol, 1 equiv.) dissolved in anhydrous toluene was added and the temperature was set to 130° C. The reaction mixture was further stirred and then the solvent was evaporated under reduced pressure. 90 mg (112%) of crude product was obtained. 1 H NMR(50 / 50):4.02(8H,m),3.5(8H,m),3.2(36H,s),2.4-1.5(28H,m),1.4-1.0(58H,m),0.94(6H,d,J=0.93Hz),0.9-0.6(16H,m). 1D 1 H NMR spectra confirmed that the expected tetraester was obtained, since the alcohol signals disappeared and the formation of the ester functionality caused a change in the chemical shifts of the protons of the TA lipid.

[0075] Functionalization of TA lipids with butanol (P 1 :P 2 :P 3 :P 4 =CO2-(CH2)3-CH3)

[0076] [ka]

[0077] A Fischer esterification was envisaged to synthesize the tetraester. The TA lipid (26.3 mg, 2.14.10-5 mol, 1 equiv.) was dissolved in 5 mL of n-butanol (nBuOH), used as both nucleophile and solvent. Methanesulfonic acid (MSA, 125 μL, 0.1 equiv.), used as acid catalyst, was added and then the mixture was stirred at 140 °C in a flask equipped with a Dean-Stark apparatus to remove the water formed during the reaction. The pressure was set at 500 mbar. After 7 h, the solvent was removed under reduced pressure to give a yellow oil. In CDCl3 1H NMR: δ=4.08(8H,2t,J=6.7Hz), 2.37-2.05(10H,m), 1.95(4H,m), 1.76(10H,m), 1.61(14H, m), 1.5-1.0(80H,m), 0.95(12H,tJ=7.2Hz),0.94(6H,d,J=7.2Hz), 0.86(20H,m), CDCI3 13 C NMR: 171.0, 170.98, 67.3, 61.5, 60.3, 50.9, 43.8, 43.0, 42.7, 42.2, 39.4, 38.3, 36.4, 35.8, 35.4, 35.0, 34.8, 34.4, 34.0, 33.5, 32.3, 32.2, 31.5, 31.2, 30.8, 30.6, 30.0, 29.9, 28.7, 28.5, 28.1, 27.8, 27.2, 23.3, 21.9, 21.5, 20.1, 17.4, 17.2, 16.6, 16.3, 16.1, 15.2, 11.2. The tetraester was isolated in 90% yield. NMR experiments confirmed that all the carboxylic acids were converted to butyl esters.

[0078] Example 6: Formulation of natural tetra-acid (TA) lipids or chemically functionalized tetra-acid (CFTA) lipids into stable liposomes called tetraacidosomes The classical method for formulating liposomes is to form a lipid film by solvent evaporation, which is then hydrated with an aqueous solution and sonicated. Although this method works well, it is not the most convenient or reproducible method. An interesting alternative exists: microfluidics. The development of new continuous processes for making matrix particles is progressing rapidly. Microfluidics is the study of the mechanism of small volumes (10 -9 ~10 -18Microfluidics is a science that studies the manipulation of the particle size distribution (L). Microfluidics is a very efficient tool to formulate liposomes, and is implemented in the examples shown below, thanks to an instrument called the NanoAssembler® Benchtop. Microfluidics offers reproducibility, particle size tailoring, scalability, and speed. All the results shown below were obtained with this platform. Ideally, liposomes with a diameter of less than 200 nm and a polydispersity index (PDI) of less than 0.3 are required to consider a formulation suitable for encapsulation of molecules. These two parameters are provided by dynamic light scattering (DLS). PDI is a dimensionless measure of the broadness of the particle size distribution and ranges from 0 to 1. If its value is greater than 1, the sample may not be suitable for measurement by DLS.

[0079] Natural TA lipid-based formulation (Procedure A) Several formulations were tested with TA or PEGylated TA, EggpC and cholesterol at defined mass percentages, some of which are shown in Table 2.

[0080] [Table 2]

[0081] Cholesterol can be combined with two other substances to modulate the stiffness of the layer. TA lipid was 4 mg.L -1 The organic phase consisted of MeOH since it was soluble enough to give a stock solution of 1 mg.mL'. Once the formulation was prepared, the organic phase was evaporated and the liposome solution was diluted to give exactly 2 grams of 1 mg.mL' 1 Solutions with theoretical concentrations of 1000 were obtained, which were then analyzed by DLS to obtain the Z-average and PDI in nm (Table 3).

