Formulations composed of cationic lipids and poly(lactic-co-glycolic acid) for delivery of polynucleotides to cells

Abstract

The present invention relates to a) at least one poly(lactic-co-glycolide); b) at least one cationic surfactant; c) at least one polynucleotide; d) optionally, at least one additive, The poly(lactide-co-glycolide) has a weight average molecular weight Mw of 1000-9500 g / mol as measured by gel permeation chromatography using polystyrene standards and chloroform. The present invention further relates to a method of forming the polynucleotide delivery particles according to the present invention, the particles being formed by nanoprecipitation or nanoemulsion techniques. The present invention further relates to oral or parenteral drug delivery compositions comprising at least one polynucleotide delivery particle according to the present invention, and their use as pharmaceuticals.

Description

[Technical field]

[0001] The present invention relates to a) at least one poly(lactic-co-glycolide); b) at least one cationic surfactant; c) at least one polynucleotide; d) optionally, at least one additive, The poly(lactide-co-glycolide) has a weight average molecular weight Mw of 1000-9500 g / mol as measured by gel permeation chromatography using polystyrene standards and chloroform. The present invention further relates to a method of forming the polynucleotide delivery particles according to the present invention, the particles being formed by nanoprecipitation or nanoemulsion techniques. The present invention further relates to oral or parenteral drug delivery compositions comprising at least one polynucleotide delivery particle according to the present invention, and their use as pharmaceuticals. [Background technology]

[0002] Polynucleotide-based drugs are a novel class of therapeutic agents that have emerged over the past 10-20 years. These drugs hold great promise for enabling novel treatment options for cancer therapy and vaccination, as well as for previously difficult-to-drug diseases.

[0003] The main obstacle to the successful application of polynucleotide therapeutics is the delivery to the site of action, i.e., cytoplasm or cell nucleus.Polynucleotides are large biomolecules, and are generally susceptible to chemical and enzymatic degradation, and do not easily enter cells.Therefore, suitable formulations must provide protection against all the hazardous environments that polynucleotide drugs inevitably encounter when applied locally, systemically, or orally.In addition, formulations must mediate the uptake of polynucleotide drugs into target cells, and ultimately promote their release from endosomal compartments into cytoplasm.

[0004] Among the many non-viral formulations known in the art, so-called lipid nanoparticles (LNPs) are currently the most advanced platform. As a result, LNPs are the delivery vehicles for the first generation of commercially approved siRNA- and mRNA-based drugs. LNPs are composed of up to four different types of surfactants (lipids) (cationic / ionizable surfactants, PEG surfactants, cholesterol, and phospholipids), which makes the formulation rather complex and expensive, especially in terms of raw material supply. Furthermore, the mandatory use of PEG surfactants raises concerns about the possibility of immunogenic reactions that may be caused by the presence of anti-PEG antibodies in a subset of the population.

[0005] Nanoparticles and microparticles formed from poly(lactic-co-glycolic acid) (PLGA) are another widely used drug delivery platform due to their excellent biocompatibility. However, PLGA itself is a rather unsuitable material for encapsulation of hydrophilic charged macromolecules, such as the case of polynucleotides, due to its charge-neutral and hydrophobic properties. Combination of PLGA with positively charged excipients, such as calcium phosphate, to increase the association with polynucleotides has been proposed as a workaround. Nevertheless, these emulsion-based strategies suffer from problems such as complex multi-step protocols, poor encapsulation efficiency, and large particle size.

[0006] Cationic / ionizable lipids and polynucleotides alone cannot coassemble into efficient nanoparticles, so helper surfactants (lipids) (cholesterol, phospholipids, PEG lipids) are a fundamental requirement for forming LNPs. Therefore, the object of the present invention was to provide PLGA-based polynucleotide delivery particles that can overcome one or more of the above-mentioned disadvantages.

[0007] In this regard, the inventors of the present invention have surprisingly found that when cationic lipids are applied as positively charged excipients, polynucleotides can be entrapped in PLGA particles using a simple mixing protocol without the need for additional surfactants or helper lipids. Furthermore, improved cell transfection can be obtained when the poly(lactide-co-glycolide) has a weight-average molecular weight Mw of 1000-9500 g / mol.

[0008] Furthermore, the absence of PEG, as required in LNPs, allows the lipid / PLGA particles to be coated with other materials, such as cell-penetrating peptides (e.g., human lactoferrin protein or fragments thereof), to tailor particle surface properties and enhance functionality.

[0009] Summary of the Invention In a first aspect, the present invention provides a method for producing a composition comprising the steps of: a) at least one poly(lactic-co-glycolide); b) at least one cationic surfactant; c) at least one polynucleotide; d) optionally, at least one additive, Here, poly(lactide-co-glycolide) refers to polynucleotide delivery particles having a weight average molecular weight Mw of 1000-9500 g / mol, preferably 2000-6800 g / mol, more preferably 4000-6800 g / mol, and most preferably 6000-6800 g / mol, as measured by gel permeation chromatography using polystyrene standards and chloroform.

[0010] A second aspect relates to a method of forming a polynucleotide delivery particle according to the invention, the particle being formed by nanoprecipitation or nanoemulsion techniques.

[0011] In a third aspect, the present invention relates to an oral drug delivery composition comprising at least one polynucleotide delivery particle according to the present invention.

[0012] In a fourth aspect, the present invention relates to a parenteral drug delivery composition comprising at least one polynucleotide delivery particle according to the present invention.

