DEVELOPMENT OF A NEW FAMILY OF NANOCARRIERS DERIVED FROM NATURAL TETRAMERIC ACID LIPIDS

Abstract

The present invention thus provides a novel family of liposomal compositions (nanocarriers), hereinafter referred to as Tetra-Acidosomes, comprising natural tetrameric acid (TA) lipids and/or novel chemically functionalized tetrameric acid (CFTA) lipids, for drug delivery applications.

Claims

1. Lipid compound having the following general formula (II): ##STR00014## wherein R.sup.A, R.sup.B, R.sup.C and R.sup.D independently represent a linear or branched, aliphatic or alicyclic, saturated, hydrocarbon group comprising from 2 to 8 carbon atoms, preferably R.sup.A, R.sup.B, R.sup.C and R.sup.D independently represent ##STR00015## in which is the point of attachment to either P.sup.1, P.sup.2, P.sup.3, and P.sup.4 or the main molecule; P.sup.1, P.sup.2, P.sup.3, and P.sup.4 are the same or different, and each represents one of the following substituents: CH.sub.2OH; COOH; COOR.sup.1, with R.sup.1 which represents an aliphatic linear or branched, saturated or unsaturated alkyl chain with a number of carbon atoms between 1 to 22, especially 4 carbon atoms; A.sup.1-CH.sub.2CH.sub.2N.sup.+(CH.sub.3).sub.3, X.sub.2, X.sub.2 representing a halogen or a sulfonate, A.sup.1 representing an ester (OC(O)) bond; A.sup.2-(PEG.sub.X1-A.sup.3).sub.n-R.sup.2, n being equal to 0 or 1, PEG.sub.x1, being a polyethylene glycol of molecular weight X.sub.1, X.sub.1 being less than or equal to 5000 daltons, A.sup.2 and A.sup.3 possibly being identical or different and representing an ester (C(O) O), an amide (C(O) NH), a triazole, R.sup.2 representing a methoxy group, a targeting agent or a probe; X and Y are independently H or CH.sub.3; provided that P.sup.1, P.sup.2, P.sup.3, and P.sup.4 are not all simultaneously COOH.

2. Lipid compound according to claim 1, wherein P.sup.1, P.sup.2, P.sup.3, and P.sup.4 are the same or different, and each represents one of the following substituents: A.sup.1-CH.sub.2CH.sub.2N.sup.+(CH.sub.3).sub.3, X.sub.2, X.sub.2 representing a halogen or a sulfonate, A.sup.1 representing an ester (OC(O)) bond; A.sup.2-(PEG.sub.x1-A.sup.3).sub.n-R.sup.2, n being equal to 0 or 1, PEG.sub.x1, being a polyethylene glycol of molecular weight X.sub.1, X.sub.1 being less than or equal to 5000 daltons, A.sup.2 and A.sup.3 possibly being identical or different and representing an ester (C(O) O), an amide (C(O) NH), a triazole, R.sup.2 representing a methoxy group, a targeting agent or a probe.

3. Lipid compound according to claim 1 having the following general formula (IIa), (IIb), (IIc), (IId), or (IIe): ##STR00016## wherein X, Y, P.sup.1, P.sup.2, P.sup.3, and P.sup.4 are as defined in claim 1.

4. Tetra-acidosome comprising a lipid compound having the following general formula (II): ##STR00017## wherein R.sup.A, R.sup.B, R.sup.C and R.sup.D independently represent a linear or branched, aliphatic or alicyclic, saturated, hydrocarbon group comprising from 2 to 8 carbon atoms, preferably R.sup.A, R.sup.B, R.sup.C and R.sup.D independently represent ##STR00018## in which is the point of attachment to either P.sup.1, P.sup.2, P.sup.3, and P.sup.4 or the main molecule; P.sup.1, P.sup.2, P.sup.3, and P.sup.4 are the same or different, and each represents one of the following substituents: CH.sub.2OH; COOH; COOR.sup.1, with R.sup.1 which represents an aliphatic linear or branched, saturated or unsaturated alkyl chain with a number of carbon atoms between 1 to 22, especially 4 carbon atoms; A.sup.1-CH.sub.2CH.sub.2N.sup.+(CH.sub.3).sub.3, X.sub.2; X.sub.2 representing a halogen or a sulfonate, A.sup.1 representing an ester (OC(O)) bond; A.sup.2-(PEG.sub.x1-A.sup.3).sub.n-R.sup.2, n being equal to 0 or 1, PEG.sub.x1, being a polyethylene glycol of molecular weight X.sub.1, X.sub.1 being less than or equal to 5000 daltons, A.sup.2 and A.sup.3 possibly being identical or different and representing an ester (C(O) O), an amide (C(O) NH), a triazole, R.sup.2 representing a methoxy group, a targeting agent or a probe; X and Y are independently H or CH.sub.3.