[0082] [Table 3]

[0083] First, liposome solutions with hydrodynamic diameters less than 200 nm were obtained, which were made from TA lipids associated with EggpC and / or cholesterol. Moreover, the PDI of each formulation was excellent, always less than 0.1, which means that monodisperse lipid vesicles could be formulated. Second, it was observed that the hydrodynamic diameter decreased with increasing concentration of TA lipid. The diameter of the liposomes could then be controlled by changing the mass percentage of TA lipid. More interestingly, these formulations were very stable. In fact, after 21 days, the average diameter and PDI of the formulations remained almost unchanged.

[0084] FIG. 4 shows the structure of a tetraacidosome of the present invention with native TA lipids.

[0085] Figures 5 and 6 show the stability of tetraacidosomes of the invention with native TA lipids, with respect to size and PDI, respectively.

[0086] Tetraol lipid-based formulation (Procedure A) Four formulations were tested using tetraol with the same mass percentage as solutions A, B, C, and D. Some differences with the TA lipid-containing liposomes could be noted. The organic phase consisted of a 1 / 1 mixture of MeOH and CHCl3, since the tetraol lipid is not soluble in MeOH. Turbidity appeared when the mass percentage of tetraol was 10 wt% or higher. The four formulations were also analyzed by DLS (Table 4).

[0087] [Table 4]

[0088] Some interesting formulations were obtained with diameters less than 200 nm and good PDI. However, there are some differences to mention. The hydrodynamic diameter no longer decreased with increasing mass percentage of tetraol. Although cholesterol-containing liposomes showed excellent PDI, formulation D, which contains only tetraol and EgGpC, has a PDI value higher than 0.1, although it can still be considered acceptable since it has a PDI value less than 0.3. Also, no liposomes with a diameter less than 100 nm were obtained.

[0089] Comparative formulations based on native or partially PEGylated TA lipids (Procedure B) Several formulations were tested with TA lipid or partially PEGylated TA lipid, EggpC, and cholesterol at distinct molar percentages, two of which are shown in Table 5.

[0090] [Table 5]

[0091] The organic phase consisted of EtOH instead of MeOH as in the case of formulations A-D to avoid potential toxicity issues induced by residual MeOH after administration of the formulation. 4 mg.L -1 A stock solution of was prepared. Once the formulation was prepared, the organic phase was removed by dialysis and the liposome solution was extracted from the dialysis. The tubes (500 μL) were then analyzed by DLS to obtain the Z-average (nm) and PDI (Table 6).

[0092] [Table 6]

[0093] First, liposome solutions with hydrodynamic diameters less than 100 nm were obtained, made from both TA lipids associated with EggpC and / or cholesterol or partially PEGylated TA lipids. Moreover, the PDI for each formulation was good, being always less than 0.2, meaning that monodisperse lipid vesicles could be formulated. Second, it was observed that the use of partially PEGylated TA lipids instead of native TA lipids promoted the formation of even smaller vesicles, which should facilitate the escape of liposomes from blood capillaries as well as the uptake of liposomes into target tissues

[21] . More interestingly, the partially PEGylated TA-based formulations were very stable. Indeed, after 21 days, the average diameter and PDI of the formulations remained almost unchanged.

