Ionizable cationic lipid and its use for delivering therapeutic molecules

Abstract

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 imaging, microscopy, CT scanning, or MRI. The present invention relates to a cationic lipid of formula (I), to a lipid nanoparticle or a liposome containing said lipid, and to a pharmaceutical composition containing same. The invention further relates to the pharmaceutical composition for use as a medicament, preferably in the delivery of negatively charged therapeutic molecules.

Description

[0001] IONIZABLE CATIONIC LIPID AND ITS USE IN DELIVERING THERAPEUTIC MOLECULES

[0002] [1] Technical field

[0003] [2] 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.

[0004] [3] 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 use as a medicinal product, preferably in the delivery of a negatively charged therapeutic molecule.

[0005] [4] State of the art

[0006] [5] Lipid nanoparticles (or LNPs according to its English acronym) are ordered mixtures of lipids and negatively charged therapeutic molecules, such as mRNA, by microfluidics.The composition of the LNPs used for SARS-CoV-2 vaccination was made public following their clinical use [1]. The three vaccines (BioNTech / Pfizer, Moderna, and CureVac) contain the following four components in the same molar percentage: 38.5% cholesterol to stiffen the LNPs and increase mRNA encapsulation; 10% DSPC (1,2-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 (polyethylene glycol) to decrease LNP aggregation and stabilize them in physiological fluids; and 50% of an ionizable lipid, which differs for each formulation and is responsible for complexing 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.

[0007] [6] Although mRNA vaccines against SARS-CoV-2 have demonstrated very high efficacy (>90%), rarely achieved by other vaccine modalities, the immune responses are not long enough 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.

[0008] [I]. This stability problem 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.

[0009] [7] 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.

[0010] [8] 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.

[0011] [9] A method of double labeling of mRNA for its tracking after administration by PET-CT and NIR imaging has recently been developed [5], However this technique is very expensive, complex and the mRNA both radiolabeled and with fluorophore is very fragile which does not allow to generalize this approach.

[0012]

[0010] 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.

[0013] [II] Detailed description of the invention

[0014]

[0012] It is to the credit of the applicants that they have proposed a new cationic lipid of formula I:

[0015] Formula I in which

[0016] - R 1 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,

[0017] - 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)-),

[0018] - Y 1 represents a secondary or tertiary amine function or an amide function (- NH-C(O)-, -C(O)-NH-, -NC(O)- or -C(O)-N-),

[0019] - a represents an integer from 1 to 2,

[0020] - b represents an integer from 1 to 4,

[0021] - R 2 represents a linear hydrocarbon chain comprising 2 to 5 carbon atoms,

[0022] - Y 2 represents a primary, secondary, tertiary or quaternary amine function or amide (-NH-C(O)- or -C(O)-NH-),

[0023] - R 3 represents independently:

[0024] °H,

[0025] ° an alkyl chain in C1 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 metallic 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,

[0026] ° an imidazole group, substituted or unsubstituted, preferably unsubstituted, and ° a thymine group, substituted or unsubstituted, preferably unsubstituted, or ° two R groups 3 form together with Y 2 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 C1 to C3 optionally substituted by an -OH group,

[0027] - c represents an integer from 1 to 3,

[0028] - HAS 1 and A 2 each represent an alkoxy group at C1 to C3, preferably methoxy, or are fused and together represent a -NL(Y) group 1 )b(R 1 )a or - NR 2 Y 2 (R 3 nc.

[0029]

[0013] In this application, the symbol “ " represents the point of attachment of the represented group to the complete molecule.

[0030]

[0014] Surprisingly, the applicants have 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 efficiency 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.

[0031]

[0015] 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-glycy Iglycine), and thus allow it to escape endosomal degradation, but also to carry the tracer enabling imaging.

[0032]

[0016] The invention is based on the innovative integration of several functions into 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 thus combine the advantages of having 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.

