IONIZABLE CATIONIC LIPID AND ITS USE IN DELIVERING THERAPEUTIC MOLECULES

Abstract

The invention relates to the therapeutic field, particularly the delivery of therapeutic molecules, and more specifically vaccines, but also to the field of medical imaging, and in particular fluorescent markers used in infrared, microscopy, CT, or MRI. The present invention relates to a cationic lipid of formula I, to a lipid nanoparticle or liposome comprising it, and to a pharmaceutical composition comprising them. The invention further relates to the pharmaceutical composition for its use as a medicinal product, preferably in the delivery of a negatively charged therapeutic molecule. [Chem 21] Formula I No figure in the abstract

Description

Title of the invention: IONIZABLE CATIONIC LIPID AND ITS USE IN DELIVERING THERAPEUTIC MOLECULES Technical field

[0001] The invention belongs to the therapeutic field, in particular the delivery of therapeutic molecules, and more particularly vaccines, but also to the field of medical imaging, and in particular fluorescent markers used in infrared, microscopy, CT or MRI.

[0002] The present invention relates to a cationic lipid of formula I, to a lipid nanoparticle or liposome comprising it, and to a pharmaceutical composition comprising them. The invention further relates to the pharmaceutical composition for its use as a medicinal product, preferably in the delivery of a negatively charged therapeutic molecule. State of the art

[0003] Lipid nanoparticles (or LNPs) are ordered mixtures of lipids and negatively charged therapeutic molecules, such as mRNA, produced by microfluidics. The composition of LNPs used for vaccination against SARS-CoV-2 was made public following their clinical use [1].The 3 vaccines (BioNtech / Pfizem, Moderna and Curevac) contain the following four constituents at the same molar percentage: 38.5% cholesterol to stiffen LNPs and increase mRNA encapsulation, 10% DSPC (l,2-distearoyl-sn-glycero-3-phosphocholine) as an auxiliary lipid whose presence induces surface irregularities favorable to fusion with cell membranes, 1.5% of a lipid carrying the hydrophilic polymer PEG (PolyEthyleneGlygol) to decrease LNP aggregation among themselves and stabilize them in physiological fluids and, 50% of an ionizable lipid which is different for each formulation and which is responsible for the complexation of mRNAs and their escape from intracellular vesicles after endocytosis [1,2]. These data demonstrate the crucial nature of the ionizable lipid, which alone allows for the patenting of a specific LNP formulation.

[0004] Although mRNA vaccines against SARS-CoV-2 have demonstrated very high efficacy (>90%), rarely achieved by other vaccine modalities, the immune responses are not of sufficient duration to warrant the need for three doses to date. The LNPs currently used have stability problems at 4°C, which have necessitated their storage at temperatures below -70°C [1]. This problem stability is due in particular to the use of ionizable lipids which contain oxygen atoms capable of forming adducts with mRNA and inactivating it and / or to the hydrolysis of ionizable lipids during their storage.

[0005] There is therefore a need to develop new ionizable cationic lipid molecules that are stable during storage in order to facilitate their handling, while maintaining the same effectiveness.

[0006] Currently, to track the fate of LNPs in a subject's body after injection, it is necessary to add a fluorescent or radiolabeled lipid, or to use radiolabeled mRNA to track LNPs in vivo after injection [3,4]. The addition of fluorescent lipids can alter the formulation, and mRNA labeling relies on numerous chemical steps that are difficult to perform under RNase-free conditions and with low yields [5]. Furthermore, the repeated use of radioactivity as a tracking modality is not feasible due to its toxicity to the patient.

[0007] A method for dual labeling of mRNA for tracking after administration by PET-CT and NIR imaging has recently been developed [5]. However, this technique is very expensive and complex, and the mRNA, both radiolabeled and fluorophore-labeled, is very fragile, which prevents the generalization of this approach.

[0008] There is therefore a need to develop new ionizable cationic lipid molecules which, in addition to solving the problems of the state of the art, and in particular the stability problems mentioned above, simultaneously allow to be tracked in the body of a patient, without the need to add tracers or to modify the sequence of the mRNAs used. Detailed description of the invention