[0013] In a fifth aspect, the present invention relates to an oral drug delivery composition according to the present invention or a parenteral drug delivery composition according to the present invention for use as a medicament. [Brief description of the drawings]

[0014] [Figure 1] Agarose gel electrophoresis of free mRNA and different DODMA:PLGA based particle samples. 1 μg of mRNA or an equal amount of particles was applied per well. [Diagram 2] Agarose gel electrophoresis of free mRNA and different DOTMA:PLGA based particle samples. 1 μg of mRNA or an equal amount of particles was applied per well. [Diagram 3] Figure 1 shows the transfection efficiency of different DODMA:PLGA-based particle samples in HeLa cells after 24 hours of incubation, 100 ng of mRNA was applied per well for each condition. [Figure 4] Figure 1 shows the transfection efficiency of different DOTMA:PLGA based particle samples in HeLa cells after 24 hours of incubation, 100 ng of mRNA was applied per well for each condition. [Diagram 5] Figure 1 shows the transfection efficiency of different DOTMA:PLGA based particle samples in HeLa cells after different incubation times, 100 ng of mRNA was applied per well for each condition. [Figure 6] Figure 1 shows the transfection efficiency of DOTMA:PLGA particles coated with different amounts of hLFF in Caco-2 cells after 24 hours of incubation. 100 ng of mRNA was applied per well for each condition. [Figure 7]FIG. 1 shows the release kinetics of DOTMA:PLGA particles after dissolution assay in 0.1 N HCl (0-120 min) and phosphate buffer pH 6.8 (120-180 min) obtained by Ribogreen assay (representative from n=2). [Figure 8] Figure 1 shows the transfection efficiency of DOTMA:PLGA particle samples in HeLa cells after different pretreatments and 24 h incubation. 100 ng of mRNA was applied per well for each condition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) at least one poly(lactic-co-glycolide) (also referred to as PLGA); b) at least one cationic surfactant; c) at least one polynucleotide; d) optionally, at least one additive, Here, poly(lactide-co-glycolide) refers to polynucleotide delivery particles having a weight average molecular weight Mw of 1000-9500 g / mol, preferably 2000-6800 g / mol, more preferably 4000-6800 g / mol, and most preferably 6000-6800 g / mol, as measured by gel permeation chromatography using polystyrene standards and chloroform.

[0016] The polynucleotide delivery particles can be nanoparticles or microparticles. In certain embodiments, the particles have a D(v,0.5) value of 50-500 nanometers and / or a z-average particle size of 1-1000 nanometers, preferably 20-500 nanometers, more preferably 20-200 nanometers.

[0017] The term "particles" as used herein preferably refers to particles having a size less than 10 μm (10,000 nm), such as in the range of about 1 nm to 25 nm, 50 nm, 100 nm, 250 nm, 500 nm, 1000 nm (1 μm), 2,500 nm (2.5 μm), 5,000 nm (5 μm), or 10,000 nm (10 μm). In some embodiments, the dry particles may be present in aggregates that are greater than 10,000 nm in diameter but disperse to particle sizes less than 10,000 nm upon addition of an aqueous fluid and mixing using techniques such as vortexing. In some embodiments, the particles described herein may be approximately spherical. In some embodiments, the particles described herein may be irregularly shaped. The particles in the compositions of the invention typically have a size distribution in aqueous fluid having a z-average and / or D(v,0.5) value in the range of less than 5,000 nm, for example, from 5,000 nm to 2,500 nm, to 1,000 nm, to 500 nm, to 250 nm, to 100 nm, to 50 nm, or to 1 nm.

[0018] As used herein, a "nanoparticle" is a particle having a size distribution in an aqueous fluid where the z-average ranges from 1 nm to 500 nm. As used herein, a "microparticle" is a particle having a size distribution in an aqueous fluid where D(v,0.5) ranges from 500 nm to 5000 nm.

[0019] Particle size can be determined (measured) using methods available in the art. For example, particle size can be determined using photon correlation spectroscopy, dynamic light scattering or quasi-elastic light scattering. These methods are based on the correlation of particle size with the diffusion properties of the particles obtained from Brownian motion measurements. Brownian motion is the random movement of particles due to collisions with the solvent molecules surrounding the particle. The larger the particle, the slower the Brownian motion. The speed is defined by the translational diffusion coefficient (D). The measurement refers to how the particle moves in the liquid (hydrodynamic diameter). The diameter obtained is the diameter of a sphere with the same translational diffusion coefficient as the particle.

[0020] Particle size can also be determined using static light scattering, which measures the intensity of light scattered by particles in a solution at a single time. Static light scattering measures light intensity as a function of scattering angle and solute concentration. Particles passing through a light source, e.g., a laser beam, scatter light at angles inversely proportional to their size. Large particles produce diffraction patterns at low scattering angles of high intensity, while small particles give rise to signals of low intensity at wide angles. Particle size distribution can be calculated when the intensity of light scattered from a sample is measured as a function of angle. The angular information is compared to a scattering model (e.g., Mie theory) to calculate the size distribution.

[0021] Generally, particle size is determined at room temperature and involves analyzing a sample of interest multiple times (eg, performing at least three replicate measurements on the same sample) to obtain an average particle size.

[0022] This value is preferably determined by dynamic light scattering, more preferably in accordance with DIN ISO 22412:2018-09.

[0023] The polynucleotide delivery particles may have a polydispersity index of 0.01 to 0.5, preferably measured by dynamic light scattering, more preferably according to DIN ISO 22412:2018-09.

[0024] In one embodiment, the polynucleotide delivery particles according to the present invention have an N / P ratio of cationic surfactant to polynucleotide of 1:1 to 50:1, preferably 5:1 to 20:1, and more preferably the N / P ratio is 8.