[0033]

[0017] Furthermore, most of the ionizable lipids used are not chiral; they are therefore racemic mixtures, with the probability of one enantiomer having greater activity than the other. To avoid the need for enantiomer purification, we will use ionizable lipids with at least one chiral center.

[0034]

[0018] 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)-).

[0035] 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 IUPAC 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.

[0036]

[0019] Advantageously, the linker L can be chosen from among the following formula linkers:

[0037]

[0038]

[0020] Advantageously, the cationic lipid can be chosen from among the cationic lipids of formula I in which the R group 1independently 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-) group and comprising at least 8 carbon atoms. Preferably, the R group 1 It can comprise from 12 to 40 carbon atoms, and even more preferably from 12 to 20 carbon atoms. The R group 1 can be a linear or branched, saturated or unsaturated hydrocarbon chain comprising 12 to 40 carbon atoms, or a group of formula -R 5 -C(O)-OR 6 in which, R 5 represents a linear hydrocarbon chain in C3 to C11 and R 6 a hydrocarbon chain, linear or branched, from C8 to C28. The R group 1 can be chosen from the following groups of formulas:

[0039]

[0021] Advantageously, the cationic lipid according to the invention can be chosen from among the cationic lipids of formula I in which the group R 2 represents a linear hydrocarbon chain comprising 2 to 5 carbon atoms. Preferably, R 2 represents a C3 alkyl chain.

[0022] Advantageously, the cationic lipid according to the invention can be selected from cationic lipids of formula I in which the R group 3 is chosen from:

[0040] - H,

[0041] - an alkyl chain in C1 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 metallic chelating group, 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,

[0042] - an imidazole group, substituted or unsubstituted, preferably unsubstituted, and

[0043] - a thymine group, substituted or unsubstituted, preferably unsubstituted.

[0044]

[0023] For the purposes of this invention, a "chelating group" is defined as a group that allows a metal to be bound 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:

[0045]

[0024] Advantageously, two R groups 3 can form together with Y 2 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 at C1 to C3 optionally substituted by an -OH group. Thus, the -Y group 2 (R 3 )2 can be chosen from the groups in which Y 2is "N" of the following formula II: Formula II in which,

[0046] - R 4 independently represents H or an alkyl in C1 to C3 optionally substituted by an -OH group.

[0047]

[0025] Advantageously, the cationic lipid according to the invention can be chosen from among the cationic lipids of formula I in which Y 1 represents a secondary or tertiary amine function or an amide function (-NH-C(O)-, -C(O)-NH-, -NC(O)- or -C(O)-N-). Preferably, Y 1 represents an amide function (-NH-C(O)- or -C(O)- NH-). For example, Y 1 will be a secondary amine when a = 1.

[0048]

[0026] Advantageously, Y 2 represents a primary, secondary, tertiary, or quaternary amine or amide function (-NH-C(O)- or -C(O)-NH-). For example, Y 2 will be a primary amine when c = 2 and R 3 = H.

[0049]

[0027] Advantageously, "a", "b" and "c" represent integers:

[0050] "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.

[0051]

[0028] Advantageously, A 1 and A 2 each represent an alkoxy group at C1 to C3, preferably methoxy, or are fused and together represent a -NL(Y) group 1 )b(R 1 )a or -NR 2 Y 2 (R 3 ) C Preferably, A 1 and A 2 each represent an alkoxy group at C1 to C3. Preferably, A 1 and A 2 each represent a methoxy (-OMe).

[0052]

[0029] Advantageously, the cationic lipid according to the invention can be chosen from compounds with the following formulas:

[0053]

[0054]

[0055]

[0030] 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 LNPs 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].

[0056]

[0031] For the purposes of this invention, a "negatively charged therapeutic molecule" is defined as a molecule whose overall charge is negative, particularly due to the addition of an excess of negative charges, in the form of excess electrons or negative ions. It is common knowledge 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 zeta potential can be measured by 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 example, a molecule chosen from among messenger RNA, DNA (such as a plasmid), or an interfering RNA (such as siRNA or miAcroRNA).