5. Tetra-acidosome according to claim 4, comprising a lipid compound having the following general formula (II) and wherein P.sup.1, P.sup.2, P.sup.3, and P.sup.4 are the same or different, and each represents one of the following substituents: A.sup.1-CH.sub.2CH.sub.2N.sup.+(CH.sub.3).sub.3, X.sub.2, X.sub.2 representing a halogen or a sulfonate, A.sup.1 representing an ester (OC(O)) bond; A.sup.2-(PEG.sub.x1-A.sup.3).sub.n-R.sup.2, n being equal to 0 or 1, PEG.sub.x1, being a polyethylene glycol of molecular weight X.sub.1, X.sub.1 being less than or equal to 5000 daltons, A.sup.2 and A.sup.3 possibly being identical or different and representing an ester (C(O) O), an amide (C(O) NH), a triazole, R.sup.2 representing a methoxy group, a targeting agent or a probe.

6. Tetra-acidosome according to claim 4, having an average size <250 nm.

7. Tetra-acidosome according to claim 4, having a polydispersity index (PDI)<0.3.

8. Tetra-acidosome according to claim 4, further comprising an encapsulated molecule of interest.

9. Use of a Tetra-acidosome according claim 4, as a carrier.

10. Use of a Tetra-acidosome according to claim 9, as a carrier to the gastrointestinal environment (GI).

11. Tetra-acidosome according to claim 4, for use as a drug.

12. Method for the synthesis of a lipid compound as defined in claim 1 comprising a step of reacting a tetrameric acid (TA) lipid with a reducing agent, an alcohol, an amine or an ester.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0067] FIG. 1 represents (FIG. 1A) an Archaeosome (encircled tetraether lipid) (FIG. 1B) the detailed structure of a tetraether lipid present in Sulfolobus acidocaldarius.

[0068] FIG. 2 represents (FIG. 2A) a simplified representation of the most abundant TA lipids with P standing for polar head (for TA lipids P.sup.1P.sup.2P.sup.3P.sup.4COOH) (FIG. 2B) a suggested structure of C.sub.81 or C.sub.82 TA lipids. The two dotted lines represent the two methyl groups that can be present to explain one or two carbons in addition to the 80 initial carbons.

[0069] FIG. 3 represents the structure of the partially esterified TA lipid (P.sup.1: P.sup.2: P.sup.3: p.sup.4COOH OR COOR.sup.1) of example 2. Carbons C.sub.17 and C.sub.17 (circle)

[0070] FIG. 4 represents (FIG. 4A) a Tetra-acidosome of the present invention with natural TA lipids (encircled TA lipid) (FIG. 4B) the detailed structure of the main TA lipids present in large quantities in petroleum calcium naphthenate deposits (=industrial waste product).

[0071] FIG. 5 represents the stability of Tetra-acidosomes of the present invention in terms of size.

[0072] FIG. 6 represents the stability of Tetra-acidosomes of the present invention in terms of PDI (PolyDispersity Index).

[0073] FIG. 7 represents PEGylation of TA lipids: asymmetrical introduction to prevent the chains from potentially filling the hydrophilic heart of Tetra-acidosomes.

[0074] FIG. 8 represents the structure of a peptide loaded Tetra-acidosome composed of TA (with different polar heads), fluorescent labelled TA (Nile Red TA) and PEGylated TA lipids.

EXAMPLES

Material and Methods

[0075] General: .sup.1H NMR spectra were recorded on a Bruker Avance III 400 spectrometer operating at 400.13 MHz for, equipped with a BBFO probe with a Z-gradient coil and a GREAT 1/10 gradient unit. All experiments were carried out at 25 C.

[0076] The noesygppr1d Bruker pulse program was used for 1D NMR with presaturation using the following parameters: TD of 64 k, a relaxation delay d1=2s and 16scans. The spectrum width was set to 18 ppm. Fourier transform of the acquired FID was performed without any apodization in most of the case.

[0077] .sup.13C NMR spectra were recorded at 100.61 MHz. Several sequences as jmod, dept135 or zgpg30 were usually used with 64 to 1024 scans depending on the concentration of the sample. TD is 64 k and a relaxation delay of 2s for a spectral width of 220 ppm was used. Fourier transform was performed after apodization with an exponential function using a LB of 0.6 Hz.

[0078] Concerning, mass spectrometry analyses, SI+, ESI and MALDI ionisation were used.

[0079] All the reagents were brought from known providers like Merck, Fischer and Alfa Aesar and were used without purification.

General Procedures for Formulation:

[0080] Procedure A: 4 mg.Math.mL.sup.1 stock solutions of EggPC, cholesterol and lipids were prepared in MeOH (HPLC grade) and CHCl.sub.3 (HPLC grade) (depending on solubility). The solutions were placed under ultrasounds for 5 mins to be certain that all lipids were well dissolved. The formulations were then prepared in 1 mL depending on the chosen mass %. Operatory conditions for NanoAssemblr were selected in a software.