[0025] In one embodiment, polynucleotide delivery particles according to the invention have a weight ratio of poly(lactide-co-glycolide) to polynucleotide of 1-200, or 2-150, or 5-100.

[0026] The polynucleotide delivery particles comprise at least one poly(lactide-co-glycolide) having a weight average molecular weight Mw of 1000-9500 g / mol, preferably 2000-6800 g / mol, more preferably 4000-6800 g / mol, and most preferably 6000-6800 g / mol, as measured by gel permeation chromatography using polystyrene standards and chloroform.

[0027] In one embodiment, the at least one poly(lactide-co-glycolide) has a number average molecular weight Mn of 1000 to 3000 g / mol, preferably 2000 to 2800 g / mol, as measured by gel permeation chromatography using polystyrene standards and chloroform.

[0028] In one embodiment, the at least one poly(lactic-co-glycolide) has a molar ratio of lactide to glycolide ranging from 40:60 to 60:40, preferably 50:50.

[0029] In one embodiment, the at least one poly(lactide-co-glycolide) has an intrinsic viscosity, measured by viscometry, of 0.05 to 0.25 dl / g, preferably 0.08 to 0.16.

[0030] In one embodiment, the at least one poly(lactide-co-glycolide) has an acid number of 20 to 30, preferably 22.5 mg KOH / g, preferably measured according to DIN EN 14104:2021-04.

[0031] In one embodiment, the at least one poly(lactic-co-glycolide) is present at 0.26 to 98.5% by weight, based on the total weight of the polynucleotide delivery particle.

[0032] Suitable poly(lactic acid-co-glycolide) polymers are commercially available under the trade name RESOMER® from Evonik Industries AG, such as, for example, RESOMER® RG501H or RESOMER® Condensate RG Polymer.

[0033] The particles include at least one cationic surfactant.

[0034] The term "surfactant" is derived from the phrase "surface active agent." Surfactants accumulate at interfaces (e.g., liquid-liquid, liquid-solid, and / or liquid-gas interfaces) and change the properties of the interface. As used herein, surfactants include detergents, dispersants, suspending agents, emulsion stabilizers, neutral lipids, ionized lipids, cationic lipids, and anionic lipids.

[0035] The cationic surfactant is provided to impart an electric charge to the particles.

[0036] In one embodiment, the at least one cationic surfactant is 1,2-di-O-octadecenyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium-propane, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide, N 4-cholesteryl-spermine, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, O,O'-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine, 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 1,2-distearoyl-sn-glycero-3-ethylphosphocholine, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, In one embodiment, the glycerol salt is selected from the group consisting of 1,2-dimyristoleyl-sn-glycero-3-ethylphosphocholine, 1,2-dimyristoleyl-sn-glycero-3-ethylphosphocholine, dimethyldioctadecylammonium, 1,2-dimyristoyl-3-trimethylammonium-propane, 1,2-dipalmitoyl-3-trimethylammonium-propane, 1,2-stearoyl-3-trimethylammonium-propane, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium and the salt of 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol.

[0037] In one embodiment, the at least one cationic surfactant is a 1,2-di-O-octadecenyl-3-trimethylammonium propane salt, preferably a 1,2-di-O-octadecenyl-3-trimethylammonium propane chloride salt.

[0038] In one embodiment, the at least one cationic surfactant is present at 0.85 to 98% by weight, based on the total weight of the polynucleotide delivery particle.

[0039] In this regard, various salt forms of the above-mentioned cationic surfactants can be provided, such as halides and hydrohalides, such as chloride, bromide, iodide, and hydrochloride.When a specific salt is listed (e.g., chloride), it should be understood that other salts (e.g., bromide, iodide, etc.) can be used as well.In one embodiment, the preferred salts of cationic surfactants are chloride and bromide, and more preferably, chloride salt.

[0040] The particles of the present invention comprise at least one polynucleotide.

[0041] As used herein, the term "polynucleotide" refers to a homopolymer or heteropolymer of at least two nucleotide units (also referred to herein as "nucleotides"). Nucleotides forming a polynucleotide as defined herein include naturally occurring nucleotides such as ribonucleotides and deoxyribonucleotides, as well as equivalents, derivatives, variants and analogs of naturally occurring nucleotides.

[0042] In one embodiment, the polynucleotide consists of 10 to 15,000 nucleotides, preferably 20 to 5,000 nucleotides, more preferably 500 to 4,500 nucleotides.

[0043] Polynucleotides can be in either single-stranded or multi-stranded form (e.g., double-stranded, triple-stranded, etc.). Polynucleotides can be in linear or non-linear form (e.g., containing circular, branched, etc. elements). Polynucleotides can be natural, synthetic, or a combination of both.

[0044] Polynucleotide can be capable of self-replicating when introduced into host cell.Thus, examples of polynucleotide include self-replicating RNA and DNA, for example, selected from replicon, plasmid, cosmid, phagemid, transposon, viral vector, artificial chromosome (e.g., bacteria, yeast, etc.) and other self-replicating species.

[0045] Polynucleotides include those that express antigenic polypeptides in host cells (e.g., polynucleotide-containing antigens). Polynucleotides include self-replicating polynucleotides that are inserted with natural or synthetic sequences (e.g., genomic DNA sequences, genomic RNA sequences, cDNA sequences, etc.) derived from eukaryotes or prokaryotes. Specific examples of self-replicating polynucleotides include, inter alia, RNA vector constructs and DNA vector constructs. Sequences that can be expressed include, inter alia, native sequences and modifications to native sequences, such as deletions, additions, and substitutions (generally conservative in nature).