[0057]

[0032] Advantageously, the molar ratio of ionizable lipid / 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.

[0058]

[0033] 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-6,1-diyl) bis(2-hexyldecanoate, CAS = 2036272-55-4).

[0059] Lipids SM-102 and ALC-0315 have the following respective formulas:

[0060]

[0061]

[0034] Advantageously, the molar ratio of ionizable lipid according to the invention / additional lipid can be within a range of 10:90 to 50:50, preferably equal to 25:75.

[0062]

[0035] 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, 1-Palmitoyl-2-oleoylphosphatidylethanolamine POPE, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine SOPC, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphate 18PA), PEGylated lipids (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] DMG-PEG2000, 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000] DSPE-PEG2000, a-[2-(ditetradecylamino)-2-oxoethyl]-œ-methoxy-poly(oxy-1,2-ethanediyl) ALC-0159).

[0063]

[0036] 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.Nucleic acid encapsulation efficiency 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®.

[0064]

[0037] 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.

[0065]

[0038] 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).

[0066]

[0039] 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).

[0067]

[0040] 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).

[0068]

[0041] 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)

[0069]

[0042] The invention covers a pharmaceutical composition according to the invention for use as a medicinal product, preferably the pharmaceutical composition according to the invention for use in the delivery of a negatively charged therapeutic molecule. Administration may be in humans or animals. Preferably, the animal is chosen from among domestic animals such as, for example, dogs, cats, cows, goats, horses, pigs, chickens, ducks, or geese.

[0070]

[0043] 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.

[0071]

[0044] 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.

[0072]

[0045] 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.

[0073]

[0046] 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.

[0074]

[0047] Brief description of the figures

[0075]

[0048] Figure 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 a mixture of the ionizable lipid ALC-0315 and lipid L6. The molar ratios are 37.5:12.5

[0076] (1.5:10:38.5 for respectively ALC-0315: L6: PEG2000-DMG:DSPC:cholesterol. Fluorescent LNP clusters are visible in the cytoplasm.)

[0077]

[0049] Examples

[0078]

[0050] The invention is further illustrated by the following examples, in a non-limiting manner.

[0079]

[0051] Example 1: Synthesis of cationic lipids

[0080]

[0052] Synthesis of the cationic lipid L6 according to the invention:

[0081]

[0053] Step 1:

[0082] 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.

[0083]

[0054] Step 2:

[0084] 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)). ESI-MS: m / z 661.3. 1 H NMR (CDCb): 3 = 1.49 (CH3, s, 18H), 2.37 (CH2, tt, 2H), 3.57 (CH2, t,

[0085] 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, 1 H), 8.26 (CH, t, H), 8.42 (CH, d, 2H).

[0086]

[0055] Step 3: 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)). ESI-MS: m / z 765.9. 1 H NMR (CDCb): 5 = 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, 1 H), 7.57 (CH, d, 1 H), 7.78 (CH, t, H), 7.81 (CH, d, 2H), 7.92 (CH, d, 2H), 7.96 (CH, d, 2H), 8.22 (CH, t, 1 H).

[0087]

[0056] Step 4:

[0088] 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

[0089]

[0057] Step 5:

[0090] 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, wedge 1085.9; [L6+H + ] 2+ 543.4, wedge 543.45.

[0091]

[0058] Synthesis of the cationic lipid L4 according to the invention:

[0092]

[0059] The synthesis comprises Step 1 and 2 identical to the synthesis of L6.

[0093] 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, SiC>2 column, DCM: MeOH 90:10 (v / v)). ESI-MS: m / z 671.4.

[0094]

[0061] Step 4:

[0095] 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 a solution of 6 in DCM in the presence of 1.1 eq of triethylamine (TEA). The mixture is left to stand for 16 h. The solvent is then evaporated and the residue is purified by chromatography (SiO2 column, DCM:MeOH 90:10 (v / v)). ESI-MS: m / z 837.4.

