Nanoparticles for nucleic acid delivery

Abstract

The present invention relates to nanoparticles comprising at least one cationic lipid, at least one neutral lipid, and at least one pH-sensitive lipid different from said cationic lipid, which can be used as a vector for the transport and release of nucleic acids within a cell.

Description

[Technical field]

[0001] The present invention relates to novel vectors for the intracellular administration of nucleic acids in in vitro or ex vivo models or for in vivo therapeutic applications. [Background technology]

[0002] prior art Single-stranded and double-stranded nucleic acids (antisense oligonucleotides, small interfering RNA, plasmid DNA and, more recently, mRNA) generally require a delivery system to reach their target intact and efficiently, which can be physical (electroporation, ultrasound-mediated delivery) or by nanometer-scale vectors. The most effective and commonly used vectors for the entry of nucleic acids into the cytoplasm of cells are cationic chemical molecules of lipid or polymer type, which bind to the nucleic acid due to their charge and allow their entry into the cell. These molecules, which are permanently charged or positively charged in response to the pH of their environment, are very commonly used to allow the entry of nucleic acids (large and small DNA or RNA) into cultured cells or cells in organisms. They are very effective in cultured cells, while in organisms they are less effective due to the phenomenon of capture by proteins and cells present in the blood. Prior art patent EP2389158 describes a composition based on anionic molecules, which can be, for example, alginic acid, and cationic lipids. This composition, which also contains cholesterol, allows the release of nucleic acids of a size smaller than 200 nucleotides. It is also known to deliver nucleic acids by liposomes formed with a combination of DOTAP and DOPE and cholesterol, optionally coated with hyaluronic acid (HA), for example in BioNTech patent application WO2013 / 143555.See Gasperini, Langmuir 31.11(2015): 3308-3317; Hattori, et al, Journal of drug targeting 21.7(2013): 639-647; Ruponen et al, Glycobiology and extracellular matrices, 276(2001): 33875-33880. Summary of the Invention

[0003] We now propose novel nanoparticles that are stable and allow for targeted intracellular release of nucleic acids. The nanoparticles of the present invention are (a) at least one cationic lipid; (b) at least one neutral lipid, and (c) at least one pH-sensitive lipid different from the cationic lipid (a); Includes.

[0004] It is understood that each type of lipid, (a), (b) and (c), can consist of a single lipid or can consist of a mixture of multiple lipids of the same type, (a), (b) and (c), respectively.

[0005] In certain embodiments, the nanoparticles are loaded with a nucleic acid. Such nanoparticles are useful for intracellular delivery of said nucleic acid.

[0006] Preferably, the nanoparticles of the invention are coated with at least one anionic polysaccharide, preferably hyaluronic acid.

[0007] Advantageously, the nanoparticles of the invention are generally free of cholesterol or even of any steroids. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a schematic diagram of uncoated lipoplex complexes (LPC) and hyaluronic acid coated lipoplex complexes (HLRC).

[0009] [Diagram 2] Figure 2 shows cryoTEM images of liposomes. Figure 2A shows the conventional morphology of unilamellar liposomes without mRNA. The interaction of liposomes with mRNA and with mRNA / HA is shown in Figure 2B and Figure 2C, respectively.

[0010] [Diagram 3]Figure 3 reports the results of transfection efficiency, cell viability and internalization of THP-1 cells after transfection with liposomes (DOTAP / DOPE / Coatsome SS-M; produced by hydrating lipid films) complexed with eGFP-mRNA at different N / P ratios. Flow cytometric analysis of transfection efficiency and fluorescence intensity of THP-1 monocytes 24 h after transfection with A) lipid nanoparticles (LRC) and B) HA-coated nanoparticles (HLRC) at different N / P ratios (1, 3, 3.5, 5). C) Cell viability was assessed by staining with propidium iodide (PI) and annexin V and observing necrosis and apoptosis at the indicated times after transfection with either LRC or HLRC at an N / P ratio of 3. Internalization of LRC D) and HLRC E) by THP-1 cells was measured 24 hours after transfection by flow cytometry using rhodamine-fluorescent nanoparticles and at the indicated times using phase contrast and fluorescence microscopy. All values ​​are means ± mean standard deviation and statistical analysis was performed using one-way ANOVA.

[0011] [Figure 4] FIG. 4 shows electrophoretic studies of formulations of mRNA complexed with DOPE / DOTAP / Coatsome SS-M liposomes at various N / P ratios (1, 5, 10 and 50) in the presence or absence of heparin, illustrating the complex formation and the presence of nucleic acids within the lipoplexes.

[0012] [Diagram 5]Figure 5 reports the results of transfection efficiency and cell viability of THP-1 cells after transfection with LRC and HLRC complexed with eGFP-mRNA (DOTAP / DOPE / Coatsome SS-M; microfluidic fabrication) at various N / P ratios. Flow cytometry analysis of transfection efficiency and fluorescence intensity of THP-1 monocytes 24 h after transfection with A) LRC, and B) HLRC at various N / P ratios (1, 2, 2.5, 3, 3.5, 5). C) Cell viability was assessed by staining with propidium iodide (PI) and Annexin V and observing necrosis and apoptosis at the indicated times after transfection with either LRC or HLRC at an N / P ratio of 2. All values ​​are means ± mean standard deviation, and statistical analysis was calculated using one-way ANOVA.

[0013] [Figure 6] FIG. 6 reports the transfection efficiency of different lipid formulations loaded with mRNA at N / P ratios of 1.5 and 3 into C2C12 myoblasts to induce GFP expression.

[0014] [Figure 7] Figure 7 reports the results on transfection efficiency into C2C12 myoblasts after transfection of LRC and HLRC (DOTAP / DOPE / Coatsome SS-M; microfluidic fabrication) complexes complexed with mRNA at an N / P ratio of 1.5 to induce GFP expression.

[0015] [Figure 8] Figure 8 shows the distribution of HLRC labeled with PE-cyanine 5 and coupled to mRNA encoding the fluorescent protein mCherry (DOTAP / DOPE / Coatsome SS-M; microfluidic fabrication) after intramuscular administration into the tibialis anterior muscle of healthy Balb / C mice.

[0016] [Figure 9]Figure 9 shows the physicochemical properties of pDNA-loaded HLRCs (DOTAP / DOPE / Coatsome SS-M; microfluidic fabrication). Average diameter and polydispersity index (A) and zeta potential values ​​(B) of pDNA-loaded HLRCs with different N / P ratios (1.5, 2, 3 or 4). Electrophoretic analysis of pDNA-loaded HLRCs to investigate the binding efficiency of DNA in HLRCs (C). CryoTEM image of pDNA-loaded HLRCs with N / P ratio of 1.5 (D).

[0017] [Figure 10] FIG. 10 shows the extent of internalization of pDNA-loaded HLRC (DOTAP / DOPE / Coatsome SS-M; microfluidic fabrication) into C2C12 myoblast cells.

[0018] [Figure 11] FIG. 11 shows the transfection efficiency of HLRC (DOTAP / DOPE / Coatsome SS-M; microfluidic fabrication) loaded with pDNA at various N / P ratios into C2C12 myoblasts to induce GFP expression.

[0019] [Figure 12] FIG. 12 reports the transfection efficiency of different lipid formulations loaded with pDNA at N / P ratios of 1.5 and 3 into C2C12 myoblasts to induce GFP expression.

[0020] [Figure 13] Figure 13 shows the results of cell viability assay in C2C12 myoblasts transfected with pDNA-loaded HLRC (DOTAP / DOPE / Coatsome SS-M; microfluidic fabrication) at various N / P ratios. Values ​​represent mean ± SD (standard deviation).

[0021] [Figure 14]FIG. 14 shows the transfection efficiency results of siRNA-HLRC (DOTAP / DOPE / Coatsome SS-M; microfluidic fabrication) at various doses into C2C12 myoblast cells to inhibit GFP expression.

[0022] [Figure 15] Figure 15 reports the transfection efficiency into C2C12 myoblasts of LRC and HLRC (DOTAP / DOPE / Coatsome SS-M; microfluidic fabrication) complexed with pDNA (encoding the protein mScarlet) and mRNA (encoding the GFP protein) in a weight ratio of 2:1, with N / P ratios of 1.5 and 3. The transfection efficiency is evaluated in the ability of the system to induce the expression of GFP, mScarlet and both at the same time at once. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description of the Invention definition "Lipoplex" refers to a complex that vectorizes a nucleic acid, in particular a complex of (i) a nucleic acid and (ii) a nanoparticle as defined herein.

[0024] "Cationic lipid" means a lipid that has a net positive charge. A cationic lipid contains a cationic polar head and one or more hydrophobic chains.

[0025] "Neutral lipid" means a lipid having a net neutral charge, and more particularly a zwitterionic lipid.

[0026] "Micelle" means a spherical aggregate of amphiphilic molecules having hydrophilic polar heads and hydrophobic chains.

[0027] "Liposome" means an artificial vesicle formed by concentric lipid bilayers that enclose an aqueous compartment between them.

[0028] "Zeta potential" describes the charge that a particle acquires when it is in suspension or solution due to the cloud of ions that surrounds it. Indeed, when said particle moves through a liquid, it is surrounded by ions organized in an "electrical double layer": - Some of the ions attach to the particles, forming a layer of attached ions called the Stern layer. -The other part of the ions forms a non-bonded layer called the diffuse layer.

[0029] A "slipping plane" defines the boundary between these two layers. The potential difference between the potential of the dispersion medium and the slipping plane defines the zeta potential. This potential represents a measure of the strength of the electrostatic repulsion or attraction between the particles. A positive zeta potential is important to ensure the efficiency of nucleic acid complexation and also affects the colloidal stability of the particles due to the electrostatic repulsion between said particles.

[0030] Cationic lipids The nanoparticles of the present invention comprise at least one cationic lipid (a). The cationic lipids are preferably chosen from the following, either alone or in mixtures: - lipopolyamines, such as 2-{3-[bis(3-aminopropyl)amino]propylamino}-N-ditetradecylcarbamoylmethyl-acetamide (compound RPR209120), 2-{3-[3-(3-aminopropylamino)propylamino]propylamino}-N,N-dioctadecyl-acetamide (RPR120535) (Byk et al, J. Med. Chem., 41, 224-235, 1998), or 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium-trifluoroacetate (DOSPA), dioctadecylamine-glycine-spermine (DOGS), dipalmitylphosphatidylethanolamine-5-carboxyspermylamide (DPPES); quaternary ammonium, such as 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DMRIE), N-(2,3-dioleyloxypropyl)-N,N,N-trimethyl ammonium chloride (DOTMA), 1,2-dioleoyloxypropyl-N,N,N-trimethyl ammonium chloride (DOTAP), dimethyldioctadecyl ammonium bromide (DDAB) or 1,2-dioleyloxypropyl-3-dimethylhydroxyethyl ammonium dioleoyl-sn-dimethylammonium bromide (DORIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (DOEPC), N-N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N-methyl-4(dioleyl)methylpyridinium chloride (SAINT-2); -stearylamine (SA), Nt-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (DiC14-amidine), O,O'-dimyristyl-N-lysyl aspartic acid (DMKD), or O,O'-dimyristyl-N-lysyl glutamic acid (DMKE); and - Lipids containing a cationic head group of the guanidine (BGTC) or imidazole (DOTIM) type.