[0094] List of References 1.Sonaje, K.;Chen,HL;Wey,SP;Juang,JHNguyen,H.-N.;Hsu,CW;Lin,KJ;Sung,HW,Biomaterials 2010,31(12),3384-3394. 2.Benvegnu,T;Lemiegre,L.;Dalencon,S.;JefJeftic,J.,Curr.Biotechnol.2013,294-303. 3. G. Kaur, T. Garg, G. Rath, AM K. Goyal, Drug Deliv., 2015, 23(7), 2497-2512. 4.Jensen,SM;Christensen,CJ;Petersen,JM;Treusch,AH;Brandl,M.,Int.J.Pharm.2015,493,(1-2),63-69. 5.Li, Z.;Chen,J.;Sun,W.;Xu,Y.,Biochem.Biophys.Res.Commun.2010,394,(2),412-417. 6.Uhl,P.;Helm,F.;Hofhaus,G.;Brings,S.;Kaufman,C.;Leotta,K.;Urban,S.;Haberkorn,U.;Mier,W.;Fricker,G.,Eur.J.Pharm.Biopharm.2016,103,159-166. 7.Maisel,K.;Ensign,L.;Reddy,M.;Cone,R.;Hanes,J.,J.Control.Release 2015,197,48-57. 8.Bode,M.I.;Buddoo,S.R.;Minnaar,S.H.;du Plessis,C.A.,Chem.Phys.Lipids 2008,154,(2),94-104. 9.Benvegnu,T.;Lemiegre,L.;Cammas-Marion,S.,Eur.J.OrgChem.2008,(28),4725-4744. 10.T.D.Baugh,K.V.Grande,H.Mediaas,J.E.Vindstad and N.O.Wolf,SPE International S ymposium on Oilfield Scale,SPE 93011 Aberdeen,United Kingdom,May 11-12,2005. 11.Mediaas,H.;Grande,K.;Hustad,B.;Rasch,A.;Rueslatten,H.;Vinstad,J.E.,5th International Symposium on Oilfiled Scale,SPE 80404,Aberdeen,United Kingdom,Jan 29-30,2003.

[0095] 12.Lutnaes,B.F.;Brandal,O.;Sjoblom,J.,Org.Biomol.Chem.2006,4,(4),616-620. 13.Lutnaes,BF;Krane,j.;Smith,BE;Rowland,SJ,Org.Biomol.Chem.2007,5,1873-1877. 14.Benvegno,T.;Rethore,G.;Brard,M.;Richter,W.,Plusquellec,D.,Chem.Commun.2005,5536-5538 15.De,S.;Girigoswami,A.,J.Coll.Int.Sci.2004,271 ,485-495. 16.Zhang,X.;Chen,G.;Zhang,T.;Ma,Z.;Wu,B.,Int.J.Nanomed.2014,9,5503-5514. 17.Arthursson,P.;Lundquist,P.,Nat.Biomed.Eng.,2020,4,12-13. 18.Simon,S.;Nordgard,E.;Bruheim,P.;Sjoblom,J.,J.Chromatogr.A 2008,1200,(2),136-143 19.Barbeau,J.,Thesis:「Archaeaosomes and archaepolyosomes:synthesis,formulation and targeted vectorization of peptides of marine origin.」Univ.Rennes 1 2009,4060. 20.M.Berchel,JPHaelters,D.Afonso,A.Maroto,L.Deschamps,P.Giamarchi,PAJaffres,Eur.J.Org.Chem.,1076-1083,2014. 21.M.Sharifi,WCCho,A.Ansariesfahani,R.Tarharoudi,H.Malekisarvar,S.Sari,S.Haj Bloukh,Z.Edis,M.Amin,JPGleghorn,TLMten Hagen,M.Falahati,Cancers,2022,14(12),2868.

Claims

1. A lipid compound having the following general formula (II): [Formula 1] During the ceremony, R Ａ , R Ｂ , R Ｃ , and R Ｄ independently represent a linear or branched, aliphatic or alicyclic, saturated hydrocarbon group containing 2 to 8 carbon atoms, preferably R Ａ , R Ｂ , R Ｃ , and R Ｄ is independent, [chemical 2] represents [C3] Is P １ , P ２ , P ３ , and P ４ or the point of attachment to the main molecule, P １ , P ２ , P ３ , and P ４ are the same or different and each represent one of the following substituents: -CH ２ O-H; -COOH; -COOR １ , R １ represents an aliphatic linear or branched, saturated or unsaturated alkyl chain having 1 to 22 carbon atoms, in particular 4 carbon atoms; -A １ -CH ２ -CH ２ -N ＋ (CH ３ ) ３ , X ２ － ;X ２ represents a halogen or a sulfonate; A １ represents an ester (OC(O)) bond; -A ２ -(PEG ｘ１ -A ３ ) ｎ -R ２ , n is 0 or 1; ｘ１ is a polyethylene glycol having a molecular weight of X1, where X1 is 5000 daltons or less; ２ and A ３ may be the same or different and represent an ester (C(O)O), an amide (C(O)NH), or a triazole; R ２ represents a methoxy group, a targeting agent, or a probe; -X and Y are independently H or CH ３ and However, P １ , P ２ , P ３ , and P ４ are all lipid compounds that are not COOH at the same time.