[0046] These modifications may be deliberate, as by site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens.

[0047] In one embodiment, the at least one polynucleotide is selected from single-stranded or multi-stranded polynucleotides, preferably from artificial messenger RNA (mRNA), chemically modified or unmodified mRNA comprising at least one coding sequence, self-replicating RNA, circular RNA, viral RNA, and replicon RNA, from linear DNA, plasmid DNA (pDNA), minicircle DNA, dog gibbon DNA (dbDNA), from small interfering RNA (siRNA), microRNA (miRNA), guide RNA, small activating RNA (saRNA), antisense oligonucleotide (ASO), or any combination thereof, most preferably from mRNA.

[0048] In one embodiment, the at least one polynucleotide is present in an amount of 0.1 to 50% by weight, preferably 0.2 to 40% by weight, and more preferably 0.3 to 35% by weight, based on the total weight of the polynucleotide delivery particle.

[0049] Polynucleotide delivery particles according to the present invention can include at least one additive.

[0050] In one embodiment, the weight ratio of the at least one additive to the at least one polynucleotide ranges from 0.01 to 50, or from 0.01 to 30, or from 0.01 to 10, or from 0.01 to 5, or from 0.01 to 2, or from 0.01 to 1, or from 0.01 to 0.1.

[0051] In one embodiment, the at least one additive, preferably a cell penetrating peptide, more preferably human lactoferrin protein or a fragment thereof, is present at 0.01-88% by weight, based on the total weight of the polynucleotide delivery particle.

[0052] Any additive known in the art is suitable, provided it is pharma-ceutically acceptable.

[0053] "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual without causing any undue undesirable biological effects in the individual or interacting in an unduly deleterious manner with any of the components of the composition in which it is contained.

[0054] The term "additive" includes, for example, buffers such as phosphate, acetate, citrate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; and other organic compounds, antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; glycine, glutamine, and the like. , asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; immune adjuvants such as metal complexes (e.g., Zn-protein complexes), vehicles, binding agents, disintegrants, cell penetrating peptides, e.g., human lactoferrin protein or fragments thereof, Tat, Ant, Rev, FHV, HSV-1 protein VP22, C6, C6M1, PF20, NAP, POD, polyarginine, polylysine, PTD-5, transportan, MAP, TP10, Pep-7, azurin p18, azurin p28, hCT18-32, Bac 7, CTP, K5-FGF, HAP-1, 293P-1, KALA, GALA, LAH4-L1, melittin, penetratin, EB1, MPG, CADY, Pep4, preferably human lactoferrin protein or fragments thereof, fillers (diluents), lubricants, flow aids (glidants), compression aids, colourants, cryoprotectants, sweeteners, suspending / dispersing agents, film formers / coatings, flavourings, printing inks, non-ionic surfactants, ionisable surfactants, lipids such as cholesterol, phospholipids, sphingolipids, ceramides, fatty acids, lipids linked to hydrophilic polymers.

[0055] In one embodiment, the polynucleotide delivery particle comprises at least one additive selected from a buffer, a cryoprotectant, an ionizable surfactant, a non-ionic surfactant, cholesterol, a lipid such as a phospholipid, a sphingolipid, a ceramide, a fatty acid, or a lipid linked to a hydrophilic polymer.

[0056] In one embodiment, the polynucleotide delivery particle solution includes at least one buffer, preferably in an amount of 0.1 mM to 1000 mM based on the total volume of the polynucleotide delivery particle solution. The at least one buffer is preferably selected from PBS, phosphate buffer, acetate buffer, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0057] In one embodiment, the polynucleotide delivery particle contains less than 0.5% by weight of a non-ionic surfactant, preferably less than 0.1% by weight of a non-ionic surfactant, and more preferably no non-ionic surfactant, based on the total weight of the polynucleotide delivery particle.

[0058] Suitable non-ionic surfactants vary widely, and a number of examples are described below: In certain preferred embodiments, the non-ionic surfactant is selected from poly(vinyl alcohol), polysorbates (e.g., polysorbate 20, polysorbate 80), and poloxamers.

[0059] In one embodiment, the polynucleotide delivery particle further comprises at least one ionizable surfactant, preferably at an N / P ratio of ionizable surfactant to polynucleotide of 1 to 50. The at least one ionizable surfactant is preferably selected from the group consisting of 1,2-distearoyl-3-dimethylammonium-propane, 1,2-dipalmitoyl-3-dimethylammonium-propane, 1,2-dimyristoyl-3-dimethylammonium-propane, 1,2-dioleoyl-3-dimethylammonium-propane, 1,2-dioleyloxy-3-dimethylaminopropane, (6Z,9Z,28Z,31Z)-heptatriacon-6,9,28,31- Tetraen-19-yl 4-(dimethylamino)butanoate, 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, N,N-dimethyl-2,2-di-(9Z,12Z)-9,12octadecadien-1-yl-1,3-dioxolane-4-ethanamine, and salts of [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate).

[0060] In certain embodiments, the particles according to the present invention can include an immune adjuvant, such as E. coli heat labile toxin, alum, phospholiposaccharide compounds, phospholiposaccharide mimetics, monophosphoryl lipid A analogs, small molecule immune enhancers, muramyl tripeptide phosphatidylethanolamine, and tocopherol.