[0031] In a preferred embodiment, the cationic lipid is selected from quaternary ammonium, eg, as described above.

[0032] In a preferred embodiment, the cationic lipid is DOTAP.

[0033] Advantageously, the cationic lipid is present in the nanoparticles in an amount of 3 mM to 15 mM, preferably 5 to 10 mM, even more preferably 5 mM, and / or the molar ratio of cationic lipid to other lipids is 1:1 to 2:1.

[0034] In a preferred embodiment, the cationic lipid (a) is DOTAP, and the molar ratio of DOTAP to all of the other lipids (b) and (c) is 1:1 to 2:1, preferably about 1:1. DOTAP is preferably present in an amount of 3 mM to 15 mM, preferably 5 to 10 mM, even more preferably 5 mM.

[0035] neutral lipid The nanoparticle of the present invention also comprises at least one neutral lipid (b).Examples of neutral lipid include 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and N'-(rac-1-[11-(F-octyl)undec-10-enyl]-2-(hexadecyl)glycero-3-phosphoethanoyl)sperminecarboxamide), which can be used alone or as a mixture.Preferably, said neutral lipid is DOPE.

[0036] A preferred nanoparticle comprises a mixture of DOTAP and DOPE, preferably in the form of a liposome or lipid nanoparticle.

[0037] Advantageously, the nanoparticles of the invention are free of cholesterol or even of any steroids.

[0038] Advantageously, the neutral lipid is present in the nanoparticles in an amount between 1 mM and 10 mM, preferably between 1 and 5 mM, preferably about 2.5 mM.

[0039] pH sensitive lipid The nanoparticles of the invention contain at least one pH-sensitive lipid (c) different from the cationic lipid (a), which becomes protonated and thus positively charged at low pH but remains neutral at physiological pH.

[0040] "pH-sensitive lipid" means an ionizable lipid, in particular a lipid that responds based on changes in the pH of the surrounding medium and that is cleavable by acid hydrolysis or contains reducible groups such as disulfide bridges.

[0041] These lipids are particularly susceptible to cleavage by cellular reducing agents such as glutathione. Lipids generally contain two hydrophobic compartments and are functionalized with two sensitive units that react to the intracellular environment. The first is a tertiary amine, which acquires a positive charge in response to an acid compartment (such as an endosome or lysosome) for membrane destabilization; the second is a disulfide bridge, which can be cleaved in response to a reducing environment (such as in the cytoplasm) for spontaneous degradation.

[0042] A preferred example of a pH-sensitive lipid is therefore known under the name ssPalm (for "SS-cleavable, pH-activated lipid mimetic").

[0043] Coatsome® products sold by NOF America are preferred pH-sensitive lipids of this type. They are described in particular in the literature by Tanaka et al, Biomaterials, 2014, 35(5): 755-1761 and Hidetaka Akita, Biol. Pharm. Bull. 2020, 43(11): 1617-1625, and also in the patent applications EP 3 315 125 by Silence Therapeutics, WO 2020 / 142725 by Oncorus and WO 2021 / 123332 by Curevac.

[0044] In a preferred embodiment, the pH sensitive lipid has the following formula (I): [ka] [During the ceremony, RCOO is a group selected from myristoyl, α-D-tocopherol succinoyl, linoleyl, and oleoyl; and X is the following structure (II), (III) or (IV): [ka] is selected from the group having the formula: is a lipid.

[0045] In particular, the pH sensitive lipid has the formula - RCOO is a myristoyl group and X is a group of formula (II) (corresponding to the product Coatsome® SS-M [SS-14 / 3AP-01]); - RCOO is an α-D-tocopherol succinoyl group and X is a group of formula (II) or (III) (corresponding to the products Coatsome® SS-E [SS-33 / 3AP-05] or Coatsome® SS-EC [SS-33 / 4PE-15], respectively); - RCOO is a linoleyl or oleoyl radical and X is a radical of formula (III) (corresponding to the products Coatsome® SS-LC [SS-18 / 4PE-13] or Coatsome® SS-OC [SS-18 / 4PE-16], respectively); or - RCOO is an oleoyl group and X is a group of formula (IV) (corresponding to the product Coatsome® SS-OP) A lipid of formula (I) as defined above.

[0046] Advantageously, the pH-sensitive lipid is a lipid of formula (I) as defined above, in which -RCOO is a myristoyl group and X is a group of formula (II). This product is known as Coatsome® SS-M.

[0047] In another embodiment, the pH sensitive lipid is the following lipid: lipids containing tertiary amine groups, such as 1,2-dilinoleyloxy-n,n-dimethyl-3-aminopropane (DLinDMA), O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) (DLin-MC3-DMA), or 2-[2,2-bis[(9Z,12Z)-octadeca-9,12-dienyl]-1,3-dioxolan-4-yl]-N,N-dimethylethanamine (DLin-KC2-DMA); [ka] or [ka] or, [ka] may be selected from:

[0048] The above pH-sensitive lipids may be used alone or in mixtures with other pH-sensitive lipids.

[0049] Advantageously, the pH-sensitive lipid is present in the nanoparticles in an amount of between 1 and 10 mM, preferably between 1 and 5 mM, even more preferably about 2.5 mM.

[0050] In a preferred embodiment, the amount of pH-sensitive lipid is substantially equal to the amount of neutral lipid.

[0051] Advantageously, the pH-sensitive lipid is a lipid of formula (I) as defined above, in which -RCOO is a myristoyl group and X is a group of formula (II), in an amount of 1 to 10 mM, preferably 1 to 5 mM, even more preferably about 2.5 mM.

[0052] Preferred nanoparticles are a) DOTAP as a cationic lipid; b) DOPE as the neutral lipid, and c) As pH-sensitive lipids, lipids of formula I as defined above, preferably lipids of formula (I) in which -RCOO is a myristoyl group and X is a group of formula (II). Contains a mixture of.

[0053] Particularly preferred nanoparticles comprise a mixture of DOTAP (in an amount of 5 mM), DOPE (in an amount of 2.5 mM) and a lipid of formula (I) as defined above in an amount of 2.5 mM, where -RCOO is a myristoyl group and X is a group of formula (II).

[0054] In another preferred embodiment, the nanoparticles are (a) DOTAP as a cationic lipid; (b) DOPE as the neutral lipid, and (c) DLin-MC3-DMA as a pH-sensitive lipid Includes.

[0055] Method for preparing nanoparticles The nanoparticles of the present invention are preferably prepared in the form of liposomes or lipid nanoparticles comprising a) a cationic lipid, b) a neutral lipid, and c) a pH-sensitive lipid different from the cationic lipid (a) in a molar ratio of 2:1:1 to 4:1:1.

[0056] Advantageously, the nanoparticles according to the invention have (a) approximately 50% cationic lipids; (b) about 25% neutral lipids, and (c) Approximately 25% of cationic lipids are pH-sensitive lipids different from those in (a). The liposome is prepared in the form of a liposome comprising:

[0057] According to a preferred embodiment of the invention, the nanoparticles according to the invention are (a) ≈50% DOTAP as the cationic lipid; (b) about 25% DOPE as the neutral lipid, and (c) about 25% of the compound of formula (I) defined above [During the ceremony, RCOO is a group selected from the group consisting of myristoyl, α-D-tocopherol succinoyl, linoleyl and oleoyl; and X is selected from the group having the structure (II), (III) or (IV) defined above. A pH-sensitive lipid selected from the lipids The compound is prepared in the form of a liposome or lipid nanoparticle comprising:

[0058] According to a preferred embodiment of the invention, the nanoparticles according to the invention are (a) ≈50% DOTAP as the cationic lipid; (b) about 25% DOPE as the neutral lipid, and (c) about 25% of the compound of formula (I) defined above [During the ceremony, - RCOO is a myristoyl group and X is a group of formula (II); - RCOO is an α-D-tocopherol succinoyl radical and X is a radical of formula (II) or (III); - RCOO is a linoleyl or oleoyl radical and X is a radical of formula (III); or - RCOO is an oleoyl group and X is a group of formula (IV). A pH-sensitive lipid selected from the lipids The compound is prepared in the form of a liposome or lipid nanoparticle comprising:

[0059] Liposomes or lipid nanoparticles are obtained using techniques known to those skilled in the art, in particular by hydrating and extruding a lipid film (generally through one or more membranes, such as polycarbonate membranes), or by microfluidic processes, these two methods being described in detail in the examples below.

[0060] Complex formation with nucleic acids According to a particular aspect of the invention, the nanoparticles are loaded with nucleic acids, in particular to form lipoplexes, which are formed in particular by complexation of the nucleic acid with lipids, in which the lipids and the nucleic acid are non-covalently associated.

[0061] The loaded nanoparticles or lipoplexes are in particular i) obtaining a mixture of lipids a), b) and c) as defined above (which makes it possible to obtain liposomes), and ii) contacting said mixture with a nucleic acid to obtain a complex or lipoplex. The composition can be obtained by a process comprising:

[0062] Step i) of this process is preferably carried out according to the method described above for forming lipid nanoparticles.

[0063] Step ii) is preferably carried out by diluting the liposomes obtained in step i) in a buffer or in an aqueous solvent, in particular RNase-free water, followed by adding a solution of the nucleic acid in the same buffer or in the same aqueous solvent, in particular RNase-free water.

[0064] According to a particular embodiment of the present invention, the nanoparticles may carry nucleic acid such that the N / P ratio is in the range of 1 to 100, preferably in the range of 1 to 60, preferably in the range of 1 to 50, preferably in the range of 1 to 40, preferably in the range of 1 to 30, even more preferably in the range of 1 to 20, preferably in the range of 1 to 10, preferably in the range of 1 to 8, even more preferably in the range of 1.5 to 8, preferably in the range of 1 to 5, even more preferably in the range of 1.5 to 5 or in the range of 1.5 to 4.

[0065] The N / P charge ratio corresponds to the ratio of the number of amine functional groups (positive charge) on the lipid of the nanoparticle to the number of phosphate molecules (negative charge) on the nucleic acid.

[0066] According to a particular embodiment of the invention, when the loaded nanoparticles are formed by a process of hydrating and extruding a lipid film, the loaded nanoparticles or lipoplexes generally have an N / P ratio of 3 or more, preferably 3-8, preferably 3-5.