Example

[0081] Volume: 3.3 mL [0082] FRR: 3:11 [0083] TFR: 8 mL.Math.min.sup.1 [0084] Left syringe: aqueous phase-3 ml syringe (dispense: 2.47 mL) [0085] Right syringe: organic phase-1 ml syringe (dispense: 0.83 mL) [0086] Star waste volume: 0.350 mL [0087] End waste volume: 0.050 mL

[0088] The aqueous phase is Dulbecco's Phosphate-Buffered Saline (DPBS). The organic phase was extracted with rotavapor: 21000 L of the liposomal solution were placed in a 10 mL flask, itself placed in 40 C. bath; Pressure was set at 300 mbar during 10 min, then it was lowered by 20 mbar every 20 s until 100 mbar was reached, and it was maintained at 100 mbar 5 additional minutes. The formulation was recovered with a syringe and was sterilized with Acrodisc, then the mass was adjusted to 2 g with DPBS. Theoretical final concentration was 1 mg.Math.L.sup.1. Each formulation was analysed by DLS with Zetasizer Malvern ZS90: 3 measures by sample corresponding to 10 runs of 20 s with 90 s of equilibration at the beginning. Temperature was set at 25 C.

[0089] Procedure B: 4 mg.Math.mL.sup.1 stock solutions of EggPC, cholesterol and lipids were prepared in EtOH (HPLC grade). The solutions were placed under ultrasounds for 5 mins to be certain that all lipids were well dissolved. The formulations were then prepared in 1 mL depending on the chosen molar %. Operatory conditions for NanoAssemblr were selected in a software.

Example

[0090] Volume: 2.4 mL [0091] FRR: 3:11 [0092] TFR: 12 mL.Math.min.sup.1 [0093] Left syringe: aqueous phase-3 ml syringe (dispense: 1.8 mL) [0094] Right syringe: organic phase-1 ml syringe (dispense: 0.6 mL) [0095] Star waste volume: 0.350 mL [0096] End waste volume: 0.050 mL

[0097] The aqueous phase is Dulbecco's Phosphate-Buffered Saline (DPBS). The organic phase (EtOH) was removed by dialysis: 1000 L of the liposomal solution were placed in a 1 mL hydrated dialysis tube (Spectrum Laboratories G235035, cut-off=100 kDa) and the filled tube was then placed in 250 mL of milliQ water with magnetic stirring at 350 rpm. The milliQ water bath was changed after one hour. 500 L of the liposomal solution inside the dialysis tube was analysed by DLS with Zetasizer Malvern ZS90: 3 measures by sample corresponding to 10 runs of 20 s with 90 s of equilibration at the beginning. Temperature was set at 25 C.

Example 1: Synthesis of Tetraol Lipid (P.SUP.1.P.SUP.2.=p.SUP.3.=P.SUP.4.CH.SUB.2.OH)

##STR00007##

[0098] Scheme 1: Synthesis of tetraol lipid derived from TA lipid

[0099] LiAIH.sub.4 (19.5 mg, 5.13.10-4 mol, 6 equiv.) used as reducing agent was dissolved in 8 mL of anhydrous THF. TA lipid (95.8 mg, 7.78.10-5 mol, 1 equiv) dissolved in 4 mL of anhydrous THF were added dropwise under inert atmosphere. The resulting mixture was then stirred at reflux for 5 h. Excess LiAlH.sub.4 was destroyed by addition of 6 mL of diethyl ether then 3 ml of water. The aqueous phase was extracted with diethyl ether, then the gathered organic phases were washed with sulfuric acid 5% in water and water. After drying with MgSO.sub.4, the solvent was removed under reduced pressure to obtain a yellow oil (90.2 mg, 99% yield). .sup.1H NMR in CDCl.sub.3: 0=2.3-1.5 (m), 1.4-1.0 (m), 0.91 (d, J=6.5 Hz), 0.89-0.83 (m), .sup.13C NMR in CDCl.sub.3: 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.

[0100] Complete reduction of the four carboxylic acids into primary alcohol functions was fulfilled in a quasi-quantitative yield. 1D NMR spectra proved that the tetraol was obtained as the signals of the carboxyl acid carbons are not present anymore and two signals corresponding to the hydroxyl functions appeared at 61.23 ppm and 62.43 ppm on the 1D 13C spectrum. Also, a general shielding of the aliphatic protons is observed on the 1D 1H spectrum, especially the doublet at 0.91 ppm which was at 0.98 ppm for the initial TA lipid. The synthesis was also confirmed by high resolution mass spectrometry since the peak for m/z 1175 was found.