[0061] Immune adjuvants may, for example, be associated (e.g., adsorbed or otherwise bound) with the surface of a particle, entrapped within the particle, or both. Immune adjuvants increase or diversify the immune response to an antigen. Thus, immune adjuvants are compounds that can enhance the immune response to an antigen. Immune adjuvants can enhance humoral immunity and / or cellular immunity.

[0062] In one embodiment, the particles have at least one additive, preferably a cell penetrating peptide, more preferably a human lactoferrin protein or a fragment thereof, adsorbed on the outer coating layer or surface of the particles.The human lactoferrin protein or a fragment thereof according to the present invention is described, for example, in WO2007076904(A1), the manufacturing process of which is incorporated by reference.

[0063] In one embodiment, the weight ratio of the cell-penetrating peptide, preferably human lactoferrin protein or a fragment thereof, to the at least one polynucleotide is in the range of 0.01-50, preferably 0.1-30.

[0064] "Carbohydrates," as defined herein, include monosaccharides, oligosaccharides, and polysaccharides, as well as substances derived from monosaccharides, for example, by reduction (e.g., alditols), oxidation of one or more terminal groups to a carboxylic acid (e.g., glucuronic acid), or replacement of one or more hydroxy group(s) with a hydrogen atom or an amino group (e.g., β-D-glucosamine and β-D-galactosamine).

[0065] As defined herein, a "monosaccharide" is a polyhydric alcohol, i.e., an alcohol that further comprises either an aldehyde group (in which case the monosaccharide is an aldose) or a keto group (in which case the monosaccharide is a ketose). Monosaccharides typically contain 3 to 10 carbons. Furthermore, monosaccharides generally have the empirical formula C n H 2n O n where n is an integer of 3 or greater, typically 3 to 10. Examples of 3-6 carbon aldoses include glyceraldehyde, erythrose, threose, ribose, 2-deoxyribose, arabinose, xylose, lyxose, allose, altose, glucose, mannose, glucose, idose, galactose, and talose.

[0066] Examples of 3-6 carbon ketones include dihydroxyacetone, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, and tagatose. Naturally occurring monosaccharides are usually found in the D-isomer as opposed to the L-isomer.

[0067] "Oligosaccharide" refers to relatively short monosaccharide polymers, i.e., those containing 2-30 monosaccharide units. "Polysaccharide" is a monosaccharide polymer that exceeds the oligosaccharide length (i.e., those containing more than 30 monosaccharide units). Furthermore, as used herein, the term "polysaccharide" also refers to a monosaccharide polymer containing two or more linked monosaccharides. To avoid ambiguity, the second definition should always be applied unless there is an explicit indication to the contrary. The term "polysaccharide" also includes polysaccharide derivatives, such as amino-functionalized and carboxyl-functionalized polysaccharide derivatives, among others. The monosaccharides are typically linked by glycosidic bonds. Specific examples include disaccharides (e.g., sucrose, lactose, trehalose, maltose, gentiobiose, and cellobiose), trisaccharides (such as raffinose), tetrasaccharides (such as stachyose), pentasaccharides (such as verbascose), and the like.

[0068] As used herein, the term "saccharide" encompasses monosaccharides, oligosaccharides, and polysaccharides. A "saccharide-containing species" is a molecule, at least a portion of which is a saccharide. Examples include saccharide cryoprotectants, saccharide antigens, antigens comprising saccharides conjugated to carrier peptides, and the like. A "polysaccharide-containing species" is a molecule, at least a portion of which is a polysaccharide.

[0069] As used herein, a "cryoprotectant" is an agent that protects a composition from experiencing adverse effects during freezing and thawing.For example, in the present invention, cryoprotectants such as polyols and / or carbohydrates, among others, can be added to prevent substantial particle aggregation from occurring when the lyophilized composition of the present invention is resuspended.

[0070] Various methods may be used to prepare the particles according to the invention, for example nanoprecipitation, i.e. mixing an aqueous phase containing the polynucleotide with a water-miscible organic phase containing excipients and additives or nanoemulsion, i.e. mixing an aqueous phase containing the polynucleotide with a water-immiscible organic phase containing excipients and additives, may be used.

[0071] In some embodiments, the particles can be formed using oil-in-water (o / w) or water-in-oil-in-water (w / o / w) solvent evaporation processes, or using nanoemulsion methods.

[0072] The w / o / w solvent evaporation process is described, for example, in O'Hagan et al., Vaccine (1993) 11:965-969, Jeffery et al., Pharm.Res. (1993) 10:362, WO 00 / 06123(A1). PLGA and cationic surfactant (e.g., selected from those listed above, among others) are dissolved in one or more organic solvent(s) to form an organic solution. The solvent or solvent mixture may include one or more organic solvent(s) selected from, for example, dichloromethane (DCM), ethyl acetate (EtOAc), chloroform, benzyl alcohol, diethyl carbonate (DMC), dimethyl sulfoxide (DMSO), methanol, propylene carbonate, isopropyl acetate, methyl acetate, methyl ethyl ketone, butyl lactate and isovaleric acid or any mixture thereof. A preferred solvent or solvent mixture may include EtOAc, DCM, EtOAc and DMSO or DCM and DMSO. The organic solution is then combined with a first volume of an aqueous solution containing at least one polynucleotide and emulsified to form a water-in-oil emulsion. The aqueous solution can be, for example, deionized water, saline, a buffer solution, such as phosphate buffered saline (PBS) or a sodium citrate / ethylenediaminetetraacetic acid (sodium citrate / ETDA) buffer solution, among others. Typically, the volume ratio of organic solution to aqueous solution ranges from about 2:1 to about 20:1, more typically about 10:1. Emulsification is performed using any equipment suitable for the task. The most common approaches include simple mechanical stirring, sonication, high shear mixing (HSM), high pressure homogenization (HPH), as well as microfluidic or millifluidic mixing such as T- or Y-mixing.