[0067] According to another embodiment, when the loaded nanoparticles are formed by a microfluidic process, the loaded nanoparticles or lipoplexes generally have an N / P ratio of 1.5 or more or 2 or more, preferably 1.5-10, preferably 1.5-5 or 2-5.

[0068] The nucleic acids may be deoxyribonucleic acids (DNA), single- or double-stranded ribonucleic acids (RNA), such as mRNA or interfering RNA, or mixtures, or hybrid DNA / RNA sequences. They may be sequences of natural or artificial origin. They may also be obtained by chemical modification of their sugar moiety, their nucleobase moiety, or their internucleotide backbone. Among the advantageous modifications in the sugar moiety, mention may be made of modifications at the 2' position of the ribose instead of the usual 2'-OH group on the ribonucleotide, such as 2'-deoxy, 2'-fluoro, 2'-amino, 2'-thio, or 2'-O-alkyl modifications, in particular 2'-O-methyl modifications, or alternatively the presence of a methylene bridge between the 2' and 4' positions of the ribose (LNA). As regards the nucleobases, in particular 5-bromouridine, 5-iodouridine, N <3> Modified bases such as 2'-methyluridine, 2,6-diaminopurine (DAP), 5-methyl-2'-deoxycytidine, 5-(1-propynyl)-2'-deoxyuridine (pdU), 5-(1-propynyl)-2'-deoxycytidine (pdC) or bases bound to cholesterol can be used. Finally, advantageous modifications of the internucleotide backbone include replacing the phosphodiester groups of this backbone with phosphorothioate, methylphosphonate, phosphorodiamidate groups, or using a backbone composed of N-(2-aminoethyl)glycine units linked by peptide bonds (PNA, peptide nucleic acid). Naturally, the various modifications (bases, sugars, backbone) can be combined to obtain modified nucleic acids of the morpholino type (bases linked to a morpholine ring and linked by phosphorodiamidate groups) or PNA type (bases linked to N-(2-aminoethyl)glycine units linked by peptide bonds).

[0069] Nucleic acids can be small (e.g., smaller than 200 nucleotides), such as, for example, antisense oligonucleotides, interfering RNA (miRNA, siRNA, etc.), or aptamers. They can also advantageously be large (e.g., larger than 200, 300, or 400 nucleotides). In a particular embodiment, the nucleic acid is a DNA plasmid (pDNA).

[0070] Examples of sizes of nucleic acids that can be carried in the lipoplexes of the invention are shown in Table 1 below. [Table 1]

[0071] In a preferred embodiment, the nucleic acid is mRNA.

[0072] In another embodiment, the nucleic acid is DNA.

[0073] In another embodiment, the nucleic acid is RNA other than mRNA, such as an interfering RNA (eg, siRNA or miRNA).

[0074] In a preferred embodiment, the nanoparticles comprise a DOPE / DOTAP and SS-Palm mixture charged with RNA (such as mRNA or interfering RNA) or DNA, preferably pDNA.

[0075] In another preferred embodiment, the nanoparticles comprise a DOPE / DOTAP and SS-Palm mixture, carrying a mixture of RNA and DNA, preferably a mixture of mRNA and pDNA, for example with an RNA:DNA ratio in the range of 1:1 to 1:10, preferably in the range of 1:1 to 1:5, preferably in the range of 1:1 to 1:3, even more preferably 1:2.

[0076] Anionic Polysaccharides The nanoparticles may be coated with one or more molecules of anionic polysaccharides. A "polysaccharide" is formed by successive sugars interconnected by glycosidic bonds, and an "anionic polysaccharide" is a polysaccharide with a net negative charge. Among the anionic polysaccharides, mention may be made of glycosaminoglycans (hyaluronic acid or its salts, alginic acid or its salts, etc.), carrageenan (sulfated red algae polysaccharide), fucan (sulfated brown algae polysaccharide), carboxymethyl benzylamide sulfonate dextran or CMDBS (a synthetic polysaccharide prepared from dextran by statistically replacing hydroxyl groups with carboxymethyl, benzylamide, sulfonic, and sulfate groups), and heparan sulfate (a complex polysaccharide belonging to the glycosaminoglycan family).

[0077] In a preferred embodiment, the anionic polysaccharide is hyaluronic acid or a salt thereof, such as the sodium salt. Alternatively, the anionic polysaccharide may be alginic acid or a salt thereof, such as the sodium salt.

[0078] In a preferred embodiment, the nanoparticles comprise a mixture of DOPE / DOTAP and SSpalm lipids of formula I, carry RNA (e.g. mRNA or siRNA type) or DNA, preferably pDNA, and are coated with molecules of hyaluronic acid or its salts, e.g. sodium hyaluronate (HA).

[0079] Coating with an anionic polysaccharide is preferably achieved by contacting the previously obtained nanoparticles / liposomes / lipoplexes with an anionic polysaccharide, such as hyaluronic acid, in an aqueous solvent, e.g. in RNase-free water.

[0080] The lipoplexes are preferably coated with an anionic polysaccharide, such as HA, in a weight ratio of cationic lipid:anionic polysaccharide of 1:2.

[0081] Coating lipoplexes with anionic polysaccharides has the particular advantage of increasing their accumulation at tumor sites or in cells, e.g., macrophages, myoblasts, and myotubes, where cellular receptors for these polysaccharides (e.g., CD44) are overexpressed.

[0082] Nucleic Acid Transfer The nanoparticles or lipoplexes of the invention can be used for the transfer of nucleic acids into cells in vivo, in vitro or ex vivo. In particular, the compositions according to the invention can be used for highly efficient transfer of nucleic acids into many cell types.

[0083] The nanoparticles according to the invention are preferably present in a culture medium containing the cells to be transfected under conditions in which the lipoplexes pass from the culture medium into the cytoplasm of the cells and the nucleic acid is then released in the cytosol and / or nucleus of the cells.

[0084] In certain embodiments, the nanoparticles may also include targeting elements that can direct the transfer of nucleic acids, such as intracellular (such as nuclear) targeting elements and / or extracellular targeting elements (targeting to specific cell / tissue types).

[0085] Another subject of the present invention is a pharmaceutical composition comprising the nanoparticles or lipoplexes defined herein and a pharma- ceutically acceptable carrier. Advantageously, the subject of the present invention is the use of the nanoparticles described herein as pharma- ceutically acceptable vectors of nucleic acids. The dose of nucleic acid used and the number of administrations can be adapted based on various parameters, in particular based on the mode of administration used, the disease in question, the nucleic acid to be administered, or other desired duration of treatment.

[0086] In a preferred embodiment, the nanoparticles of the present invention are useful in gene therapy applications. The nucleic acid may preferably be a DNA encoding a functional protein, in particular a protein that is not functionally produced in the patient.

[0087] In some embodiments, the nucleic acid can be the mRNA that encodes the protein that is not functionally produced in the patient, or the protein that is of therapeutic or vaccine-related interest.According to another embodiment, the nucleic acid selected can inhibit the expression of the protein that is expressed by the subject, and thus can be, for example, an iRNA or an antisense oligonucleotide that is complementary to the target mRNA sequence.Therefore, the mRNA can be modified, in particular by exon skipping or inclusion, by acting on the splicing step.

[0088] In other embodiments, the nanoparticles of the invention are useful in ex vivo applications, particularly for cell therapy purposes, e.g., to modify cells. A particular therapeutic application relates to treating muscle or musculoskeletal disorders.

[0089] Also described herein is a method for releasing a nucleic acid into the muscle of a subject, preferably a human patient suffering from a muscular or musculoskeletal disorder, by intramuscular administration of nanoparticles described herein loaded with a nucleic acid, e.g., an mRNA encoding a protein of therapeutic interest.

[0090] The examples and figures illustrate the invention without limiting its scope. EXAMPLES

[0091] Example 1 : Preparation of liposomes (lipoplexes) as carriers for intracellular mRNA administration. material and method material The cationic lipid 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and the co-lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids. Coatsome® SS-M was obtained from NOF America Corporation. GFP-encoding mRNA and Viromer were provided by BioNTech. Sodium hyaluronate (HA, weight-average molar mass, Mw = 20000 g mol -1 ) was purchased from Lifecore Biomedical (Minnesota, USA). Fixable Live / Dead® was provided by Sigma-Aldrich (Saint-Quentin-Fallavier, France).

[0092] Liposome and Lipoplex Formulations Liposome formulation Liposomes were prepared using the thin film method followed by extrusion. Lipids were dissolved in chloroform to prepare solutions of the final desired concentration and added to a 10 mL round-bottom flask to a final lipid concentration of 10 mM after hydration according to the molar ratios shown in Table 2. Organic solvents were removed using a rotary evaporator at 40°C under reduced pressure. The round-bottom flask was left in a desiccator overnight to completely remove the chloroform. HEPES buffer 0.1 M was used to rehydrate the pH 5.5 lipid film. After vortexing for 5 min, multilamellar vesicles were extruded through a large polycarbonate membrane (400 nm) using an Avanti mini extruder (Avanti Polar Lipids), followed by a second extrusion through a 100 nm membrane to obtain unilamellar vesicles. Fluorescent liposomes were prepared using rhodamine-labeled DOPE (Rho-PE) embedded in the liposomal membrane. Rho-PE was added to the lipid solution at a ratio of 2% w / w, followed by a drying step. For radiolabeling purposes, DTPA liposomes were prepared using DSPE-DTPA (PE-DTPA). 0.5% mol / mol of PE-DTPA was added to the lipid solution, followed by a drying step. [Table 2]

[0093] Lipoplex formulation Lipoplexes were prepared by diluting 50 μL of liposomes in RNase-free water to a final concentration of 1.8 mM, followed by the addition of 50 μL of various concentrations of mRNA (0.03-0.15 mg / mL) in RNAse-free water, resulting in complexes with N / P ratios ranging from 1 to 5, as shown in Table 3. Lipoplexes coated with hyaluronic acid were prepared by adding 50 uL of a 0.6 mg / mL HA solution in RNAse-free water to the already prepared liposomes. For the purpose of in vivo use, lipoplexes were diluted in a 10% glucose solution or coated with HA dissolved in 10% glucose to obtain the same final concentration and good osmolarity. [Table 3]

[0094] Physicochemical analysis of lipoplexes Particle size distribution and surface charge were measured using a Nano ZS Zetasizer® from Malvern (Malvern Instruments SA, Worcestershire, UK). Hydrodynamic diameter and polydispersity index (PDI) were measured by dynamic light scattering (DLS) using the cumulant method, and these measurements were performed at 25°C and a detection angle of 173°. Zeta (ζ) potential was measured by electrophoretic techniques. All samples were diluted with 1 mM NaCl and analyzed in triplicate. mRNA binding efficiency was measured using Sybr Green® agarose gel electrophoresis. Samples were diluted with HEPES, pH 5.5, to a final amount of RNA of 0.125 μg. After adding mRNA gel loading stain, samples were run on a 1% (w / v) agarose gel at 80 V for 30 min. The mRNA bands were analyzed using Gel DocTM The cells were visualized and imaged using an EZ imaging device (Bio-Rad).