Example 2: Synthesis of Partially Esterified Ta Lipid (at Least One P.SUP.1.: P.SUP.2.: p.SUP.3.: P.SUP.4.COOR.SUP.1.)

##STR00008##

[0101] Scheme 2: Selective esterification of a TA lipid using an acidic resin

[0102] The selective esterification of a TA lipid was performed through the use of an acidic resin. The TA lipid was dissolved in a large excess of a mixture of octane and butyl formate, and a reaction of transesterification occurred at the interface of a strongly acidic ion-exchange resin which is Dowex 50W-X.sub.2 (50-100 mesh). The explanation for the selectivity was based on the great solubility of tetracarboxylic acid (TA) lipids in butyl formate compared to octane. The transesterification occurred at the resin and butyl formate interface due to the acidic aqueous characteristic of the resin. Then, the resulting partially esterified TA lipid, which is far more soluble in octane, has not been as reactive and its interaction with the resin was reduced. Several experiments were carried out by changing reaction time or solvent ratio. The results are shown in Table 1 below with estimated total esterification rates and esterification rates of the right and left sides calculated depending on the time of reaction or solvent ratio.

[0103] Three signals in the .sup.1H NMR spectrum allowed the calculation of both the global esterification rate and the esterification rate of the right side of the lipid. The doublet found at 0.94 ppm and the doublet at 0.98 ppm correspond to the CH.sub.3 of carbons C.sub.17 and C.sub.17 (FIG. 3). If the polar head is a carboxylic acid, the doublet is found at 0.98 ppm whereas if it is a butyl ester, the doublet is found at 0.94 ppm. However, a triplet corresponding to the terminal CH.sub.3 of the butyl ester was found at 0.95 ppm and was superimposed with the doublet at 0.94 ppm. With a line fitting of the region between 1.01 and 0.92 ppm, it was possible to integrate separately the three signals.

[0104] There is a mathematical relationship between the integration of these three signals. As each C.sub.17 and C.sub.17 (FIG. 3) always correspond to CH.sub.3, the sum of the integration of the two doublets is always equal to 6. As for the triplet, its integration can range between 0 and 12 protons since a lipid can possess between 0 and 4 butyl ester functions, i.e. between 0 and 4 terminal CH.sub.3 of butyl esters. With a cross product, it is possible to determine the integration of the triplet and compare it with the theoretical maximum, which is 12, i.e. global esterification rate=(integration.sub.triplet0.95 ppm/12)*100. The esterification rate of only the right side of the molecule is given by (integration.sub.doublet0.94 ppm/6)*100.

TABLE-US-00001 TABLE 1 Estimated Deduced Solvent Reaction Estimated esterification esterification ratio time esterification on the right on the left Entry (n.sub.1/n.sub.2) (h) rate (%) side (%) side (%) 1 90/10 2 3 0 6 2 90/10 6 8 0 16 3 90/10 14 11 3 19 4 90/10 24 12 4 22 5 90/10 72 61 20 100 6* 50/50 24 57 14 100 7 50/50 120 84 73 100 *After silica gel column chromatography, a fraction was isolated with a total esterification rate of 51%, an esterification rate of 5% at the right side and an esterification rate of 97% at the left side. n.sub.1 = octane; n.sub.2 = butyl formate.

[0105] Entries 1-5 in Table 1 gave information on the reaction time needed to transform the carboxylic acids into butyl esters with a 90/10 solvent ratio. Within 24 h, the conversion was limited as only 13% was reached. It was still possible to get satisfactory conversion rate since 61% was reached after 3 days (entry 5). Then, the effect of solvent ratio was important. The esterification rate has been multiplied by 4.5 for the same reaction time, by increasing the proportion of butyl formate (entry 4 and entry 6). It was possible to almost convert the carboxylic acids completely into butyl esters after a longer reaction time and with enough butyl formate (entry 7). In addition, the esterification was selective for all the conditions that were tested (entries 1-7). Indeed, the esterification rate for the right side of the molecule increased slowly compared to the global esterification. Therefore, the conditions of this reaction have been optimised to obtain TA lipids with the number of ester functions controlled, with the left side of the lipids more substituted than the right side. Indeed, the more the reaction time increased, 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 appeared to be more reactive for this reaction. It may be due to the geometry of approach of the lipids towards the resin which could be more favourable for one side of the molecule.

[0106] In conclusion, a 50/50 solvent ratio and a reaction time of 24 h (entry 6) were the most appropriate conditions for an asymmetrical esterification of TA lipids since the esterification rate at the right side was low (14%) compared to the left side (100%). Furthermore, the removal of the tetraester compound (Fraction 1) by column chromatography allowed to isolate partially esterified TA lipids (Fraction 2) with total esterification rate of 51%, an esterification rate of 5% at the right side and an esterification rate of 97% at the left side (see below).