[0009] It is to the credit of the applicants that they have proposed a new cationic lipid of formula I: [Chem 1] Formula I in which - R1 independently represents a hydrocarbon chain, linear or branched, saturated or unsaturated, optionally interrupted by an ester function (-C(O)-O- or -OC(O)-) or phosphate (-OP(O)(OH)-O-), and comprising at least 8 carbon atoms, preferably from 18 to 40 carbon atoms, and even more preferably from 12 to 20 carbon atoms, - L represents a trivalent, tetravalent or pentavalent linker, preferably trivalent, comprising from 3 to 18 carbon atoms, at least one heteroatom chosen from nitrogen and oxygen, optionally an aryl or heteroaryl group, and optionally one or more ester functions (-C(O)-O- or -OC(O)-), - Y1 represents a secondary or tertiary amine function or an amide function (-NH-C(O)-, -C(O)-NH-, -NC(O)- or -C(O)-N-), - a represents an integer from 1 to 2, - b represents an integer from 1 to 4, - R2 represents a linear hydrocarbon chain comprising 2 to 5 carbon atoms, - Y2 represents a primary, secondary, tertiary or quaternary amine or amide function (-NH-C(O)- or -C(O)-NH-), - R3 represents independently: °H, ° a C5 Cl alkyl chain, said alkyl chain being optionally substituted by at least one group selected from -OH, -NH2, -COOH, a polyethylene glycol comprising from 1 to 5 units, a group, and a metal chelating group preferably comprising a metallic atom chosen from Gd, Y, La, Ce, Pr, Nd, Sm, Eu, Tb, Dy, Ho, Er, Tm, Yb, Lu, Cu, Zn, Mn, Ga, In and Tl, of 6 or 30 carbon atoms, one or more heteroatoms chosen from N and O and optionally one or more aryls or heteroaryls, ° an imidazole group, substituted or unsubstituted, preferably unsubstituted, and ° a thymine group, substituted or unsubstituted, preferably unsubstituted, or ° two R3 groups together with Y2 form a ring comprising 5 to 7 links, said ring being optionally aromatic, and optionally comprising at least one N or O heteroatom, said ring being optionally substituted by at least one hydrocarbon chain in Cl to C3 optionally substituted by an -OH group, - c represents an integer from 1 to 3, - A1 and A2 each represent an alkoxy group in Cl to C3, preferably methoxy, or are fused and together represent a -NL(Y1)b(R1)a or -NR2Y2(R3)C group.

[0010]

[0011]

[0012]

[0013]

[0014]

[0015] In this application, the symbol » represents the attachment point of the group represented in the complete molecule. Surprisingly, the applicants identified and developed novel ionizable cationic lipids based on an intrinsically fluorescent molecular platform, but one that is smaller than conventional fluorescent probes. These fluorescent lipids integrate well into LNPs while maintaining the same efficacy as existing alternatives and allow LNPs to become part of cells. The use of amides for coupling aliphatic chains significantly reduces their hydrolysis during storage. In existing technologies, groups or molecules with an imaging modality are added to existing formulations. The ionizable cationic lipid according to the invention makes it possible both to complex negatively charged molecules, for example negatively charged nucleic acid due to the presence of phosphate groups (e.g. siRNA, mRNA, miRNA or DNA), the negatively charged therapeutic molecule or negatively charged polymers (e.g. polyacrylic acid (or their copolymers) or polymethacryloyl-glycylglycine), and thus allow it to escape endosomal degradation, but also to carry the tracer enabling imaging. The invention is based on the innovative integration of several functions within a single cationic lipid molecule, thereby simplifying the formulation and delivery of negatively charged therapeutic molecules, while also enabling improved in-cell and in-vivo monitoring. The compounds according to the invention combine the advantages of a fluorescent, positively charged core, possible variations in the hydrophobic portion, and an ionizable polar portion to which a metallic complex for medical imaging can also be added. This synergistically contributes to the encapsulation of molecules, increases the amount of tracer lipid, and thus ensures better in-vivo monitoring while simplifying the formulation and delivery of negatively charged therapeutic molecules. Furthermore, most of the ionizable lipids used are not chiral; they are racemic mixtures, with one enantiomer likely to have greater activity than the other. To avoid the need for enantiomer purification, we will use ionizable lipids with at least one chiral center. Advantageously, the cationic lipid can be chosen from among the cationic lipids of formula I in which the linker is a trivalent, tetravalent or pentavalent linker, comprising from 3 to 18 carbon atoms, at least one heteroatom chosen from nitrogen and oxygen, optionally an aryl or heteroaryl group and optionally one or more ester functions (-C(O)-O- or -OC(O)-). The term "aryl" refers to a group derived from arenes by the removal of a hydrogen atom from a carbon atom in the ring. By analogy, "heteroaryl" refers to a group similarly derived from heteroarenes (according to the IUP AC definition [6]). Preferably, the aryl or heteroaryl group is a heteroaryl, preferably a triazine. Preferably, the linker L may comprise from 3 to 12 carbon atoms. Preferably, the linker L is trivalent.