[0095] Analysis of mRNA-loaded liposomes by cryo-electron microscopy (cryoTEM) The morphology of lipid nanoparticles was evaluated using transmission cryo-electron microscopy. Briefly, 3.5 μL of the formulation was added to a CF-2 / 1-3Cu-50 copper mesh and immersion frozen using a Vitrobot (Thermoscientific) to generate vitreous ice. Samples were stored in liquid nitrogen and imaged using a JEM-1400Flash electron microscope operating at 120 kV.

[0096] Cellular studies in THP-1 Transfection studies The reverse transfection method was used to amplify and extend gene expression. Freshly prepared lipoplexes with N / P ratios of 1, 3, 3.5 and 5 were placed in 24-well plates. THP-1 cells were added to the lipoplexes at a density of 150000 cells in a total volume of 600 μL of complete RPMI medium supplemented with 15% FBS, 1 mM sodium pyruvate, 10% HEPES buffer, 1% L-glutamine, 1% penicillin / streptomycin. The volume of the lipoplexes was adjusted and the cells were transfected with 3 μg of eGFP mRNA for each condition. A solution of Viromer Red containing eGFP mRNA was used as a positive control for transfection; 3 μg of e-GFP mRNA was placed in the plate. The cells were then incubated at 37°C for 24 h. Four hours after transfection, the cell medium was replaced. After transfection, cells were washed and stained with Fixable Live / Dead Violet solution before measurements were taken using a BD LSR 2 flow cytometer. Data were analyzed using FlowJo software and statistical analysis was performed using GraphPad Prism v8.1 software.

[0097] Viability test THP-1 cells and the lipoplexes were plated as described above. Lipoplexes were freshly prepared and the required amount was added to each well to achieve 3 μg of eGFP mRNA. Treatment with 50 μM cisplatin was used as a positive control for cell death. Cells were then incubated at 37° C., 5% CO 2 The cells were incubated with 100 mM NaCl for 4, 24, and 48 h. The cell medium was replaced 4 h after transfection. The cells were then washed and stained with Annexin V Pacific Blue. TM After staining with and diluting with 1:50 propidium iodide, measurements were taken using a BD LSR 2 flow cytometer. Data were analyzed using FlowJo software and statistical analysis was performed using GraphPad Prism v8.1 software.

[0098] Internalization Test Freshly prepared lipoplexes of liposomes conjugated with PE-Rho at an N / P ratio of 3 were plated in 24-well plates. THP-1 cells were then added at a density of 150000 cells in a total volume of 600 μL of complete RPMI medium. The volume of lipoplexes was adjusted and cells were transfected with 3 μg of eGFP mRNA for each condition. Cells were incubated at 37°C for 2, 4, 24, and 48 hours. The cell medium was changed 4 hours after transfection. After transfection, cells were washed and stained with Fixable Live / Dead Violet solution and then stained with BD TM Measurements were taken using an LSR II flow cytometer. Data were analyzed using FlowJo software and statistical analysis was performed using GraphPad Prism v8.1 software.

[0099] In vivo biodistribution studies In vivo biodistribution of radiolabeled liposomes 4.17 μmol (500 μL) of liposomes (DOTAP-DOPE-Coatsome-SS-M) were added to 85.8 MBq (100 μL) of 111The liposome surface was radiolabeled by incubation with In at 60°C for 30 min. This resulted in a radioactivity of 20.58 GBq / mmol. The mole / activity ratio was 0.24 nmol PE-DTPA / MBq. Radiolabeling efficiency was assessed by instant thin-layer chromatography on silica gel strips (iTLC-SG, Biodex Medical Systems, Shirley, USA) using 100 mM citrate buffer at pH 5 as the mobile phase. Free and labeled 111 In activity was evaluated in an ionization chamber (Capintec, Florham Park, USA). Lipoplexes were prepared with an N / P ratio of 3. Briefly, 1.25 μmol (150 μL) of radiolabeled liposomes (0.63 μmol DOTAP) were incubated with 0.2 nmol mRNA (equivalent to 0.244 μmol phosphate) at ambient temperature for 30 min each. The mRNA solution was prepared and transferred to the NanoDrop TM (Ozyme, Saint-Cyr-l'Ecole, France) was used to measure the osmolarity. Then, 10% glucose solution or 0.025 μmol of hyaluronic acid dissolved in 10% glucose was added to the lipoplexes to adjust the osmolarity.

[0100] The formulations were injected intravenously into BALB / c_Rj mice anesthetized with 3% isoflurane. 2.07 ± 0.82 MBq chelated to DTPA lipoplexes, either as positively charged lipoplexes LRC or negatively charged lipoplexes HLRC, were administered intravenously. 111Mice were injected with In (specific activities of 20.58 and 25.77 Gbq / mmol total lipid; 50 nmol total lipid / mouse in 100 μL, n=18). For each formulation, groups of three mice were euthanized by cervical dislocation 6, 24, and 48 hours after injection. Mice were then dissected, organs were harvested, and quantitative analysis of cumulative activity was performed. Ex vivo quantification of organ radioactivity was performed using a Wizard 3” gamma counter (Perkin Elmer, Waltham USA). For each formulation, one mouse from the 48-h group was imaged in prone position after 6, 24 and 48 h. All live animal images were acquired with a NanoSPECT / CTTM in vivo animal imager (Bioscan Inc., Washington D·C, USA). During imaging, animals were anesthetized with 1.5% isoflurane and respiration was monitored with a Model 1025T small animal monitoring and gating system (SA Instruments Inc., Stony Brook, NY, USA). The images were acquired using a preclinical SPECT / CT multiplexed with a multiplexed multi-pinhole aperture. The SPECT system acquired 24 projections of 256 × 256 pixels spaced 15° apart. The scan time was 100 seconds per projection. Reconstruction was performed with the manufacturer's software HiSPECT using an ordered subsets expectation maximization (OSEM) algorithm with 9 iterations and 4 subsets, with an image voxel size of 0.6 mm.

[0101] result Characterization of lipoplexes Preparation of liposomes and complex formation with IVT mRNA Cationic liposomes (LP) were prepared using a lipid film hydration method and then extruded through a 100 nm MWCO membrane. The hydrodynamic diameter of the liposomes measured by DLS was 140.8 ± 3.4 nm with a PDI less than 0.2. The stability of the formulation was evaluated over a 4-month period, which showed that the size and PDI did not change during the entire duration of the experiment. Similar results were obtained with rhodamine-labeled liposomes (Rho-LP) and DTPA liposomes (DTPA-LP), which had hydrodynamic diameters of 151 ± 2.3 nm and 139.1 ± 2.9 nm, respectively, with both PDIs less than 0.2. The PDI has a significant impact on the stability and bioavailability of liposomes. To be stable, safe, and effective, liposome preparations must be homogenous. A liposome formulation acceptable for administering pharmaceuticals should have a PDI value less than 0.3. The surface charge of all prepared liposomes was positive, ranging from +40 to +55 mV (Table 4). [Table 4]

[0102] The amount of positive charge present in the formulation is crucial if electrostatic complexation with a precise amount of negative charge is to occur. For this reason, a method was developed to quantify the charge carried by lipids using RP-HPLC. The final formulation contained about 95% DOTAP and DOPE, while the yield was lower (about 60%) for SS-Palm. This could be due to the pH of the formulation, which could cause partial protonation of the tertiary amines, thereby precipitating some of the lipids. Two different types of complexes were prepared (Figure 1). To obtain liposome-RNA complexes (LRC), mRNA was mixed with liposomes at N / P ratios ranging from 1 to 5. Seven different N / P ratios (1, 1.5, 2, 2.5, 3, 3.5 and 5) were tested for their physicochemical properties and nucleic acid retention capacity. With the aim of obtaining systems with similar properties in terms of size but opposing properties in terms of surface charge, the same set of lipoplexes was coated with the anionic polymer hyaluronic acid (HA) to form hybrid liposome-RNA complexes (HLRC). At physiological pH and at the pH used for liposomal formulation (5.5), the phosphate groups exist mainly in anionic form, allowing good complexation with cationic lipid particles. The amount of positive charge in the lipid was determined based on information provided by the manufacturer. Seven different N / P ratios, ranging from 1 to 5, were tested for physicochemical properties and nucleic acid retention capacity. [Table 5] [Table 6]

[0103] As can be seen from the average size and polydispersity values ​​reported in Tables 5 and 6, for both LRC and H-LRC, N / P ratios between 1.5 and 2.5 showed the greatest instability in terms of complex formation. On the other hand, for N / P ratios equal to 1 and above 3, the size became smaller than 300 nm with a good PDI of about 0.2, which is important for the stability and bioavailability of the system. For LRC, the surface charge was positive for all N / P ratios except for N / P ratio 1, which resulted in negatively charged particles due to the excess of mRNA relative to the amount of lipid. Interestingly, LPC with N / P ratio 1 proved to be stable at a negative ζ potential initially after preparation, but drifted towards positive values ​​after 24 h, probably due to the formation of aggregates due to the instability of the system. For all other N / P ratios, the surface charge was similar to that of liposomes alone and decreased after the addition of mRNA. This could be the result of electrostatic interactions between the cationic lipids and the negatively charged nucleic acid backbone. On the other hand, the zeta potential of HLRC was negative at all tested N / P ratios due to the presence of hyaluronic acid: indeed, at neutral pH, the pKa of the carboxyl groups of the polymer is about 3 to 4. We obtained negatively charged lipoplexes using this coating with the aim of observing their different behavior in vitro and in vivo.

[0104] CRYOTEM LRCs and HLRCs do not consist of an ordered nucleic acid phase surrounded by an outer lipid bilayer, but rather are partially condensed nucleic acid complexes with ordered and irregular morphology. CryoTEM techniques are methods for detecting single particles and are used to characterize the morphology of liposomes. Figure 2A shows the morphology of conventional unilamellar liposomes. These are spherical vesicles with an aqueous internal compartment bounded by a single bilayer membrane. The interactions of liposomes with mRNA and mRNA / HA are shown in Figures 2B and 2C, respectively. The deformation and rearrangement of the liposome membrane results from the strong electrostatic interactions between the cationic lipid head groups and the nucleic acid phosphate groups. Due to the presence of mRNA, which acts as a bridge between liposomes, liposomes that adsorb to each other to form paired membranes can be observed in an ordered manner. Indeed, the bilayer interface has a higher contrast than the outer edges, suggesting that electron-dense nucleic acid molecules are sandwiched between the membranes. In other cases, as already reported in the literature for cationic liposomes and DNA, multilamellar systems are formed in which the nucleic acid and the liposomes alternate in a structure similar to a sandwich type. This structural modification is said to be beneficial for the stability of the nucleic acid: bilayer packing promotes protection of the mRNA molecules from degradation rather than simple surface binding, which is susceptible to degradation by serum nucleases.