Representative Reaction for Partial Esterification of TA Lipid:

[0107] TA lipids (107 mg, 0.086 mmol, 1 equiv.) were 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 eliminated by filtration. The solvent was removed under reduced pressure to obtain a yellow oil (114 mg). Two fractions were isolated after flash chromatography (CH.sub.2Cl.sub.2/MeOH: 100/0 to 90/10). Fraction 1 (10 mg) was identified as tetrabutylester (P.sup.1P.sup.2P.sup.3P.sup.4CO.sub.2Bu), fraction 2 (83 mg, 72%) was composed of partially esterified TA lipids (total esterification rate: 51%; esterification rate of 5% at the right side and esterification rate of 97% at the left side). .sup.1H NMR in CDCl.sub.3: 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=6.7 Hz), 2.4-1.9 (14H, m), 1.9-1.5 (20H, m), 1.4-1.0 (80H, m), 0.98 (6H, d, J=7.0 Hz), 0.95 (6H, t, J=7.1 Hz), 0.9-0.7 (20H, m).

Example 3: Total or Partial Pegylation of a Ta Lipid (at Least One P.SUP.1.: p.SUP.2.: p.SUP.3.: p.SUP.4.=A.SUP.2.-(PEG.SUB.x1.-A.SUP.3.) n-R.SUP.2 .OR COOR.SUP.1.)

[0108] The goal here was to introduce PEG chains on the lipids to give mucus-inert properties to Tetra-acidosomes. The strategy of PEGylation was based on peptide coupling reactions between the carboxylic acids of the lipid and the amine of the PEG. TBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate) and DIPEA (N-N-diisopropylethylamine) were selected since they are known for their efficiency in coupling reactions with other lipids [19]. The use of a uronium salt like TBTU allowed by itself the formation of HOBt (1-Hydroxybenzotriazole hydrate) while DIPEA ensured that the carboxylic acids were deprotonated. Also, to avoid the formation of secondary products, the amine was added after at least 20 min of stirring of the TA lipid with TBTU and DIPEA so that the carboxylate functions were completely activated as esters of HOBt.

Synthesis of tetrazidoPEGylated (N.sub.3PEG.sub.500) 4-TA Lipids Using N.sub.3-PEG.sub.500-NH.sub.2

[0109] The first coupling reaction was performed between N.sub.3-PEG.sub.500-NH.sub.2 and the initial TA lipid to obtain tetrazidoPEGylated (N.sub.3PEG.sub.500) 4-TA lipid (scheme 3) that would serve in the copper catalysed cycloaddition described in the next part related to the functionalization of PEG chains (scheme 5).

##STR00009##

[0110] Scheme 3: Synthesis of the tetrazidoPEGytated (N.sub.3PEG) 4-TA lipid with N.sub.3-PEG.sub.500-NH.sub.2 TBTU (75 mg, 0.23 mmol, 4.8 equiv.), DIPEA (4.8 equiv.) and TA lipids (60 mg, 0.049 mmol, 1 equiv.) were dissolved in 9 ml of anhydrous CH.sub.2Cl.sub.2 under inert atmosphere. DIPEA (30 mg, 0.23 mmol, 4.8 equiv) was added in the mixture. After 20 minutes, N.sub.3-PEG.sub.500-NH.sub.2 (102 mg, 0.23 mmol, 4.8 equiv.) dissolved in 3 mL of anhydrous CH.sub.2Cl.sub.2 was added, and the mixture was brought to reflux for 48h. Then, DIPEA was neutralized by adding some drops of 4% HCl solution. The organic phase was washed with distilled water (32 mL). The organic phase was dried with MgSO.sub.4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography (CH.sub.2Cl.sub.2/MeOH: 100/0 to 90/0) to obtain a tetrazidoPEGylated (N.sub.3PEG.sub.500) 4-TA lipid as a yellow oil in excellent yield (92%). .sup.1H NMR in CDCl.sub.3: 0=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=6.2 Hz), 0.78 (16H, m). The .sup.1H NMR spectrum confirmed the structure as integrations are correct, and a shielding resulting from the formation of amides is observed and is more important for protons located near these amides.

Representative PEGylation of a Partially Esterified TA Lipid Using MeO-PEG.sub.2000-NH.sub.2

[0111] In addition, partially esterified TA lipids (46%) with known esterification rate were then engaged in the coupling reaction but with MeO-PEG.sub.2000-NH.sub.2.

##STR00010##

[0112] Scheme 4: Synthesis of the Partially esterfied TA with MeO-PEGn-NH.sub.2.