[0096]

[0062] Step 5:

[0097] Compound 7 is dissolved in 500 µl DCM. Add 200 µl 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 725.3.

[0063] Step 6:

[0098] 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, wedge. 1223.9.

[0099]

[0064] Example 2: Synthesis of lipid nanoparticles

[0100]

[0065] LNPs were prepared with the same lipid composition as the Moderna COVID19 vaccine: ionizable lipid, PEG2000-DMG (1-monomethoxypolyethyleneglycol2000-2,3-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.

[0101]

[0066] Example 3: Stability test

[0102]

[0067] 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], one week.

[0103] Table 2: Transfection efficiency of LNP HeLa cells stored at different temperatures for one week.

[0104] 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 similar stability to existing at this temperature and thus offers all the advantages related to imaging, without sacrificing the stability of the LNPs.

[0105]

[0068] Example 4: Fluorescence test

[0106]

[0069] HeLa cells were seeded onto glass slides 24 h 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 h of incubation, the cells were fixed with paraformaldehyde and the nuclei labeled with DAPI (diaminophyenylindole). The slides were then imaged by confocal imaging. The dots in the cytoplasm correspond to the intrinsic fluorescence of lipid L6 (see Figure 1).

[0107] The incorporation of L6, and more generally of cationic lipids according to the invention, into LNPs allows for the detection of said LNPs after transfection of HeLa cells by confocal microscopy. The compounds according to the invention provide efficient encapsulation, maintain stability, and offer fluorescence for tracking.

[0108]

[0070] List of references

[0109] [1] Verbeke R et al, The dawn of mRNA vaccines: The COVID-19 case, J. Control. Release, 2021 , 333, 511-520 Rel

[0110] [2] Uchida, S., Perche, F., ét al., Nanomedicine-based approaches for mRNA delivery. Molecular Pharmaceutics, 2020. 17(10): p. 3654-3684.

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Claims

Demands

1. Cationic lipid of formula I: Formula I in which, - R 1 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)-), - Y 1 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, - R 2 represents a linear hydrocarbon chain comprising 2 to 5 carbon atoms, - Y 2 represents a primary, secondary, tertiary or quaternary amine function or amide (-NH-C(O)- or -C(O)-NH-), - R 3 represents independently: °H, ° an alkyl chain from C1 to C5, said alkyl chain being optionally substituted by at least one group selected from -OH, -NH2, -COOH, a polyethylene glycol comprising 1 to 5 units, a group , and a metallic 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 groups R 3 form together with Y 2 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 C1 to C3 optionally substituted by an -OH group, - c represents an integer from 1 to 3, - HAS 1 and A 2 each represent an alkoxy group at C1 to C3, preferably methoxy, or are fused and together represent a group - NL(Y 1 ) b (R 1 )a or -NR 2 Y 2 (R 3 ) C .

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

3. Cationic lipid according to claim 1 or 2, wherein the group R 1 is chosen from the following groups of formulas:

4. Cationic lipid according to any one of the preceding claims, selected from compounds of the following formulas:

5. Lipid nanoparticle comprising at least one cationic lipid according to any one of the preceding claims.

6. Liposome comprising at least one cationic lipid according to any one of claims 1 to 4.

7. Pharmaceutical composition comprising at least one lipid nanoparticle according to claim 5 or one liposome according to the claim 6 and at least one negatively charged therapeutic molecule.

8. A pharmaceutical composition according to the preceding claim, further comprising a pharmaceutical excipient.

9. A pharmaceutical composition according to claim 7 or 8 for use as a medicinal product, preferably in the delivery of a negatively charged therapeutic molecule.

10. Pharmaceutical composition according to any one of claims 7 to 8, for use in regenerative medicine, in replacement therapy for deficient or walled-off proteins, or as a vaccine.

11. Lipid nanoparticle according to claim 5, for its use in medical imaging.

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