2. P １ , P ２ , P ３ , and P ４ are the same or different and each represent one of the following substituents: -A １ -CH ２ -CH ２ -N ＋ (CH ３ ) ３ , X ２ － , X ２ represents a halogen or a sulfonate; A １ represents an ester (OC(O)) bond, -A ２ -(PEG ｘ１ -A ３ ) ｎ -R ２ , n is 0 or 1; ｘ１ is a polyethylene glycol having a molecular weight of X1, where X1 is 5000 daltons or less; ２ and A ３ may be the same or different and represent an ester (C(O)O), an amide (C(O)NH), or a triazole; R ２ The lipid compound of claim 1 , wherein represents a methoxy group, a targeting agent, or a probe.

3. It has the following general formula (IIa), (IIb), (IIc), (IId), or (IIe): [C4] During the ceremony, X, Y, P １ , P ２ , P ３ , and P ４ is as defined in claim 1, claim 1 or 3. A lipid compound according to claim 2.

4. A tetraacidosome comprising a lipid compound having the following general formula (II): [C5] During the ceremony, R Ａ , R Ｂ , R Ｃ , and R Ｄ each independently represents a linear or branched, aliphatic or alicyclic saturated hydrocarbon group containing 2 to 8 carbon atoms, preferably R Ａ , R Ｂ , R Ｃ , and R Ｄ is independent, [C6] represents [C7] Is P １ , P ２ , P ３ , and P ４ or the point of attachment to the main molecule; P １ , P ２ , P ３ , and P ４ are the same or different and each represent one of the following substituents: -CH ２ O-H; -COOH; -COOR １ , R １ represents an aliphatic linear or branched, saturated or unsaturated alkyl chain having 1 to 22 carbon atoms, in particular 4 carbon atoms; -A １ -CH ２ -CH ２ -N ＋ (CH ３ ) ３ , X ２ － ;X ２ represents a halogen or a sulfonate; A １ represents an ester (OC(O)) bond; -A ２ -(PEG ｘ１ -A ３ ) ｎ -R ２ , n is 0 or 1; ｘ１ is a polyethylene glycol having a molecular weight of X1, where X1 is 5000 daltons or less; ２ and A ３ may be the same or different and represent an ester (C(O)O), an amide (C(O)NH), or a triazole; R ２ represents a methoxy group, a targeting agent, or a probe; X and Y are independently H or CH ３ That is, the tetraacidosome.

5. P １ , P ２ , P ３ , and P ４ are the same or different and each represents one of the following substituents: -A １ -CH ２ -CH ２ -N ＋ (CH ３ ) ３ , X ２ － ;X ２ is halogen or sulfonate represents the １ represents an ester (OC(O)) bond; -A ２ -(PEG ｘ１ -A ３ ) ｎ -R ２ , n is 0 or 1; ｘ１is a polyethylene glycol having a molecular weight of X1, where X1 is 5000 daltons or less; ２ and A ３ may be the same or different and represent an ester (C(O)O), an amide (C(O)NH), or a triazole; R ２ The tetraacidosome of claim 4, comprising a lipid compound having the following general formula (II):

6. Tetraacidosomes according to claim 4 or 5, having an average size of less than 250 nm.

7. Tetraacidosomes according to claims 4 to 6, having a polydispersity index (PDI) of less than 0.

3.

8. The tetraacidosome of any one of claims 4 to 7, further comprising an encapsulated molecule of interest.

9. Use of a tetraacidosome according to any one of claims 4 to 8 as a carrier.

10. 10. Use of the tetraacidosomes according to claim 9 as carriers for the gastrointestinal environment (GI).

11. A tetraacidosome according to any one of claims 4 to 8 for use as a medicament.

12. A method for the synthesis of a lipid compound according to any one of claims 1 to 3, comprising reacting a tetramer acid (TA) lipid with a reducing agent, an alcohol, an amine or an ester.

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