[0073] A volume of the water-in-oil emulsion is then combined with a larger second volume of an aqueous solution, which may contain an emulsion stabilizer, such as a non-charged surfactant (e.g., PVA (polyvinyl alcohol), povidone (also known as polyvinylpyrrolidone or PVP), sorbitan esters, polysorbates or poloxamers, among others) or an anionic or cationic surfactant (e.g., selected from those listed above, among others). The volume ratio of aqueous solution to water-in-oil emulsion is typically in the range of about 2:1 to 20:1, more typically about 4:1. The mixture is then homogenized to produce a stable w / o / w double emulsion. The organic solvent is then evaporated to obtain the particles.

[0074] Nanoprecipitation, also referred to as solvent displacement, is another example of a suitable method for forming particles for use in the present invention.See, for example, EP 0274961(B1), entitled "Process for the preparation of dispersible colloidal systems of a substance in the form of nanocapsules", Devissaguet et al., U.S. Pat. No. 5,049,322 of the same name, Fessi et al., U.S. Pat. No. 5,118,528, entitled "Process for the preparation of dispersible colloidal systems of a substance in the form of microparticles", and Wendorf et al., WO 2008 / 051245(A1), entitled "Nanoparticles for use in Immunogenic compositions".In this technique, for example, at least one PLGA and at least one cationic surfactant (e.g., selected from those listed above, among others) may be dissolved in one or more organic solvent(s) (e.g., hydrophilic organic solvents such as acetone, ethanol, DMSO, or any mixture thereof). The resulting organic solution may then be combined with a non-solvent for the polymer, typically an aqueous solution, but with an additional solvent that is miscible with the organic solvent. The aqueous solution may be, for example, deionized water, saline, a buffer solution, such as phosphate buffered saline (PBS), acetate buffer or sodium citrate / ethylenediaminetetraacetic acid (sodium citrate / EDTA) or (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution. The organic solution and the aqueous solution may then be combined in a suitable relative volume, typically 1:9 to 9:1. For example, the organic solution may be injected and dripped into the non-solvent, or vice versa, while stirring or homogenizing or shaking. Mixing of the two solutions may also be achieved by standard T / Y tube mixing techniques, microfluidic mixing, millifluidic mixing, turbulent mixing, trituration mixing, or combinations thereof.By selecting a system in which the polymer is soluble in the organic solvent but significantly less soluble in a miscible blend of the organic solvent and non-solvent, a suspension of particles can be formed substantially instantaneously, after which the organic solvent can be removed from the suspension by, for example, evaporation, dialysis, or diafiltration.

[0075] As mentioned above, in certain embodiments, it may be desirable to provide one or more additives (in addition to PLGA), which may be associated with the interior (e.g., entrapped) and / or surface of the particle (e.g., by adsorption, covalent attachment, co-lyophilization, etc.), or may be unassociated with the particle. Such additional species may include, for example, agents for adjusting isotonicity or pH, cryoprotectants, immune adjuvants, antigens, etc.

[0076] Such additional species may be provided during the particle formation process. Thus, in the above-mentioned particle formation techniques (e.g., w / o / w solvent evaporation, o / w solvent evaporation, nanoprecipitation, etc.), the organic and / or aqueous solutions used may further contain various additives as desired. For example, these additives may be added (a) to the organic solution if in oil-soluble or oil-dispersible form, or (b) to the aqueous solution if in water-soluble or water-dispersible form.

[0077] In some embodiments, one or more additives may be added after particle formation (typically after removal of the organic solvent, as well as after a washing step or a step in which the particles are dialyzed against water, if present). These additives are often added to the particles as an aqueous solution or dispersion. These additives may, for example, be in solution and / or may accumulate at the particle-solution interface, e.g., may be adsorbed to the particle surface.

[0078] Once a suitable composition has been formed (eg, using the techniques described above or other techniques), it may be lyophilized for subsequent use.

[0079] Polynucleotide delivery particles according to the present invention can be included in oral drug delivery compositions or parenteral, preferably injectable, drug delivery compositions.

[0080] Once formulated (and resuspended as necessary), the polynucleotide delivery particles of the present invention can be administered parenterally, for example by injection (which may be needleless), among other routes of administration.In this regard, the particle composition is typically provided and lyophilized in a vial or other container that is provided with a septum or other suitable means for providing a resuspension medium (e.g., water for injection) and for collecting the resulting suspension.A suitable syringe can also be provided for injection.The composition can be injected, for example, subcutaneously, intradermally, intramuscularly, intravenously, intraarterially, or intraperitoneally.

[0081] Other modes of administration include nasal, mucosal, ocular, rectal, vaginal, oral and pulmonary administration, as well as transdermal or transepidermal application.

[0082] For oral administration, the polynucleotide delivery particles can be contained in a capsule, preferred capsules being described, for example, in WO 2019096833 A1, WO 2020229178 A1, WO 2020229192 A1 and European Patent Application No. 21175704.2.

[0083] In some embodiments, the composition of the present invention can be used for site-specific targeted delivery.For example, intravenous administration of the composition can be used for targeting the lung, liver, spleen, blood circulation or bone marrow.In addition, oral administration of the composition can be used for gastrointestinal targeted delivery.