[0113] Partially esterified TA lipids (see Fraction 2 of Example 2) (10 mg, 0.008 mmol, 1 equiv.), TBTU (6.2 mg, 0.019 mmol, 2.4 equiv: 1.2 equiv. for each equiv. of carboxylic acids) were dissolved in 2 mL of anhydrous CH.sub.2Cl.sub.2 under inert atmosphere. DIPEA (6.7 mg, 0.019 mmol, 2.4 equiv: 1.2 equiv. for each equiv. of carboxylic acids) was added in the mixture. After 20 minutes, MeO-PEG.sub.2000-NH.sub.2 (38 mg, 0.019 mmol, 2.4 equiv: 1.2 equiv. for each equiv. of carboxylic acids) dissolved in 5 mL of anhydrous CH.sub.2Cl.sub.2 was added and the mixture was brought to reflux for 48 h. Then, DIPEA was neutralized by adding some drops of 4% HCl solution. The organic phase was washed with distilled water (32 mL). The organic phase was dried with MgSO.sub.4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography (CH.sub.2Cl.sub.2/MeOH: 100/0 to 90/0) to obtain a yellow oil (42 mg). .sup.1H NMR in CDCl.sub.3: 0=3.8-3.2 (HPEG), 2.4-1.9 (m), 1.9-1.4 (m), 1.4-0.95, 0.95-0.70 (m). NMR spectra proved that the remaining carboxylic acids of the TA lipids are quasi quantitively PEGylated. The initial goal of controlling where the PEG chains are grafted to the TA lipids could then be achieved in excellent yield with prior selective protection of the right carboxylic acids (FIG. 7).

[0114] Thereafter, to confirm that previous selective esterification of the TA lipids is necessary to obtain diPEGylated lipid with PEG chains on the same side of the molecule, a coupling reaction with (non-protected) initial TA lipids was performed. TBTU (2 equiv.), DIPEA (2 equiv.) and TA lipids (1 equiv.) were added in anhydrous CH.sub.2Cl.sub.2, before adding MeO-PEG.sub.2000-NH.sub.2 (2 equiv.). We got diPEGylated TA lipids in a quasi-quantitative yield but integrations on the .sup.1H NMR spectrum showed that the PEG chains were statistically attached to the right or left part of the lipids. Thus the way to control where the PEG chains will be attached is to protect selectively the carboxylic acids beforehand, as previously described.

Example 4: Functionalization of the Pegylated Ta Lipids (P.SUP.1.: P.SUP.2.: p.SUP.3.: P.SUP.4.=A.SUP.2.-(PEG.SUB.x1.-A.SUP.3.) n-R.SUP.2 .OR COOR.SUP.1.)

[0115] The introduction of a specific function (targeting agent, probe) at the terminal ends of the PEG chains could be achieved through esterification reactions, peptidic coupling reactions or click chemistry reactions (for example using Copper catalysed Huisgen cycloaddition). As an example, the introduction of a probe at the four terminal ends of the tetrazidoPEGylated (N.sub.3PEG.sub.500) 4-TA lipids was performed for biopharmaceutical characterizations. Copper catalysed Huisgen cycloaddition is now popular to easily add probes or specific molecules to lipids for example. However, it is fundamental to find the right conditions, i.e. right solvent and catalyst, as yields can easily drop. Classically, copper sulphate with ascorbic acid as additive are used in polar solvents like water or alcohols.

[0116] Given the aliphatic structure of TA lipids, it looked relevant to try the reaction with other conditions.

##STR00011##

[0117] Scheme 5: Synthesis of the Nile Red labelled tetraPEGylated (Nile Red-PEG.sub.500) 4-TA lipids by click reaction

[0118] TetrazidoPEGylated (N.sub.3PEG.sub.500) 4-TA lipids (50 mg, 61.9 mmol, 1 equiv.) and 9-diethylamino-2-(prop-2-ynyloxy)-5H-benzo[a]phenoxazine-5-one (Nile Red probe: 23 mg, 61.9 mmol, 4equiv.) were dissolved in a mixture of 9 mL of two organic solvents: anhydrous toluene and anhydrous methanol (MeOH) in a 2/1 ratio (scheme 5). The catalyst copper iodide (Cul) which is directly at an oxidation degree of I (13.6 mg, 40% mol) was added, followed by DIPEA, and the reaction mixture was stirred at 60 C. overnight. The mixture was cooled and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (CH.sub.2Cl.sub.2/MeOH: 100/0 to 90/0) to obtain a dark purple oil (57 mg, 78%) corresponding to the tetra-Nile-Red functionalized (Nile Red-PEG.sub.500) 4-TA lipid. 1H NMR in CDCl.sub.3: 0=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 triazoles was evidenced in .sup.1H NMR by the presence of a singlet at 7.9 ppm corresponding to 4 protons, and the loss of the signal at 2.58 ppm of the alkynes.