[0016] Advantageously, the linker L can be chosen from the following formula linkers: [Chem 2]

[0017] Advantageously, the cationic lipid can be chosen from among the cationic lipids of formula I in which the R1 group independently represents a chain A hydrocarbon chain, linear or branched, saturated or unsaturated, optionally interrupted by an ester (-C(O)-O- or -OC(O)-) or phosphate (-OP(O)(OH)-O-) group, and comprising at least 8 carbon atoms. Preferably, the R1 group may comprise from 12 to 40 carbon atoms, and even more preferably from 12 to 20 carbon atoms. The R1 group may be a hydrocarbon chain, linear or branched, saturated or unsaturated, comprising from 12 to 40 carbon atoms, or a group of formula -R5-C(O)-O-R6 in which R5 represents a linear hydrocarbon chain from C3 to C1 and R6 a linear or branched hydrocarbon chain from C8 to C28. The R1 group may be selected from the following formula groups: [Chem 3]

[0018] Advantageously, the cationic lipid according to the invention can be selected from cationic lipids of formula I in which the R2 group represents a linear hydrocarbon chain comprising 2 to 5 carbon atoms. Preferably, R2 represents a C3 alkyl chain.

[0019] Advantageously, the cationic lipid according to the invention can be chosen from among the cationic lipids of formula I in which the R3 group is chosen from: - H, - a Cl-5 alkyl chain, said alkyl chain being optionally substituted by at least one group selected from -OH, -NH2, -COOH, a polyethylene glycol comprising from 1 to 5 units, a group, and a metal chelating group, HAS. »ss-i said metallic chelating group preferably comprising a metallic atom selected from Gd, Y, La, Ce, Pr, Nd, Sm, Eu, Tb, Dy, Ho, Er, Tm, Yb, Lu, Cu, Zn, Mn, Ga, In and Tl, of 6 or 30 carbon atoms, one or more heteroatoms selected from N and O and optionally one or more aryls or heteroaryls, - an imidazole group, substituted or unsubstituted, preferably unsubstituted, and - a thymine group, substituted or unsubstituted, preferably unsubstituted.

[0020] For the purposes of this invention, a "chelating group" is defined as a group that allows a metal to be fixed by at least two coordination bonds (preferably via nitrogen or oxygen atoms). The chelating group may be cyclic or non-cyclic and may bear carboxylic acid functional groups. The metal is in ionic form, coordinated to the rest of the group. The chelating group may be selected from the following chelating groups: [Chem 4]

[0021] Advantageously, two R3 groups can together with Y2 form a ring comprising 5 to 7 links, said ring being optionally aromatic, and optionally comprising at least one N or O heteroatom, said ring being optionally substituted by at least one hydrocarbon chain in Cl to C3 optionally substituted by an -OH group. Thus, the group -Y2(R3)2 can be chosen from among the groups in which Y2 is "N" of the following formula II: [Chem 5] NW- Formula II in which, - R4 independently represents H or an alkyl in Cl to C3 optionally substituted by an -OH group.

[0022] Advantageously, the cationic lipid according to the invention can be selected from cationic lipids of formula I in which Y1 represents a secondary or tertiary amine function or an amide function (-NH-C(O)-, -C(O)-NH-, -NC(O)- or -C(O)-N-). Preferably, Y1 represents an amide function (-NH-C(O)- or -C(O)-NH-). For example, Y1 will be a secondary amine when a = 1.

[0023] Advantageously, Y2 represents a primary, secondary, tertiary or quaternary amine or amide function (-NH-C(O)- or -C(O)-NH-). For example, Y2 will be a primary amine when c = 2 and R3 = H.

[0024] Advantageously, "a", "b" and "c" represent integers: "a" represents an integer from 1 to 2, "b" represents an integer from 1 to 4, preferably 2, and c represents an integer from 1 to 3, preferably from 1 to 2.

[0025] Advantageously, A1 and A2 each represent a C3 Cl alkoxy group, preferably methoxy, or are fused and together represent a -NL(Y*)b (R )a or -NR Y (R )c group. Preferably, A and A each represent a C3 Cl alkoxy group. Preferably, A1 and A2 each represent a methoxy (-OMe).