[0105] Assay of mRNA-liposome binding and stability To evaluate the stability of the complex formation of LRC and HLRC, electrophoretic assays were performed at all tested N / P ratios. Nucleic acids migrate in the agarose gel matrix under the action of an electric field depending on their charge, size and morphology. In the case of LRC, a weak signal corresponding to free mRNA is observed, which means that a part of the nucleic acid is not constantly bound to the cationic particles. Moreover, the presence of a hyaluronic acid coating prevents this release and no fluorescence can be observed.

[0106] In vitro cell studies in THP-1 Transfection studies To investigate whether and to what extent LRC and HLRC have the ability to transfect cells, they were incubated with THP-1 cells after complex formation with IVT mRNA, which has the ability to induce eGFP expression. The protocol was optimized around the stability of the complex, the amount of IVT mRNA, the number of cells, and the volume, by quantifying eGFP expression along with cell viability using flow cytometry. Indeed, the entry of nucleic acids into cells, in conjunction with the use of transfection agents such as our lipid system, often causes considerable stress and may ultimately affect cell viability. A commercially available material composed of a polycationic core of polyethyleneimine was used as a positive control.

[0107] As shown in Figure 3 (A and B), eGFP transfection was obtained in all lines, with no significant difference between the uncoated and HA-coated lines at the same N / P ratio. The highest transfection efficiency was achieved at an N / P ratio of 3 in both LRC and HLRC lines. The N / P ratio was maintained in other tests.

[0108] Internalization Test To investigate the ability of nanoparticles to transfect cells and their internalization kinetics, 2% rhodamine-DOTAP lipid was used in their formulation. To monitor the release and translation of RNA in the cytoplasm of THP-1 cells, a monocytic cell line, rhodamine-LRC and HLRC were complexed with eGFP mRNA at an N / P ratio of 3. As shown in Figure 3(D), two fluorescent signals were observed using flow cytometry at 2, 4, 24, and 48 hours after transfection. After 2 hours, rhodamine-LRC and HLRC were present in each cell. This fluorescent rhodamine signal slightly decreased in both LRC and HLRC after 48 hours, but was always strongly present at each time; this can be interpreted as the start of nanoparticle externalization from the cells.

[0109] Viability test The Annexin-PI test was chosen for the viability of LPC and HLRC at an N / P ratio of 3. As shown in Figure 3(C), 4 and 24 h incubation of nanoparticles did not affect cell viability as occurred with the Viromer control, whereas at 48 h, LRC and HLRC were less toxic than Viromer.

[0110] In vivo testing In vivo biodistribution To investigate the in vivo targeting properties of the liposomal formulations, 111 In-labeled liposomes were injected intravenously into BALB / c_Rj mice after complexation with mRNA at a selected N / P ratio of 3. SPECT / CT imaging was performed 6, 24, and 48 hours after injection. Results were expressed as the percentage of the total administered LRC or HLRC dose accumulated per gram of tissue. Nanoparticle biodistribution did not differ between LRC and HLRC. After 6 hours, it was detected mainly in the liver and spleen in both systems. The percentage detected in the lung was less than 10%. At 24 hours, the radiation dose in the spleen increased, reaching approximately 60% of the injected dose, while in the liver the percentage decreased to approximately 30%.

[0111] No significant difference in accumulation was evident between LRC and HLRC, decreasing over time in the liver while remaining high in the spleen, peaking at 24 h. Indeed, for LRC, 29.5% of the radioactivity was detected in the liver after 24 h, whereas 58.9% was detected in the spleen. Similar results were obtained for HLRC, i.e., 37.2% was detected in the liver and 52.4% in the spleen.

[0112] Example 2 : Microfluidically fabricated liposomes as carriers for intracellular mRNA delivery material and method material The same materials were used here as in Example 1. The GFP mRNA used for the tests in THP-1 in vitro and in vivo after intravenous administration was provided by BioNTech, whereas that used for the tests in C2C12 or CSPi in vitro or in vivo after intramuscular administration was purchased from Tebuo (Le Perray-en-Yvelines, France).

[0113] Formulation of lipid nanoparticles and lipoplexes Briefly, cationic lipids (DOTAP or DOTMA), neutral lipids (DOPE) and pH-sensitive lipids (Coatsome® SS-M or DLin-MC3-DMA) were dissolved in ethanol in a molar ratio of 2:1:1 to a final concentration of 10 μmol of total lipids. TM A microfluidic platform (Precision NanoSystems Inc, Vancouver, Canada) was used with a flow ratio of 3:1 (v / v) and a total flow rate of 15 mL.min -1Liposomes were formed by mixing the lipid solution with HEPES at pH 5.5. The system was then dialyzed using a 5 kDa dialysis membrane to remove residual alcohol from the formulation. Fluorescent lipid nanoparticles were obtained by adding PE-rhodamine to the lipid mixture. Lipoplexes were prepared by diluting 50 μL of liposomes to a final concentration of 1.8 mM with RNase-free water and adding 50 μL of various concentrations of mRNA (0.06-0.3 mg / mL) in RNAse-free water to obtain complexes with N / P ratios ranging from 1 to 50. Lipoplexes coated with hyaluronic acid were prepared by adding 50 μL of a 0.6 mg / mL solution in RNase-free water to the already prepared liposomes. For the purpose of in vivo use, lipoplexes were diluted with 10% glucose solution or coated with HA dissolved in 10% glucose to obtain the same final concentration and good osmolarity. For comparison purposes, formulations consisting of neutral lipids and cationic or ionizable lipids only were tested. To this end, formulations of DOTAP / DOPE or DOPE / Coatsome SS-M were prepared under the same conditions as described above.

[0114] Physicochemical analysis of lipoplexes Here, the same analysis as in Example 1 was used.

[0115] In vitro transfection studies in THP-1 Here, the same analysis as in Example 1 was used.

[0116] In vitro transfection study in C2C12 C2C12 myoblasts (immortalized mouse muscle cell line purchased from ATCC® CRL-1772) were cultured in GlutaMax Dulbecco's Modified Eagle's Medium supplemented with 10% (v / v) fetal bovine serum (FBS), 0.5% (v / v) and 100 units / mL penicillin-streptomycin (Gibco) at 37° C. in a humidified atmosphere with 5% CO2. To induce differentiation into myotubes, myoblasts were cultured for 6 days in GlutaMax Dulbecco's Modified Eagle's Medium supplemented with 1% (v / v) fetal horse serum (FHS) and 100 units / mL penicillin-streptomycin (Gibco).

[0117] Transfection and viability assay For transfection efficiency testing, cells were seeded in flat-bottom 12-well plates at a density of 15000 cells per well. Myoblasts were then treated with 3 μg of mRNA complexed with different nanosystems at N / P ratios of 1.5 and 3. Viromer transfection reagent (Lipocalyx®) was used as a transfection control, according to the manufacturer's instructions. All treatments were left for 2 h before being transferred to fresh medium and internalization efficiency was evaluated after 24 h of incubation. Cells were then enzymatically detached using Trypsin-EDTA (0.5%, Gibco) and stained with Fixable Live / Dead Violet solution to assess cell viability. Data were acquired using a BD LSR 2 flow cytometer and analyzed using FlowJo software. Statistical analysis was performed using GraphPad Prism v8.1 software.

[0118] Internalization Test For this confocal microscopy analysis, myoblasts were seeded at a density of 10000 cells / well in 24-well multiwell plates on 12 mm glass slides, whereas to obtain myotubes, myoblasts were seeded at a density of 40000 cells / well on a 4-compartment Labtek culture chamber and induced to differentiate for 6 days. Myoblasts and myotubes were treated with 1 μg of cyanine 5-labeled mRNA and complexed with PE-rhodamine-labeled HLRC (DOTAP / DOPE / Coatsome SS-M) at an N / P ratio of 1.5 for 30 min, 2 h, and 24 h. For longer incubation times, cells were incubated with the treatment for 2 h before receiving fresh medium. After each time point, cells were fixed with 4% (v / v) paraformaldehyde in PBS for 15 min at ambient temperature. For myoblasts, the cell cytoplasm was stained with Phalloidin-Atto 488 (Sigma) diluted 1:20 in PBS for 1 h at ambient temperature, whereas the cell nuclei were counterstained with DAPI (20 mM stock, diluted 1:2000) for 1 h at ambient temperature. For myotubes, only the cell nuclei were stained with DAPI under the same conditions. Samples were finally mounted in Floromount mounting medium (Invitrogen) and imaged with a Zeiss LSM800 confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany) using a 63X lens.

[0119] Testing in vitro transfection into human induced pluripotent stem cells 37°C, 5% CO 2iPSC cells were cultured in a humid atmosphere at 37 °C with the medium StemMACSTM PSC-Brew XF (Miltenyi biotec). For the test of transfection efficiency, iPSCs were seeded in 24-well plates at a density of 50,000 cells per well. Then, iPSCs were treated with 3 μg of mRNA-LRC or mRNA-HLRC (DOTAP / DOPE / Coatsome SS-M) at an N / P ratio of 1.5. The transfection reagent Lipofectamine2000 (invitrogene®) was used as a transfection control for RNA experiments and was used according to the manufacturer's instructions. After 2 hours, the medium was replaced. 24 hours after transfection, cells were enzymatically detached using Tryple (Gibco), after which cells were stained with Live / Dead Violet solution to evaluate the percentage of dead cells, and fixed with 4% PFA solution at ambient temperature for 10 minutes. For flow cytometric analysis, data were acquired using a BD LSR 2 flow cytometer and analyzed using FlowJo software. Statistical analysis was performed using GraphPad Prism v8.1 software.

[0120] In vivo testing Intravenous Here, the same analysis as in Example 1 was used. Intramuscular To test the efficacy of the liposomal formulation in transfecting muscle fibers, 5 μg of mRNA-HLRC (DOTAP / DOPE / Coatsome SS-M) was injected into the tibialis anterior muscle of BALB / c mice at an N / P ratio of 1.5. To investigate the distribution of lipid nanoparticles and the protein expression of mRNA-HLRC, HLRC was labeled with PE-cy5 and an mRNA sequence expressing the fluorescent protein mCherry was selected. After 2 h, mice were sacrificed, tibialis anterior muscles were explanted and frozen in isopentane, and sections of these muscles were taken in a cryostat. The membranes of muscle fibers were labeled with anti-laminin 488 immunofluorescence labeling, and cell nuclei were counterstained with DAPI. Samples were finally mounted in Floromount mounting medium (Invitrogen) and observed with a Zeiss LSM800 confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany) using a 40X lens.