[0084] Treatment may be according to a single dose schedule or a multiple dose schedule. A multiple dose schedule is one in which a first course of administration is given, for example, in 1-10 separate doses, followed by other administrations at subsequent time intervals selected to maintain and / or enhance the therapeutic response, for example, a second administration at 1-4 months, and subsequent dose(s) may be given several months later, if necessary. The administration regimen will also be determined, at least in part, by the needs of the subject and will be dependent on the physician's judgment.

[0085] Moreover, where prevention of disease is desired, the compositions are generally administered prior to the onset of the primary occurrence of the infection or disorder of interest, Where other forms of treatment, such as reducing or eliminating symptoms or recurrences, are desired, the compositions are generally administered after the onset of the primary occurrence of the infection or disorder of interest. EXAMPLES

[0086] Example 1: Testing of DODMA:PLGA for encapsulation of mRNA and transfection of mRNA into cells (Comparative Example). In this example, the ability of the ionizable lipid DODMA to act as an ionizable surfactant in combination with low molecular weight PLGA is investigated. Particle formation, encapsulation of mRNA, and transfection of mRNA into cells are evaluated.

[0087] Preparation of DODMA:PLGA particles [Table 1]

[0088] 99 μL ribonuclease-free water, 16.5 μL 100 mM acetate pH 4 buffer, and 16.5 μL firefly luciferase-encoding mRNA (FLuc mRNA, 1 g / L) together formed the aqueous phase, which was added to a sterile 1.5 mL safe-lock tube. The aqueous phase was vortexed and spun down. 10.98 μL DMSO containing a 20 g / L DODMA stock solution, 4.39 μL DMSO containing a 50 g / L RG501H stock solution, and 14.63 μL DMSO together formed the organic phase, which was added to a second 1.5 mL safe-lock tube. For DODMA-only particles, the respective volumes of RESOMER® RG 501H solution were replaced with DMSO. The organic phase was vortexed (Scientific Industries SI™ Vortex-Genie™ 2) and spun down. 120 μL of the aqueous phase was removed from the first tube and added to the second tube in one vigorous movement with strong pipetting. The two phases were further mixed by frequent pipetting. The resulting mRNA-loaded DODMA:PLGA particles were stored in solution at 4° C. until further use.

[0089] Characterization of DODMA:PLGA particles Particle size was measured in a Malvern Zetasizer Nano ZS using water as the dispersant at an mRNA concentration of 10 ng / μL.

[0090] Gel electrophoresis was performed with 1 μg of mRNA per well using an Invitrogen™ E-Gel™ Power Snap electrophoresis system.

[0091] Luciferase assay was performed with human epithelial cells (HeLa). One day before transfection, 10,000 cells per well were seeded in a 96-well plate and cultured at 37°C and 5% CO2 for 24 hours. On the second day, the old medium was removed and 90 μL of fresh medium was added to the cells. Samples were adjusted to an mRNA concentration of 10 ng / μL using ribonuclease-free water for dilution. 10 μL of each diluted sample was added to the cells equivalent to an amount of 100 ng of mRNA per well in a total volume of 100 μL. The cells were further incubated at 37°C and 5% CO2 for 24 hours. On the third day, transfection efficiency was determined using a luciferase kit system according to the manufacturer's protocol (Promega GmbH). Luminescence signals were quantified by a multiplate reader (Plate Reader Infinite® 200 PRO, Tecan).

[0092] result [Table 2]

[0093] DODMA and mixtures of DODMA and RG501H form defined nanoparticles when the organic phase is mixed with the aqueous phase containing the mRNA. The addition of RG501H results in a significant increase in particle size.

[0094] However, agarose gel electrophoresis (Figure 1) shows that mRNA is not encapsulated in the particles formed by either DODMA or the mixture with RG501H. Correspondingly, neither of the two compositions tested achieved substantial transfection when incubated with HeLa cells (Figure 3).

[0095] Therefore, this experiment clearly demonstrates that ionizable surfactants alone in combination with PLGA are insufficient for encapsulation and transfection of mRNA.

[0096] Example 2: Testing of DOTMA:PLGA for encapsulation and transfection of mRNA into cells (example of the invention). In this example, the ability of the cationic lipid DOTMA to act as an ionizable surfactant in combination with low molecular weight PLGA is investigated. Particle formation, encapsulation of mRNA, and transfection of mRNA into cells are evaluated.

[0097] Preparation of DOTMA:PLGA particles [Table 3]

[0098] 66 μL of ribonuclease-free water, 11 μL of 100 mM acetate pH 4 buffer, and 11 μL of FLuc mRNA (1 g / L) collectively formed the aqueous phase, which was added to a sterile 1.5 mL safe-lock tube. The aqueous phase was vortexed and spun down. 7.91 μL of DMSO with 20 g / L DOTMA stock solution, 6.33 μL of RG501H at 50 g / L, and 5.76 μL of DMSO collectively formed the organic phase, which was added to a second 1.5 mL safe-lock tube. For DOTMA-only particles, the respective volume of PLGA solution was replaced with DMSO. The organic phase was vortexed and spun down. 80 μL of the aqueous phase was removed from the first tube and added to the second tube in one vigorously movement with vigorous pipetting. The two phases were further mixed by frequent pipetting. The resulting mRNA-loaded DOTMA:PLGA particles were stored in solution at 4° C. until further use.

[0099] Characterization of DOTMA:PLGA particles Particle size was measured in a Malvern Zetasizer Nano ZS using water as the dispersant at an mRNA concentration of 10 ng / μL.

[0100] Gel electrophoresis was performed with 1 μg of mRNA per well using an Invitrogen™ E-Gel™ Power Snap electrophoresis system.