[0026] Advantageously, the cationic lipid according to the invention can be chosen from compounds of the following formulas: [Chem 6] 16

[0027] The invention also relates to a lipid nanoparticle comprising at least one cationic lipid according to the invention. The lipid nanoparticle according to the invention may further comprise at least one negatively charged therapeutic molecule. For the purposes of the invention, a "lipid nanoparticle" or "LNP" means assemblies of lipids and nucleic acids formed by a controlled mixture of a lipid solution in a solvent (e.g., ethanol) and a solution of negatively charged therapeutic molecules (e.g., nucleic acids) in an acidic buffer. The core of the LNP mainly comprises lipids and small hydrophilic water pockets formed by ionizable lipids and negatively charged therapeutic molecules (e.g., nucleic acids) in their inner core, surrounded by a lipid monolayer or a lipid bilayer membrane [7].

[0028] For the purposes of this invention, a "negatively charged therapeutic molecule" is understood to be a molecule whose overall charge is negative, in particular due to the addition of an excess of negative charges, in the form of excess electrons or negative ions. It is common practice for a person skilled in the art to implement techniques for measuring the overall charge of a molecule. A negative overall charge is understood to be a negative zeta potential value. It is obvious to a person skilled in the art that The profession requires that the zeta potential be measured using well-known techniques such as conductimetry, potentiometric electrophoresis, or acid-base titration. For example, a nucleic acid is negatively charged due to the presence of phosphate groups (siRNA, mRNA, miRNA, DNA). This could be, for instance, a molecule chosen from messenger RNA, DNA (such as a plasmid), or an interfering RNA (such as siRNA or miAcroRNA).

[0029] Advantageously, the molar ratio of ionizable lipid to negatively charged therapeutic molecule can be within a range of 1 to 1000, preferably 1 to 500, even more preferably 1 to 200, or 1 to 100 or 1 to 50 and finally preferably equal to 6.

[0030] Advantageously, the lipid nanoparticle according to the invention may further comprise an additional ionizable lipid. The additional ionizable lipid may be selected from lipids commonly used to form lipid nanoparticles. Preferably, the additional ionizable lipid is selected from SM-102 (heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy)hexyl)amino)octanoate, CAS = 2089251-47-6 [9]), ALC-0315 ([(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate, CAS = 2036272-55-4). Lipids SM-102 and ALC-0315 have the following respective formulas: ALC-0315.

[0031] Advantageously, the molar ratio of ionizable lipid according to the invention / additional lipid can be within a range from 10:90 to 50:50, preferably equal to 25:75.

[0032] Advantageously, the lipid nanoparticle according to the invention may further comprise a pharmaceutically acceptable excipient selected from sterols (e.g., cholesterol, stigmasterol, or beta-sitosterol), phospholipids (e.g., distearoylphosphatidylcholine DSPC, dioleoylphosphatidylethanolamine DOPE, l-Palmitoyl-2-oleoylphosphatidylethanolamine POPE, l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine SOPC, 1,2-di-(9Z-octadecenoyl)-OT-glycero-3-phosphate 18PA) PEGylated lipids (l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] DMG-PEG2000, l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] DSPE-PEG2000, a-[2-(ditetradecylamino)-2-oxoethyl]-co-methoxy-poly(oxy-l,2-ethanediyl) ALC-0159).

[0033] Advantageously, the lipid nanoparticle according to the invention has a diameter in the range of 70 to 150 nm, with a polydispersity index of less than 0.2. The "polydispersity index" is understood to be a measure of the heterogeneity of a sample as a function of its size. In the context of the invention, the diameter and the polydispersity index can be measured by laser granulometry. The lipid nanoparticle according to the invention can have an encapsulation efficiency of negatively charged therapeutic molecules (e.g., mRNA) greater than 80%, and a neutral charge. Encapsulation efficiency is understood to mean the amount of negatively charged therapeutic molecule (e.g., mRNA) present in the LNPs.The encapsulation efficiency of nucleic acids can be measured using an intercalator by comparing the amount of free negatively charged therapeutic molecule (e.g., mRNA) and the amount of negatively charged therapeutic molecule (e.g., mRNA) present in LNPs dissociated with TirtonX-100®.

[0034] The invention also relates to a liposome comprising at least one cationic lipid according to the invention. For the purposes of the invention, "liposome" means an aqueous buffer self-assembly consisting of one or more lipid bilayers designed to trap large quantities of aqueous buffer within the membrane of the lipid bilayer, often called the aqueous core of the liposomes. The liposome according to the invention may further comprise at least one negatively charged therapeutic molecule.

[0035] Advantageously, the liposome according to the invention may further comprise a pharmaceutically acceptable excipient selected from sterols (cholesterol, stigmasterol, beta-sitosterol), phospholipids (DSPC, DOPE, POPE, SOPC, 18PA), and PEGylated lipids (DMG-PEG2000, DSPE-PEG2000).