[0121] result Characterization of mRNA-loaded LRCs and HLRCs (DOTAP / DOPE / Coatsome SS-M or DOTAP / DOPE / DLin-MC3-DMA or DOTMA / DOPE / Coatsome SS-M) Complexation was optimized based on the nitrogen / phosphate (N / P) ratio, defined as the ratio of the amine groups of the lipid, which can be positively charged, to the phosphate groups of the nucleic acid, which can be negatively charged. All showed a hydrodynamic diameter of about 200 nm, with a positive surface charge in the LRC and a negative surface charge in the HLRC due to the hyaluronic acid coating. mRNA complexation was evaluated using electrophoresis (Figure 4). At all N / P ratios tested, mRNA complexed effectively with all systems. The higher the N / P ratios tested, the stronger the mRNA complexed, as shown by the decrease in the mRNA signal. To show that the weak mRNA signal at high N / P ratios was due to strong complexation of the mRNA and not its degradation, the formulations were incubated with heparin, a large anionic molecule, to decomplex the mRNA. When incubated with heparin, all the various formulations showed that the mRNA was indeed present.

[0122] Internalization and transfection efficiency of fluorescently labeled DOTAP / DOPE / Coatsome SS-M into THP-1 We developed a protocol using THP-1 cells treated with LRC-RNA encoding GFP at various N / P ratios compared to a commercially available transfection agent. THP-1 refers to a spontaneously immortalized monocyte-type cell line obtained from peripheral blood from a childhood acute monocytic leukemia case. THP-1 cells, including their genetically modified derivatives, represent an important tool in studying the structure and function of monocytes in both healthy and non-healthy subjects and are widely used in translation assays. We designed experiments aimed at investigating the translation efficiency, uptake and internalization of GFP transfection complexes. First, we optimized the transfection protocol and quantified GFP expression together with cell viability using a flow cytometer. Indeed, the exogenous introduction of nucleic acids into cells, in conjunction with the use of transfection agents, often induces considerable stresses that can ultimately affect cell viability. Important parameters were tested: i) LRC stability; ii) transfection agent and mRNA concentrations; iii) toxicity profile of the nanosystem; and iv) incubation period. As shown in Figure 5, all the lines were transfected with GFP. At the selected N / P ratio of 2, we achieved a transfection efficiency (approximately 70%) comparable to the commercial agent without significant effect on cell viability. Lines that were retained for further testing were prepared at an N / P ratio of 2 for both LRC and HLRC. The transfection efficiency and mean fluorescence intensity (MFI) were much greater than those at N / P ratios of 2.5, 3, 3.5, and 5. On the other hand, an N / P ratio of 1 was not retained due to its instability. We hypothesized that the increase in mRNA expression by these lipid mixtures could be due to increased uptake of mRNA upon complexation with LRC. This hypothesis was tested by the internalization kinetics of LRC using fluorescently labeled lines. These lines were complexed with GFP RNA at an N / P ratio of 2, which was the ratio that resulted in the highest transfection efficiency. Two fluorescent signals, one derived from lipid and one derived from GFP signal, were observed using flow cytometry at 2, 4, 24 and 48 hours after transfection.After 2 hours, lipid fluorescence was quantified in all cells: the signal was maintained up to 24 hours, with only a slight decrease observed after 48 hours.

[0123] Internalization and transfection efficiency of various nanosystems in C2C12 Transfection studies Transfection efficiency was tested by flow cytometry to assess the percentage of myoblasts expressing GFP (Figure 6). The data showed that in the DOTAP / DOPE / Coatsome SS-M and DOTAP / DOPE / Dlin-MC3-DMA formulations with N / P ratios of 1.5 and 3, approximately 90% of myoblasts expressed GFP, indicating a transfection efficiency similar to that of the commercial transfection agent used. In the DOTMA / DOPE / Coatsome SS-M formulation, this level was reduced to approximately 40%. In contrast, formulations composed of only neutral and cationic lipids (DOTAP / DOPE) or only pH-sensitive lipids (Coatsome SS-M / DOPE) showed only low transfection levels of approximately 20% under conditions similar to those of the previous formulations. Cells treated with mRNA alone did not show any signs of transfection, with a percentage similar to that of untreated cells.

[0124] Viability test To test the cytotoxicity induced by complexes with different N / P ratios, a Live / Dead viability assay was performed. Our results show that cells incubated with lipoplexes showed only a slight decrease in cell viability compared to the control, with up to 90% viable cells in all conditions, whereas the commercial product causes a decrease in cell viability of about 80%.

[0125] Internalization Test The intracellular distribution of mRNA complexed with HLRC (DOTAP / DOPE / Coatsome SS-M) in C2C12 myoblasts and myotubes was examined using confocal microscopy. In myoblasts, fluorescently labeled HLRC was shown to rapidly penetrate into the cells in a time-dependent manner. After 2 h of incubation, mRNA-HLRC accumulated in the cytoplasm in the perinuclear region without penetrating into the nucleus. After 24 h of incubation, HLRC appeared in the form of larger fluorescent clusters with a more diffuse signal, probably due to lipids being exchanged with the cells. The intracellular release of mRNA was also examined by assessing the colocalization between HLRC and mRNA signals. After only 30 min, mRNA was already found free in the cytoplasm and complexed with HLRC, whereas after 2 h, virtually no mRNA complexed with HLRC was found in the cytoplasm. After 24 h of incubation, no mRNA was found anymore in the intracellular medium. In myotubes, only a few HLRC were found in the cytoplasm after 24 h of incubation, even though myotubes were effectively transfected as indicated by GFP expression.More interestingly, GFP expression was more prominent in myotubes than in myoblasts, indicating promising efficacy for treating skeletal muscle.

[0126] Transfection efficiency of various nanosystems in iPSCs Transfection efficiency was tested by flow cytometry to evaluate the percentage of iPSCs expressing GFP (Figure 7). The results showed that LRC and HLRC of the DOTAP / DOPE / Coatsome SS-M composition had high transfection efficiency into iPSCs, with approximately 80% of cells expressing GFP, similar to the commercially available in vitro transfection agent Lipofectamine. Furthermore, no decrease in cell viability was observed.

[0127] In vivo testing of radiolabeled nanoparticles in healthy mice Intravenous Long circulatory time is required to maximize targeting of monocytes, which reside primarily in the blood compartment, bone marrow and spleen.Radiolabeling techniques were used to quantitatively measure the biodistribution of nanoparticles after systemic administration in healthy mice. 111 In-labeled liposomes were complexed with mRNA at a selected N / P ratio of 2 and then injected intravenously into BALB / c_Rj mice. SPECT / CT imaging was performed 6, 24 and 48 hours after injection. The dose of the injected radioactive agent was 3.30 MBq in LRC and 3.59 in HLRC. In both cases, significant particle accumulation was observed in the liver and spleen, with no difference between the two strains. The amount of radioactivity in each animal organ was quantified using a gamma counter, and the results were normalized and expressed in the form of %DI / g (injected amount per gram of tissue). It was observed that there was no significant difference in accumulation between LRC and HLRC, and this accumulation decreased over time in the liver and remained at higher values ​​in the spleen, reaching a peak 24 hours after IV injection. Indeed, in LRC, 34% of the DI / g was detected in the liver after 24 hours, whereas 60% was detected in the spleen. Similar results were obtained in HLRC, i.e. 39.1% were detected in the liver and 51.7% in the spleen.

[0128] Intramuscular After injection into the tibialis anterior muscle of healthy mice, the in vivo distribution of lipid nanoparticles and the protein expression of mRNA-HLRC were evaluated (Figure 8). After observing the sections using a confocal microscope, the images showed that HLRC was mainly distributed between muscle fibers and diffused into adjacent fibers 2 hours after administration. Moreover, slight expression of the protein encoded by the mRNA complexed to HLRC was already seen at the location of HLRC after only 2 hours of incubation, indicating the efficiency of this formulation in targeting and delivering mRNA to muscle fibers. No cellular or morphological damage was observed in the harvested sections.

[0129] Example 3 : Microfluidically fabricated liposomes as carriers for intracellular DNA delivery material and method material The same materials were used here as in Examples 1 and 2. pCAGIG plasmid DNA was obtained from Addgene (plasmid #11159).

[0130] Complexation of pDNA within liposomes Here, we followed the same protocol and the same formulation as described in Example 2. Plasmid DNA (pCAGIG) encoding the expression of green fluorescent protein (GFP) was complexed by mixing the appropriate amount of DNA with various liposomes to obtain lipoplexes with N / P ratios of 1.5 and 3. The lipoplexes were finally coated with HA (HLRC) at a DOTAP:HA weight ratio of 1:2.

[0131] Physicochemical analysis of pDNA-loaded liposomes Here, the same analysis as in Example 1 was used.

[0132] Analysis of pDNA-loaded liposomes by cryo-TEM The morphology of DOTAP / DOPE / Coatsome SS-M lipid nanoparticles was evaluated by transmission cryo-electron microscopy. Briefly, 3.5 μL of the formulation was added to a CF-2 / 1-3Cu-50 copper mesh and immersion frozen using a Vitrobot (Thermoscientific) to generate vitreous ice. Samples were stored in liquid nitrogen and observed using a JEM-1400Flash electron microscope operating at 120 kV.

[0133] In vitro transfection assay C2C12 myoblasts were cultured as previously described in Example 2.

[0134] Internalization Test The internalization efficiency of pDNA-HLRC (DOTAP / DOPE / Coatsome SS-M) into myoblasts was evaluated using flow cytometry and confocal microscopy. For flow cytometry analysis, cells were seeded at a density of 20,000 cells per well in flat-bottom 12-well plates. Myoblasts were then treated with PE-rhodamine-labeled HLRC loaded with 7.5 μg of pDNA at N / P ratios of 1.5 and 2. The internalization efficiency was evaluated after 2, 4, 24, and 48 hours of incubation. After 2 hours of post-incubation, the medium containing the treatment was replaced with fresh medium. After each incubation period, cells were washed, enzymatically detached using trypsin-EDTA (0.5%, Gibco), and stained with Fixable Live / Dead Violet solution to test cell viability. Data were acquired using a BD LSR 2 flow cytometer and analyzed using FlowJo software. Statistical analysis was performed using GraphPad Prism v8.1 software. For confocal microscopy analysis, the same conditions as in Example 2 were used.