Example 5: Functionalization of the Ta Lipids with Cationic Moieties or with Butanol

Functionalization of the TA Lipids with Choline (P.sup.1: P.sup.2: P.sup.3: P.sup.4=A.sup.1-CH.sub.2CH.sub.2N.sup.+(CH.sub.3).sub.3, X with XCl)

[0119] Esterification of the TA lipids was achieved in quasi quantitative yield using MSA as catalyst in a two-step reaction.

##STR00012##

[0120] Scheme 6: Synthesis of tetracholine ester lipids derived from TA lipids

[0121] Firstly, choline chloride (27 mg, 0.19 mmol, 4.8 equiv.) was put in a flask with a Dean-Stark 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 counter ion of the choline became mesylate while HCl was released.

[0122] The second step was the esterification using the hydroxyle function of choline in acidic conditions and at a temperature of 130 C. TA lipids 1 (50 mg, 0.040 mmol, 1 equiv.) dissolved in anhydrous toluene was added and the temperature was set at 130 C. The mixture was stirred overnight then the solvent was removed under reduced pressure. 90 mg (112%) of the crude product was obtained. .sup.1H NMR in CDCl.sub.3/CD.sub.3OD (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=0.93 Hz), 0.9-0.6 (16H, m). 1D .sup.1H NMR spectrum confirmed that the expected tetraester was obtained as the signal of the alcohol disappears and the formation of the ester function caused a change of chemical shifts for the protons of TA lipids.

Functionalization of the TA Lipids with Butanol (P.sup.1: P.sup.2: P.sup.3: P.sup.4CO.sub.2(CH.sub.2).sub.3CH.sub.3)

##STR00013##

[0123] Scheme 7 Synthesis of tetrabutyl ester lipids derived from TA lipids

[0124] Fisher esterification was envisaged to synthesize tetraester. TA lipids (26.3 mg, 2.14.10-5 mol, 1 equiv.) was dissolved in 5 mL of n-butanol (nBuOH) used both as nucleophilic ragent and solvent. Methane sulfonic acid (MSA, 125 L, 0.1 equiv.) used as acidic catalyst was added, then the mixture was stirred at 140 C. in a flask with Dean-Stark apparatus to eliminate water formed during the reaction. The pressure was set at 500 mbar. After 7 h, the solvent was removed under reduced pressure to obtain a yellow oil. .sup.1H NMR in CDCl.sub.3: 0=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), .sup.13C NMR in CDCl.sub.3: 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. Tetraester was isolated in a 90% yield. NMR experiments confirmed that all carboxylic acids were transformed into butyl esters.

Example 6: Formulation of Natural Tetra-Acids (Ta) Lipids or Chemically Functionalized Tetra-Acids (Cfta) Lipids into Stable Liposomes Called Tetra-Acidosomes

[0125] The classic method to formulate liposomes is to form a lipid film by solvent evaporation which is then hydrated with an aqueous solution and sonicated. This method can work but is not the most convenient and reproducible one. An interesting alternative exists which is microfluidics. Development of new continuous processes to elaborate matrix particles is burgeoning. Microfluidics is the science which studies the manipulation of small volumes (from 10-9 to 10-18 L) in microscopic canals (from 10 to 1000 m). Microfluidics is a very efficient tool to formulate liposomes and it is performed in the examples given hereafter thanks to an equipment called NanoAssemblr Benchtop. It offers reproducibility, tuning of particle sizes, scalability, and speed. All the results presented below were obtained using this platform. Ideally, liposomes with diameters lower than 200 nm and with Polydispersity Index (PDI) lower than 0.3 are required to consider that the formulations are suitable for encapsulation of molecules. These two parameters are provided by Dynamic Light Scattering (DLS). The PDI is a dimensionless measure of the broadness of the particle size distribution, and it ranges from 0 to 1. If its value exceeds 1, it may be that the sample is not suitable for measurement by DLS.

Formulations Based on Natural TA Lipids (Procedure A)

[0126] Several formulations were tested using TA or PEGylated TA, EggPC and cholesterol with well-defined mass percentages and some of them are presented in Table 2.

TABLE-US-00002 TABLE 2 Composition of the different organic solutions prepared for formulations of Tetra-Acidosomes mass % of mass % of Formulation name EggPC mass % of TA cholesterol A 67 3 30 B 63 7 30 C 60 10 30 D 85 15 0

[0127] Cholesterol can be associated with the two other lipids to modulate the rigidity of the layer. The organic phase was constituted of MeOH as TA lipids were soluble enough to get stock solutions of 4 mg.Math.L.sup.1. Once the formulations were produced, the organic phase was evaporated, and the liposomal solutions was diluted to get precisely two grams of solution of theorical concentration of 1 mg.Math.mL.sup.1. They were then analysed by DLS to get the Z-average in nm and the PDI (Table 3).