[0036] Advantageously, the liposome has a diameter in the range of 100 to 150 nm, with a polydispersity index of less than 0.2. The liposome according to the invention can have an encapsulation efficiency of negatively charged therapeutic molecule (e.g. mRNA) greater than 80%, and a positive charge (> + 20 mV).

[0037] The invention further relates to a pharmaceutical composition comprising at least one lipid nanoparticle according to the invention or one liposome according to the invention. The pharmaceutical composition according to the invention further comprises at least one negatively charged therapeutic molecule. Preferably, the negatively charged therapeutic molecule is selected from messenger RNA, DNA (such as a plasmid), or interfering RNA (such as siRNA or miAcroRNA).

[0038] Advantageously, the pharmaceutical composition according to the invention can further be a pharmaceutically acceptable excipient, preferably the pharmaceutically acceptable excipient selected from among sterols (cholesterol, stigmasterol, beta-sitosterol) phospholipids (DSPC, DOPE, POPE, SOPC, 18PA) PEGylated lipids (DMG-PEG2000, DSPE-PEG2000, ALC-0159)

[0039] The invention covers a pharmaceutical composition according to the invention for its use as a medicinal product, preferably, the pharmaceutical composition according to the invention for its use in the delivery of a negatively charged therapeutic molecule.

[0040] An object of the invention covers a pharmaceutical composition according to the invention for its use in regenerative medicine, in replacement therapy for deficient or mutated proteins, or as a vaccine, preferably for its use as a vaccine against an infectious disease, against cancer, preferably SARS-CoV2.

[0041] An object of the invention is a lipid nanoparticle according to the invention, for its use in medical imaging. For the purposes of this invention, medical imaging means infrared imaging, microscopy, CT scans, or MRI.

[0042] The invention relates to a method for forming a lipid nanoparticle according to the invention, comprising a step of controlled mixing of a negatively charged therapeutic molecule solution in an acidic buffer and a lipid solution in an organic solvent (for example, ethanol) by microfluidics. The lipids in the organic solvent are then protonated in the acidic buffer and complex with the negatively charged therapeutic molecule. The change of medium (organic solvent to acidic buffer) causes the lipids to interact with each other to form spherical particles with a dense core surrounded by a lipid monolayer or bilayer. Preferably, the organic solvent is an alcohol, for example, ethanol.

[0043] Finally, the invention relates to a method for forming a liposome according to the invention, comprising a step of forming a lipid film comprising at least one cationic lipid according to the invention, followed by a step of hydrating the lipid film followed by an extrusion step to obtain uniform unilamellar vesicles. Liposomes are formed by self-assembly in aqueous buffer and consist of one or more lipid bilayers designed to trap large quantities of aqueous buffer within the membrane of the lipid bilayer, often called the aqueous core of the liposomes. Brief description of the figures

[0044] [Fig. 1] shows: (left) confocal microscopy imaging of HeLa cells transfected with LNPs formed with the ionizable lipid ALC-0315, PEG2000-DMG, DSPC, cholesterol at molar ratios of 50:1.5:10:38.5 with no fluorescence of the LNPs; and (right) confocal microscopy imaging of HeLa cells transfected with LNPs formed with the mixture of the ionizable lipid ALC-0315 and lipid L6. The molar ratios are 37.5:12.5:1.5:10:38.5 for ALC-0315:L6:PEG2000-DMG:DSPC:cholesterol, respectively. Clusters of fluorescent LNPs are visible in the cytoplasm.

[0045] Examples

[0046] The invention is further illustrated by the following examples, in a non-limiting manner.

[0047] Example 1: Synthesis of cationic lipids

[0048] Synthesis of the cationic lipid L6 according to the invention:

[0049] Step 1: [Chem 8] Place 100 mg of precursor (1; tris(dimethoxyphenyl)carbenium tetrafluoroborate) and 500 µL of 1,3-diaminopropane (DAP) in a round-bottom flask and shake overnight at room temperature. Then evaporate the DAP, add dichloromethane (DCM) and water, and extract the red product (2) in the organic phase. Wash the aqueous phase with DCM twice more. Combine the extracts, evaporate the DCM using a rotary evaporator, and then evaporate it using a vacuum pump until dry.