[0135] Transfection and viability assay Transfection efficiency was tested by flow cytometry to evaluate the percentage of myoblasts expressing GFP. To this end, cells were seeded in flat-bottom 12-well plates at a density of 15000 cells per well. For a first detailed test of our main formulation, myoblasts were then treated with HLRC (DOTAP / DOPE / Coatsome SS-M) loaded with 5 μg, 7.5 μg and 10 μg of pDNA at N / P ratios of 1.5, 2 and 3. Jet-prime transfection reagent (Polyplus transfection®) was used as transfection control according to the manufacturer's instructions. All treatments were left overnight and then replaced with fresh medium. After 48 hours, cells were treated and analyzed as described above. For a second comparative test of the different formulations, myoblasts were then treated with 3 μg of pDNA complexed with the different nanosystems at N / P ratios of 1.5 and 3. The commercial transfection reagent Jet-Prime (Polyplus®) was used as a transfection control according to the manufacturer's instructions. All treatments were left for 2 h before being transferred to fresh medium and internalization efficiency was assessed after 48 h of incubation. Cells were then enzymatically detached using Trypsin-EDTA (0.5%, Gibco) and stained with Fixable Live / Dead Violet solution to assess cell viability. Data were acquired using a BD LSR 2 flow cytometer and analyzed using FlowJo software. Statistical analysis was performed using GraphPad Prism v8.1 software.

[0136] result Characterization of pDNA-loaded HLRCs (DOTAP / DOPE / Coatsome SS-M or DOTAP / DOPE / DLin-MC3-DMA or DOTMA / DOPE / Coatsome SS-M) pDNA-loaded HLRCs were obtained by complexation of pDNA with microfluidically fabricated liposomes. All lipoplexes showed an average diameter of about 200 nm and a polydispersity index of less than 0.2 at the various N / P ratios tested, and showed a negative surface charge (about -20 mV) due to the phosphate and carboxyl groups of DNA and HA (Figure 9, A and B). pDNA complexation was evaluated using electrophoretic measurements. At all N / P ratios tested, pDNA was effectively and completely complexed with all systems. As in Example 2, the higher the N / P ratio tested, the stronger the mRNA was complexed, as shown by the decrease in the mRNA signal (Figure 9C for DOTAP / DOPE / Coatsome SS-M formulation). Morphological analysis performed using cryoTEM (Figure 9D for DOTAP / DOPE / Coatsome SS-M formulation) showed that pDNA-loaded HLRCs formed vesicles with a multilamellar structure in which the nucleic acid was trapped between different lipid bilayers, thereby ensuring its protection.

[0137] Internalization efficiency The internalization efficiency of pDNA-loaded HLRC (DOTAP / DOPE / Coatsome SS-M) into muscle cells was examined by flow cytometry (Figure 10). The percentage of cells labeled with PE-rhodamine and pDNA-loaded HLRC was analyzed to quantitatively examine the internalization efficiency (Figure 10). Flow cytometry analysis showed rapid and efficient internalization of pDNA-loaded HLRC for two N / P ratios, with virtually all cells being positive for rhodamine signal after only 2 hours of incubation. The degree of internalization remained constant up to 48 hours, implying that pDNA-loaded HLRC was not completely expelled from cells, and nanoparticles were transferred from cell to cell during cell proliferation. The intracellular distribution of pDNA-loaded HLRC in C2C12 myoblasts was examined using confocal microscopy. In myoblasts, HLRC labeled with PE-rhodamine and loaded with pDNA was shown to rapidly penetrate into cells in a time-dependent manner. After 2 h of incubation, HLRC loaded with pDNA accumulated in the cytoplasm in the perinuclear region without penetrating into the nucleus. After 24 h of incubation, lipoplexes are observed in the form of larger fluorescent clusters with a more diffuse signal, probably due to partial exchange of lipids with the cells.

[0138] Cell transfection efficiency Transfection efficiency was tested by flow cytometry to assess the percentage of myoblasts expressing GFP. For the first detailed test of our lead formulation, DOTAP / DOPE / Coatsome SS-M (Figure 11), the data showed that the transfection efficiency was dependent on the N / P ratio, with the lowest N / P ratio increasing the transfection percentage of cells. Furthermore, the data showed that, similar to commercial in vitro transfection agents, more than 60% of cells expressed GFP in HLRCs loaded with pDNA at an N / P ratio of 1.5, indicating the efficiency of this formulation in targeting and delivering pDNA into myoblasts. Interestingly, varying the treatment dose only slightly increased the transfection efficiency. Cells treated with pDNA alone did not show any signs of transfection, and the transfection percentage was similar to that of untreated cells. Furthermore, slight expression of the protein encoded by the mRNA complexed to the HLRC was already seen at the location of the HLRC after only 2 hours of incubation, indicating the efficiency of this formulation in targeting and delivering mRNA to muscle fibers. No cellular or morphological damage was observed in the harvested sections. For the second comparative study of the various formulations (Figure 12), the data showed that in the DOTAP / DOPE / Coatsome SS-M and DOTMA / DOPE / Coatsome SS-M formulations with N / P ratios of 1.5 and 3, approximately 30% of myoblasts expressed GFP, indicating a transfection efficiency similar to that of the commercial in vitro transfection agent used. For the DOTAP / DOPE / DLin-MC3-DMA formulation, this level increased to approximately 60%, thereby indicating a superior efficiency to the commercial in vitro transfection agent used. In contrast, formulations composed of only neutral and cationic lipids (DOTAP / DOPE) or only pH-sensitive lipids (Coatsome SS-M / DOPE) showed very low transfection levels of less than 10% under similar conditions to the previous formulations. Cells treated with pDNA alone did not show any signs of transfection, with rates similar to those of untreated cells.

[0139] Cell viability assay To test the cytotoxicity induced by pDNA-HLRC(DOTAP / DOPE / Coatsome SS-M) complexes at various N / P ratios tested in the first transfection study, a viability assay (Live / Dead) was performed (Figure 13). Our results showed that cells incubated with lipoplexes displayed a decrease in cell viability (50-75%) compared to the control. The commercial transfection agent Jet-prime showed the same trend, with a cell viability of about 65%. This decrease in cell viability correlated with the overnight treatment period, suggesting that shorter incubation times may improve cell viability without negatively affecting transfection efficiency. In the second transfection study, our results showed that cells incubated with lipoplexes for 2 h displayed only a slight decrease in cell viability, with up to 90% viable cells in each condition.

[0140] Example 4 : Microfluidically fabricated liposomes as carriers for intracellular delivery of antisense oligonucleotides material and method material Here, the same materials were used as in the previous examples: the antisense oligonucleotide ASO-Cyanine 5 supplied by Eurogentec, which has 14 nucleotides.

[0141] Antisense oligonucleotide (ASO) complex formation Here, the same protocol was followed as described in Example 2. The antisense oligonucleotide tested is ASO-Cyanine 5, designed to target a specific DNA sequence and induce its cleavage. The sequence ASO-Cyanine 5 was complexed by mixing an appropriate amount of ASO with lipid nanoparticles (DOTAP / DOPE / Coatsome SS-M) to obtain lipoplexes with N / P ratios of 2, 4, 8 and 10. The lipoplexes were finally coated with HA (HLRC) at a DOTAP:HA weight ratio of 1:2.

[0142] Physicochemical analysis of ASO-liposomes Here, the same analysis as in Example 1 was used.

[0143] cell culture C2C12 myoblasts and myotubes were cultured as previously described in Example 2. Uptake and intracellular distribution of ASO-HLRC The intracellular distribution of ASO-HLRC was examined by confocal microscopy. To this end, myoblasts were seeded at a density of 10000 cells / well in 24-well multiwell plates on 12 mm glass slides, whereas myoblasts were seeded at a density of 40000 cells / well on a 4-compartment Labtek culture chamber to obtain myotubes and induced to differentiate for 6 days. Myoblasts were treated with 1 μg ASO-Cyanine5-HLRC for 30 min, 2 h and 24 h, and myotubes were treated with 1 μg ASO-Cyanine5-HLRC at an N / P ratio of 10 for 2 h and 48 h. For longer incubation times, cells were incubated for 2 h before being fed with fresh medium. After each time point, cells were fixed with 4% (v / v) paraformaldehyde in PBS for 15 min at ambient temperature. The cell cytoplasm was stained with Phalloidin-Atto 488 (Sigma) diluted 1:20 in PBS for 1 h at ambient temperature, whereas the cell nuclei were counterstained with DAPI (20 mM stock, diluted 1:2000) for 1 h at ambient temperature. For myotubes, only the cell nuclei were stained with DAPI under the same conditions. Samples were finally mounted in Floromount mounting medium (Invitrogen) and imaged with a Zeiss LSM800 confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany) using a 63X lens.

[0144] result Characterization of ASO-Cyanine5-HLRC ASO-cyanine5-HLRC lipoplexes were obtained by complexation of ASO-cyanine5 with microfluidically fabricated liposomes. The lipoplexes showed an average diameter of about 150 nm and a polydispersity index of less than 0.2 for all N / P ratios. The surface potential was negative (about -20 mV) due to the ASO sequence and the phosphate and carboxyl groups of HA. Gel electrophoresis was used to test the complexation of ASO with the HLRC nanosystem. At N / P ratios of 2 to 8, ASO was partially complexed with liposomes. This complexation was enhanced at an N / P ratio of 10.

[0145] Internalization and subcellular distribution of ASO-Cyanine5-HLRC The intracellular distribution of ASO-Cyanine5-HLRC in C2C12 myoblasts and myotubes was examined using confocal microscopy. In myoblasts, HLRC labeled with fluorescent lipids was shown to rapidly penetrate into the cells in a time-dependent manner. After 2 h of incubation, ASO-Cyanine5-HLRC accumulated in the cytoplasm in the perinuclear region without penetrating into the nucleus. After 24 h of incubation, ASO-Cyanine5-HLRC appeared in the form of larger fluorescent clusters with a more diffuse signal, probably due to lipid exchange with the cells. The intracellular release of ASO was also examined by assessing the colocalization between HLRC and ASO signals. After 30 min of incubation, ASO was virtually completely complexed with HLRC, whereas after 24 h of incubation, ASO was no longer complexed with HLRC in the cytoplasm. The same overall trend was observed for myotubes. After 2 h of incubation, ASO-Cyanine5-HLRC was observed to enter myotubes and the ASO was fully complexed with HLRC. After 24 h of incubation, HLRC was found in free form in the cytoplasm and the ASO was no longer complexed with HLRC, demonstrating the efficiency of HLRC nanoparticles in delivering ASO sequences to myotubes and paving the way for their application in skeletal muscle treatment.

[0146] Example 5: Microfluidically fabricated liposomes as carriers for intracellular siRNA delivery material and method material Here, the same materials were used as in the previous examples. The interfering RNA is an RNA with 21 nucleotides provided by Eurogentec. siRNA complex formation Here, we followed the same protocol as described in Example 2. Interfering RNA was complexed by mixing an appropriate amount of siRNA with lipid nanoparticles (DOTAP / DOPE / Coatsome SS-M) to obtain lipoplexes with N / P ratios of 2, 4, 8 and 10. The lipoplexes were finally coated with HA (HLRC) at a DOTAP:HA weight ratio of 1:2.