TABLE-US-00003 TABLE 3 Z-average (nm) and PDI measured by DLS for the Tetra-Acidosomes formulations with natural TA lipids at J and J + 21 days Formulation name A B C D Average size (nm) - J 169.5 102.9 67.6 58.7 Average size (nm) - J + 21 177.0 156.2 75.4 63.6 Average PDI - J 0.042 0.059 0.047 0.05 Average PDI - J + 21 0.046 0.095 0.062 0.034

[0128] Firstly, liposomal solutions made from TA lipids associated with EggPC and/or cholesterol were obtained with hydrodynamic diameters lower than 200 nm. Moreover, the PDI for each formulation is excellent as it is always inferior to 0.1, which means that we were able to formulate monodisperse lipidic vesicles. Secondly, it was observed that the more the concentration of TA lipid increased, the more the hydrodynamic diameter decreased. Then, the diameter of the liposomes can be controlled by varying the mass percentage of TA lipids. More interestingly, these formulations were very stable. Indeed, after twenty-one days, the average diameters and PDIs of the formulations remained almost unchanged.

[0129] FIGS. 4A-4B illustrate the structure of a Tetra-acidosome of the present invention with natural TA lipids.

[0130] FIGS. 5 and 6 illustrate the stability of Tetra-acidosomes of the present invention with natural TA lipids in terms of size and PDI, respectively.

Formulations Based on Tetraol Lipids (Procedure A)

[0131] Four formulations were tested with tetraol with the same mass percentages as solutions A, B, C and D. Some differences that could be noted from TA lipids-containing liposomes. The tetraol lipids are not soluble in MeOH so the organic phase was constituted of a mixture of MeOH and CHCl.sub.3 1/1. A turbidity appeared when the mass percentage of tetraol was equal or superior to 10 wt. %. The four formulations were also analysed by DLS (Table 4).

TABLE-US-00004 TABLE 4 Z-average (nm) and PDI measured by DLS for the formulations of Tetra-acidosomes with tetraol 2. Formulation name A B C D Average size (nm) 153.1 104.4 149.2 130.8 Average PDI 0.067 0.085 0.076 0.172

[0132] Some interesting formulations were obtained with diameters lower than 200 nm and good PDIs. However, there are some differences to mention. The hydrodynamic diameter did not decrease anymore as the mass percentage of tetraol increased. Cholesterol-containing liposomes showed excellent PDIs, but the formulation D, which only contained tetraol and EggPC, has a PDI value higher than 0.1, even though it can still be considered acceptable as it is lower than 0.3. Also, liposomes with diameters lower than 100 nm were not obtained.

Comparative Formulations Based on Natural TA Lipids or Partially PEGylated TA Lipids (Procedure B)

[0133] Several formulations were tested using TA lipids or partially PEGylated TA lipids, EggPC and cholesterol with well-defined molar percentages and two of them are presented in Table 5.

TABLE-US-00005 TABLE 5 Composition of the different organic solutions prepared for formulations of TA-based (formulation E) or Partially PEGylated TA-based (formulation F) Tetra-Acidosomes molar % of molar % of molar % of Partially molar % of Formulation EggPC TA PEGylated TA cholesterol name (mass %) (mass %) (mass %) (mass %) E 50 5 0 45 (62) (10) (0) (28) F 50 0 5 45 (47) (0) (32) (21)

[0134] The organic phase was constituted of EtOH instead of MeOH as for formulations A-D in order to avoid potential toxicity problems induced by residual MeOH after the administration of formulations. Stock solutions of 4 mg.Math.L.sup.1 were prepared. Once the formulations were produced, the organic phase was removed by dialysis, and the liposomal solutions extracted from the dialysis. Tube (500 L) were then analysed by DLS to get the Z-average in nm and the PDI (Table 6).

TABLE-US-00006 TABLE 6 Z-average (nm) and PDI measured by DLS for the Tetra- Acidosomes formulations with natural TA lipids or partially PEGylated TA lipids at J and J + 21 days Formulation name E F Average size (nm) - J 65.31 39.93 Average size (nm) - J + 21 n.d. 44.21 Average PDI - J 0.172 0.162 Average PDI - J + 21 n.d. 0.127 (n.d. = non-determined)

[0135] Firstly, liposomal solutions made from both TA lipids or partially PEGylated TA lipids associated with EggPC and/or cholesterol were obtained with hydrodynamic diameters lower than 100 nm. Moreover, the PDI for each formulation is good as it is always inferior to 0.2, which means that we were able to formulate monodisperse lipidic vesicles. Secondly, it was observed that the use of partially PEGylated TA lipids instead of natural TA lipids promoted the formation of even smaller vesicles, which should facilitate the liposome escape from the blood capillaries as well as the liposomal uptake into the targeted tissue [21]. More interestingly, partially PEGylated TA-based formulation was very stable. Indeed, after twenty-one days, the average diameter and PDI of the formulation remained almost unchanged.

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