[0050] Step 2: IChem 9] In a 25 mL round-bottom flask, introduce 50 mg of 2, 180 µl of t-butyl bromoacetate, 150 µl of DIPEA, and 500 µl of anhydrous DMF. Shake at room temperature overnight. Evaporate the solvents (DMF) using a vacuum pump at 50°C. The product is purified by flash chromatography (PuriFlash Interchim, SiO2 column, DCM:MeOH 93:7 (v / v)). ESLMS: m / z 661.3. 1 H NMR#(CDC13): ô#= 1.49 (CH3, s, 18H), 2.37 (CH2, tt, 2H), 3.57 (CH2, t, 2H), 3.57 (CH3, s, 6H), 3.55 (CH3, s, 6H), 5.55 (CH2, t, 2H), 6.65 (CH, d, 2H), 7.00 (CH, d, 2H), 7.36 (CH, t, 1H), 8.26 (CH, t, H), 8.42 (CH, d, 2H).

[0051] Step 3: [Chem 10] In a round-bottom flask, introduce 30 mg of compound 3 and heat to 100°C under nitrogen for 4 hours with stirring. Evaporate using a vacuum pump at 50°C. Product 4 is purified by flash chromatography (PuriFlash Interchim, SiO2 column, DCM:MeOH 90:10 (v / v)). ESLMS: m / z 765.9. *H NMR#(CDC13): ô#= 1.43 (CH3, s, 18H), 2.05-2.14 (CH2, tt, 2H), 2.54-2.61 (CH2, tt, 2H), 2.91-2.96 (CH2, t, 2H), 3.44 (CH2, s, 4H), 3.53 (CH3, t, 2H), 3.67 (CH3, s, 3H), 3.68 (CH3, s, 3H), 4.58-4.84 (CH2, t, 2H), 4.7-4.92 (CH2, t, 2H), 6.73 (CH, d, 2H), 6.75 (CH, d, 2H), 7.54 (CH, d, 1H), 7.57 (CH, d, 1H), 7.78 (CH, t, H), 7.81 (CH, d, 2H), 7.92 (CH, d, 2H), 7.96 (CH, d, 2H), 8.22 (CH, t, 1H).

[0052] Step 4: [Chem 11] Compound 4 is dissolved in 500 µL of DCM. Add 200 µL of TFA and stir for 6 h at 30°C. Evaporate using a water pump and then a vacuum pump. Verify hydrolysis with TLC. ESI-MS: m / z 653.7

[0053] Step 5: [Chem 12] . po" . L6 Introduce 5 into a round-bottom flask, add 2.2 eq of HATU and 2.2 eq of HoBt. Place the flask under N2 and then add DIPEA (2.2 eq), DMF, and oleylamine (3 eq). Shake for 2 days at 30°C. Evaporate the solvents using a vacuum pump at 50°C. Purify L6 by flash chromatography (PuriFlash Interchim, SiO2 column, DCM: MeOH 90:10 (v / v)). L6: ESI-MS: m / z L6+ 1086.0, sq 1085.9; [L6+H+]2+ 543.4, sq 543.45.

[0054] Synthesis of the cationic lipid L4 according to the invention:

[0055] The synthesis comprises Step 1 and 2 identical to the synthesis of L6.

[0056] Step 3: [Chem 13] In a round-bottom flask, introduce 30 mg of compound 3 and heat to 100°C under nitrogen for 2 hours with stirring. Evaporate using a vacuum pump at 50°C. Product 4 is purified by flash chromatography (PuriFlash Interchim, SiO2 column, DCM:MeOH 90:10(v / v)). ESI-MS: m / z 671.4.

[0057] Step 4: [Chem 14] 1.1 eq of thymin-N-methylacetic acid is activated in the presence of 1.2 eq of DCC and 1.2 eq of NHS in DCM for 12 h at 30°C. This mixture is then added to the solution of 6 in DCM in the presence of 1.1 eq of triethylamine (TEA). The Everything is mixed for 16 hours. Then, the solvent is evaporated and the residue is purified by chromatography (SiO2 column, DCM:MeOH 90:10(v / v)). ESI-MS: m / z 837.4.

[0058] Step 5: [Chem 15] Compound 7 is dissolved in 500 µL of DCM. Add 200 µL of TFA and stir for 6 hours at 30°C. Evaporate using a water pump and then a vacuum pump. Verify hydrolysis with TLC. ESI-MS: m / z 725.3.

[0059] Step 6: [Chem 16] Introduce 8 into a round-bottom flask, add 2.2 eq of HATU and 2.2 eq of HoBt. Place the flask under N2 and then add DIPEA (2.2 eq), DMF, and oleylamine (3 eq). Shake for 2 days at 30°C. Evaporate the solvents using a vacuum pump at 50°C. Purify by flash chromatography (PuriFlash Interchim, SiO2 column, DCM: MeOH 90:10 (v / v)). ESI-MS: m / z L4+ 1224.0, slab 1223.9.