[0147] Physicochemical analysis of siRNA-liposomes Here, the same protocol as described in Example 1 was followed.

[0148] In vitro transfection assay C2C12 myoblasts were cultured as previously described in Example 2.

[0149] Transfection efficiency test Transfection efficiency was tested by flow cytometry to assess the percentage of myoblasts expressing GFP. Myoblasts were seeded at a density of 15000 cells per well in flat-bottom 12-well plates. First, myoblasts were treated with 0.5 μg, 1 μg and 2 μg of siRNA-HLRC at an N / P ratio of 10, followed by pCAGIG treatment (plasmid DNA encoding GFP) using Jet-prime transfection reagent (Polyplus transfection®) according to the manufacturer's instructions. All treatments were left for 2 hours before transferring to fresh medium. After 48 hours, cell culture medium was harvested and cells were enzymatically detached using trypsin-EDTA (0.5%, Gibco). Data were acquired using a BD LSR 2 flow cytometer and analyzed using FlowJo software. Statistical analysis was performed using GraphPad Prism v8.1 software.

[0150] Viability test After 48 hours of incubation, cells were stained with Fixable Live / Dead Violet solution to assess cell viability. Data were acquired using a BD LSR 2 flow cytometer and analyzed using FlowJo software. Statistical analysis was performed using GraphPad Prism v8.1 software.

[0151] result Characterization of siRNA-HLRC siRNA-HLRC was obtained by complexation of siRNA with microfluidically fabricated liposomes (DOTAP / DOPE / Coatsome SS-M). The lipoplexes showed an average diameter of about 150 nm and a polydispersity index of less than 0.2 for all N / P ratios. The surface potential was negative (about -20 mV) due to the phosphate and carboxyl groups of RNA and HA. A gel electrophoresis study was used to test the complexation of siRNA with the HLRC nanosystem. At N / P ratios of 2 to 8, siRNA was partially complexed with the nanoparticles. This complexation was enhanced at an N / P ratio of 10, as indicated by the absence of RNA signal running on the gel.

[0152] Cell transfection efficiency Transfection efficiency was tested by flow cytometry to assess the percentage of myoblasts expressing GFP (Figure 14). Using a commercial agent to induce GFP expression, myoblasts were transfected with pCAGIG pDNA to test the inhibitory effect of siRNA sequences complexed with HLRC on GFP expression. Data showed that cells transfected with pCAGIG appeared to be approximately 70% GFP+ cells. The percentage of cells expressing GFP was lower in cells treated with siRNA-HLRC than in cells transfected with siRNA using the commercial transfection agent Jet-prime, indicating that the HLRC formulation is more efficient in delivering siRNA than the commercial agent. The transfection efficiency of HLRC showed a dose-dependent profile, with less than 30% GFP+ cells treated with the highest dose of siRNA complexed with HLRC. Thus, it was demonstrated that siRNA complexed with HLRC can halve the percentage of cells expressing GFP.

[0153] Cell viability A viability assay (Live / Dead) was performed to test the cytotoxicity induced by the complexes at various N / P ratios. Our results showed that cells incubated with all different doses of siRNA-HLRC did not exhibit any decrease in cell viability.

[0154] Example 6 : Microfluidically fabricated liposomes as carriers for intracellular co-administration of mRNA and pDNA material and method material The same materials were used here as in the previous examples: GFP mRNA used in this example was purchased from Tebuo (Le Perray-en-Yvelines, France) and the mScarlet_pcDNA3.1 plasmid DNA encoding the fluorescent protein mScarlet was obtained from Genscript.

[0155] Complex formation of mRNA and pDNA Here, we followed the same protocol as described in Example 2. Two different nucleic acids were complexed with the LRC (DOTAP / DOPE / Coatsome SS-M) by mixing the liposomes with appropriate amounts of mRNA and pDNA to obtain lipoplexes with N / P ratios of 1.5 and 3 and a mRNA:pDNA weight ratio of 1:2.

[0156] In vitro transfection assay C2C12 myoblasts were cultured as described above in Example 2. Transfection efficiency was tested by flow cytometry to assess the percentage of myoblasts expressing GFP and mScarlet simultaneously. Myoblasts were seeded at a density of 15000 cells per well in flat-bottom 12-well plates. Cells were treated with mRNA-pDNA-LRC at an N / P ratio of 1.5 and 3, in an amount equivalent to 3 μg of mRNA and 6 μg of pDNA per well. All treatments were left for 2 hours before transferring to fresh medium. After 48 hours, cells were enzymatically detached using trypsin-EDTA (0.5%, Gibco) and stained with Fixable Live / Dead Violet solution to assess cell viability. Data were acquired using a BD LSR 2 flow cytometer and analyzed using FlowJo software. Statistical analysis was performed using GraphPad Prism v8.1 software.

[0157] result Characterization of mRNA-pDNA-LRCs Complexation of mRNA and pDNA with microfluidically fabricated liposomes yielded mRNA-pDNA-LRC. The lipoplexes showed an average diameter of approximately 150 nm and a polydispersity index of less than 0.2 for all N / P ratios. Gel electrophoresis also demonstrated that mRNA and pDNA were efficiently complexed in the same system at N / P ratios of 1.5 and 3.

[0158] Cell transfection efficiency Transfection efficiency was tested by flow cytometry to evaluate the total percentage of myoblasts expressing GFP, mScarlet or both fluorescent proteins simultaneously (Figure 15). Although the cells were carrying two nucleic acids in LRC, about 80% of myoblasts expressed GFP induced by mRNA as in Example 2. Regarding pDNA-induced mScarlet expression, about 30% of myoblasts were positive for this protein. Even more interestingly, about 30% of myoblasts expressed both GFP and mScarlet, indicating the efficiency of this formulation in delivering different nucleic acids simultaneously, paving the way for gene editing applications such as CRISPR / Cas9.

Claims

1. (a) at least one cationic lipid; (b) at least one neutral lipid; (c) at least one pH-sensitive lipid different from the cationic lipid (a); Nanoparticles comprising:

2. The nanoparticle of claim 1, which is loaded with a nucleic acid.

3. 2. The nanoparticles according to claim 1, which are coated with at least one anionic polysaccharide, preferably hyaluronic acid.

4. 10. The nanoparticles of claim 1, which do not contain cholesterol or even any steroids.

5. 2. The nanoparticles of claim 1, wherein the molar ratio of the cationic lipid to the other lipid is in the range of 1:1 to 2:

1.

6. Cationic lipids include, alone or in mixtures, 1,2-dioleoyloxypropyl-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), N-(2,3-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride (DOTMA), dimethyldioctadecylammonium bromide (DDAB), 1,2-dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DDAB), and 1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium bromide (DDAB). quaternary ammonium such as DORIE, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (DOEPC), N-N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), and N-methyl-4(dioleyl)methylpyridinium chloride (SAINT-2); The cationic lipid is preferably DOTAP. The nanoparticles of claim 1.

7. the neutral lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and N'-(rac-1-[11-(F-octyl)undec-10-enyl]-2-(hexadecyl)glycero-3-phosphoethanoyl)sperminecarboxamide), alone or in mixture; The neutral lipid is preferably DOPE. The nanoparticles of claim 1.

8. The pH-sensitive lipid comprises at least one of formula (I): 【Chemistry 1】 [During the ceremony, RCOO is a group selected from myristoyl, α-D-tocopherol succinoyl, linoleyl, and oleoyl; and X is the following structure (II), (III) or (IV): 【Chemistry 2】 is selected from groups having the formula: The nanoparticle of claim 1, wherein the nanoparticle is a lipid.

9. The pH-sensitive lipid is at least one lipid of formula (I), wherein: RCOO is a myristoyl group and X is of formula (II); RCOO is an α-D-tocopherol succinoyl group and X is a group of formula (II) or formula (III); RCOO is a linoleyl or oleoyl group and X is a group of formula (III); or RCOO is an oleoyl group and X is a group of formula (IV) The nanoparticles according to claim 8.

10. The pH-sensitive lipid is at least one lipid of formula (I), wherein RCOO is a myristoyl group and X is a group of formula (II): 【Transformation 3】 9. The nanoparticle of claim 8, wherein the group is

11. (a) at least DOTAP as a cationic lipid; (b) at least DOPE as a neutral lipid; (c) at least one pH-sensitive lipid represented by the formula (I) wherein RCOO is a myristoyl group and X is a group of formula (II): 【Chemistry 4】 is a group represented by the formula: of lipids and 11. The nanoparticle of claim 10, comprising:

12. The nanoparticle of claim 1 , wherein the pH-sensitive lipid comprises a tertiary amine group.

13. 13. The nanoparticles of claim 12, wherein the pH-sensitive lipid is selected from 1,2-dilinoleyloxy-n,n-dimethyl-3-aminopropane (DLinDMA), O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) (DLin-MC3-DMA), and 2-[2,2-bis[(9Z,12Z)-octadeca-9,12-dienyl]-1,3-dioxolan-4-yl]-N,N-dimethylethanamine (DLin-KC2-DMA), alone or in mixture.

14. (a) at least DOTAP as a cationic lipid; (b) at least DOPE as a neutral lipid; (c) At least DLin-MC3-DMA as pH-sensitive lipid 14. The nanoparticle of claim 13, comprising:

15. Nanoparticles according to any one of claims 1 to 14, carrying a nucleic acid which is RNA.

16. 16. The nanoparticle of claim 15, carrying a nucleic acid that is mRNA.

17. 16. The nanoparticle of claim 15, carrying a nucleic acid that is an interfering RNA.

18. Nanoparticles according to any one of claims 1 to 14, carrying a nucleic acid which is DNA.

19. 15. Nanoparticles according to any one of claims 1 to 14, loaded with a mixture of RNA and DNA, preferably in a weight ratio of RNA:DNA in the range of 1:1 to 1:10, preferably in the range of 1:1 to 1:5, preferably in the range of 1:1 to 1:3, even more preferably 1:

2.

20. 20. The nanoparticle of claim 19, which carries a mixture of plasmid DNA (pDNA) and mRNA.

21. 15. The nanoparticles according to any one of claims 1 to 14, wherein the nanoparticles are loaded with nucleic acid such that the N / P ratio is in the range of 1 to 100, preferably in the range of 1 to 60, preferably in the range of 1 to 50, preferably in the range of 1 to 30, even more preferably in the range of 1 to 20, preferably in the range of 1 to 10, preferably in the range of 1 to 8, even more preferably in the range of 1.5 to 8, preferably in the range of 1 to 5, even more preferably in the range of 1.5 to 4.

Images