[0060] Example 2: Synthesis of lipid nanoparticles

[0061] LNPs were prepared with the same lipid composition as the Moderna COVID19 vaccine: ionizable lipid, PEG2000-DMG (1-monomethoxypolyethylene glycol 2000-2,3-dimyristylglycerol), DSPC, cholesterol in molar ratios of 50:1.5:10:38.5. For the reference LNPs, the ionizable lipid was SM-102. For the LNPs according to the invention, comprising L4 and L6, 25% of the amount of SM-102 was replaced by L4 or L6, giving the molar ratios 37.5:12.5:1.5:10:38.5 for SM-102:L4 (or L6) / DMG-PEG:DSPC:cholesterol respectively.

[0062] Example 3: Stability test

[0063] The LNPs of Example 2 were either used on the same day to transfect human HeLa cancer cells or stored for one week at 4°C, -20°C or -80°C. The mRNA encapsulation efficiency, size and polydispersity and transfection efficiency were measured according to the methods described in reference Medjmedj et al [8]. [Tables 1] LNP Size Fresh Stored 4C Size PDI Size SM-102 95 / 2 0.08 / 0.02 87 / 1 0.103 / 0.008 SM-102 + L4 98 / 5 0.102 / 0.05 96 / 0.4 0.102 / 0.008 SM-102 + L6 98 / 5 0.102 / 0.05 97 / 0.5 0.108 / 0.006 Table 1: Size and polydispersity of LNPs stored at different temperatures for one week. [Tables 2] Fresh Stored 4C Encapsulation Encapsulation SM-102 97% 99% SM-102 + L4 78% 99% SM-102 + L6 80% 95% Table 2: Transfection efficiency of LNP HeLa cells stored at different temperatures for one week.

[0064] The results presented in Tables 1 and 2 demonstrate that the addition of L4 and L6 lipids according to the invention does not decrease the stability of LNPs stored at 4°C and therefore offers a stability similar to existing at this temperature and thus offers all the advantages related to imaging, without sacrificing the stability of the LNPs.

[0065] Example 4: Fluorescence test

[0066] HeLa cells were seeded onto glass slides 24 hours prior to transfection with LNPs prepared either with the ionizable lipid ALC-0.15 or with a mixture of two ionizable lipids (75% ionizable lipid ALC-0315 and 25% ionizable lipid L6). After 4 hours of incubation, the cells were fixed with paraformaldehyde and the nuclei labeled with DAPI (diaminophyenylindole). The slides were then imaged by confocal microscopy. The dots in the cytoplasm correspond to the intrinsic fluorescence of lipid L6 (see [Fig. 1]). The incorporation of L6, and more generally of cationic lipids according to the invention, into LNPs makes it possible to detect said LNPs after transfection of HeLa cells by confocal microscopy. The compounds according to the invention make it possible to obtain encapsulation efficiency, to maintain stability, while benefiting from fluorescence for tracking.

[0067] List of references [1] Verbeke R et al, The dawn of mRNA vaccines: The COVID-19 case, J. Control. Release, 2021, 333, 511-520 Rel [2] Uchida, S., Perche, F., et al., Nanomedicine-based approaches for mRNA delivery. Molecular Pharmaceutics, 2020. 17(10): p. 3654-3684. [3] Ci L. et al Biodistribution of Lipid 5, mRNA, and Its Translated Protein Following Intravenous Administration of mRNA-Encapsulated Lipid Nanoparticles in Rats, Drug Metab Dispos, 2023, Jul;51(7):813-823. [4] Pateev I et al, Biodistribution of RNA Vaccines and of Their Products: Evidence from Human and Animal Studies, Biomedicines, 2024, 12, 59. [5] Lindsay KE et al., Visualization of early events in mRNA vaccine delivery in non-human primates via PET-CT and near-infrared imaging, Nat. Biomed. Eng, 2019. 3(5): p. 371-380 [6] PAC, 1995, 67, 1307. (Glossary of class names of organic compounds and reactivity intermediates based on structure (IUPAC Recommendations 1995)) on page 1320 [7] Wei PS et al, Enhancing RNA-lipid nanoparticle delivery: Organ- and cell-specificity and barcoding strategies Journal of Controlled Release 375 (2024) 366-388 [8] Medjmedj A. et al., In Cellulo and In Vivo Comparison of Cholestérol, Beta-Sitosterol and Dioleylphosphatidylethanolamine for Lipid Nanoparticle Formulation of mRNA Nanomaterials (Basel), 2022 Jul 17; 12(14):2446 [9] Szebeni J. eta al., Applying lessons leamed from nanomedicines to understand rare hypersensitivity reactions to mRNA-based SARS-CoV-2 vaccines Nat Nanotechnol, 2022 Apr;17(4):337-346.

Claims

1. Demands Cationic lipid of formula I: [Chem 17] Formula I in which, - R1 independently represents a hydrocarbon chain, linear or branched, saturated or unsaturated, optionally interrupted by an ester (-C(O)-O- or -OC(O)-) or phosphate (-OP(O)(OH)-O-) function, and comprising at least 8 carbon atoms, preferably from 18 to 40 carbon atoms, and even more preferably from 12 to 20 carbon atoms, - L represents a trivalent, tetravalent or pentavalent linker, comprising from 3 to 18 carbon atoms, at least one heteroatom chosen from nitrogen and oxygen, optionally an aryl or heteroaryl group, and optionally one or more ester functions (-C(O)-O- or -OC(O)-), - Y1 represents a secondary or tertiary amine function or an amide function (-NH-C(O)-, -C(O)-NH-, -NC(O)- or -C(O)-N-), - a represents an integer from 1 to 2, - b represents an integer from 1 to 4, - R2 represents a linear hydrocarbon chain comprising 2 to 5 carbon atoms, - Y2 represents a primary, secondary, tertiary or quaternary amine or amide function (-NH-C(O)- or -C(O)-NH-), - R3 represents independently: °H, ° an alkyl chain in Cl to C5, said alkyl chain being optionally substituted by at least one group selected from -OH, -NH2, -COOH, a polyethylene glycol comprising from 1 to 5 units, a group, and a metal chelating group preferably comprising a metal atom selected from Gd, Y, La, Ce, Pr, Nd, Sm, Eu, Tb, Dy, Ho, Er, Tm, Yb, Lu, Cu, Zn, Mn, Ga, In, and Tl, of 6 or 30 carbon atoms, one or more heteroatoms selected from N and O and optionally one or more aryls or heteroaryls, an imidazole group, substituted or unsubstituted, preferably unsubstituted, and a thymine group, substituted or unsubstituted, preferably unsubstituted, or two R3 groups together with Y2 form a ring comprising 5 to 7 members, said ring being optionally aromatic, and optionally comprising at least one N or O heteroatom, said ring being optionally substituted by at least one C3 Cl hydrocarbon chain optionally substituted by an -OH group, -c represents a integers from 1 to 3, - A1 and A2 each represent an alkoxy group in Cl to C3, preferably methoxy,or are merged and together represent a group -NLCY^bCR^a or -NR2Y2(R3)C.,

2. Cationic lipid according to claim 1, wherein the linker is selected from: [Chem 18]

3. Cationic lipid according to claim 1 or 2, wherein the group R1 is chosen from the following groups of formulas: [Chem 19]

4. Cationic lipid according to any one of the preceding claims, selected from compounds of the following formulas: [Chem 20] c L12 > N XSX UX / C X-7 j / / f XV \ N 1 CT L14 x / 'OO - O -UXXI^ U c O lîj "y^c* v ' ' XJ XX QO / x XfAx. v 0 f 0 XX / X°°xx 'Nb h B Zvr}] < h 'X v X^' ^■X CX-< / X fj ;i XL IJ >r "X-^v JX ) HN ^xZ'^xx^x'Xx^sx CH s HH ù ' . / ^s / X °s / \ / SZ\ / '. 0 i » V..-X..X .X ■ - . . J°xx^n^xx'^-^0-Xx^x2x.^^ l--^>ÿo L2 H „ N ^^svx^xz^^^xa^z^.zxxx^*s .■" u.^ î p ^n3 XJ b NH 6 16 o « H L4.x'x / x^^^Z^Z -> M^XZYZSZV—X -^X^x. ,0H3 UN • JH L7 O ü f3x.xx_xx.xTM Axxxx~xx,X <s h

5.

6.

7.

8.

9.

10.

11. Lipid nanoparticle comprising at least one cationic lipid according to any one of the preceding claims. Liposome comprising at least one cationic lipid according to any one of claims 1 to 4. Pharmaceutical composition comprising at least one lipid nanoparticle according to claim 5 or one liposome according to claim 6 and at least one negatively charged therapeutic molecule. Pharmaceutical composition according to the preceding claim, further comprising a pharmaceutical excipient. Pharmaceutical composition according to claim 7 or 8 for its use as a medicinal product, preferably in the delivery of negatively charged therapeutic molecule. Pharmaceutical composition according to any one of claims 7 to 8, for use in regenerative medicine, in replacement therapy for deficient or blocked proteins, or as a vaccine. Lipid nanoparticle according to claim 5, for its use in medical imaging.< / s>

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