[0101] Luciferase assay was performed with human epithelial cells (HeLa). One day before transfection, 10,000 cells per well were seeded in 96-well plates in DMEM medium and cultured at 37°C and 5% CO2 for 24 hours. On the second day, the old medium was removed and 90 μL of fresh medium was added to the cells. Samples were adjusted to an mRNA concentration of 10 ng / μL using RNAse-free water for dilution. 10 μL of each diluted sample was added to the cells equivalent to an amount of 100 ng of mRNA per well in a total volume of 100 μL. The cells were further incubated at 37°C and 5% CO2 for 24 hours. On the third day, transfection efficiency was determined using a luciferase kit system according to the manufacturer's protocol (Promega GmbH). Luminescence signals were quantified by a multiplate reader (Plate Reader Infinite® 200 PRO, Tecan).

[0102] result [Table 4]

[0103] DOTMA and mixtures of DOTMA and RG501H form defined nanoparticles when the organic phase is mixed with the aqueous phase containing the mRNA. In contrast to DODMA, for DOTMA, the particle size is in the same range for all tested mixtures, and the addition of RG501H does not cause a significant size increase. Without being bound by any theory, it is hypothesized that this may be due to the permanent cationic charge of DOTMA, which results in better colloidal stabilization of the particles than DODMA.

[0104] According to agarose gel electrophoresis (Figure 2), the mRNA is fully encapsulated in the particles formed in all compositions tested. Without being bound by any theory, it is hypothesized that this may also be due to the cationic charge imparted to the particles by the use of DOTMA.

[0105] In the case of transfection efficiency (FIG. 4), significantly better performance is observed for the DOTMA:RG501H mixed particles compared to DOTMA alone and compared to all compositions tested so far containing DOTMA.

[0106] Therefore, the experiment clearly shows that the combination of cationic surfactant and the specific PLGA of the present invention is well suited for encapsulating and transfecting mRNA.Furthermore, these results show that when combined with cationic surfactant, the specific PLGA of the present invention has beneficial effect on the efficiency of particles described in the present invention.

[0107] Example 3: Comparison of low and medium molecular weight PLGA for mRNA encapsulation and transfection of mRNA into cells (Example of the present invention). In this example, two PLGA polymers with low and medium molecular weights are applied in combination with DOTMA to encapsulate FLuc mRNA and form particles. The transfection efficiency and kinetics of the particles according to the applied PLGA are evaluated.

[0108] Preparation of DOTMA:PLGA particles [Table 5]

[0109] 75.17 μL of ribonuclease-free water, 5.5 μL of 200 mM HEPES pH 7 buffer, and 11 μL of FLuc mRNA (1 g / L) collectively formed the aqueous phase, which was added to a sterile 1.5 mL safe-lock tube. The solution was vortexed and spun down. 7.91 μL of DMSO containing 20 g / L DOTMA stock solution, 6.33 μL of DMSO containing either 50 g / L RG501H stock solution or 50 g / L RG503H stock solution, and 2.42 μL of DMSO collectively formed the organic phase, which was added to a second 1.5 mL safe-lock tube. The solution was vortexed and spun down. 83.33 μL of the aqueous phase was removed from the first tube and added to the second tube in one vigorously movement with vigorous pipetting. The two phases were further mixed by frequent pipetting. The resulting mRNA-loaded DOTMA:PLGA particles were stored in solution at 4° C. until further use.

[0110] Characterization of DOTMA:PLGA particles Particle size was measured in a Malvern Zetasizer Nano ZS using water as the dispersant at an mRNA concentration of 10 ng / μL.

[0111] Luciferase assay was performed with human epithelial cells (HeLa). One day before transfection, 10,000 cells per well were seeded in 96-well plates in DMEM medium and cultured at 37°C and 5% CO2 for 24 hours. On the second day, the old medium was removed and 90 μL of fresh medium was added to the cells. Samples were adjusted to an mRNA concentration of 10 ng / μL using ribonuclease-free water for dilution. 10 μL of each diluted sample was added to the cells equivalent to an amount of 100 ng of mRNA per well in a total volume of 100 μL. The cells were further incubated at 37°C and 5% CO2 for 24 hours. On the third day, transfection efficiency was determined using a luciferase kit system according to the manufacturer's protocol (Promega GmbH). Luminescence signals were quantified by a multiplate reader (Plate Reader Infinite® 200 PRO, Tecan).

[0112] result [Table 6]

[0113] Mixtures of DOTMA with the low molecular weight RESOMER® RG501H and the medium molecular weight RESOMER® RG503H form defined nanoparticles upon mixing with an organic phase and an aqueous phase containing mRNA. However, the particles obtained with RG503H are larger than those obtained with RG501H.

[0114] The in vitro transfection efficiency of either RG501H or RG503H-containing particles was evaluated by luciferase assay at different time points of cell incubation (Figure 5). Luciferase assay clearly shows the functionality of DOTMA:PLGA particles in transfecting cells with encapsulated FLuc mRNA. Importantly, RG501H-containing particles perform better than RG503H-containing particles at all test time points. Without being bound by any theory, it is hypothesized that this may be due to the low molecular weight of RG501H compared to RG503H, which promotes particle degradation and release of encapsulated mRNA. Ultimately, the enhanced mRNA release leads to faster transfection kinetics of the corresponding particles as well as an increase in overall transfection efficiency. Thus, the efficiency of the particles described in this invention directly correlates with the PLGA used within the composition.

[0115] Example 4: Coating of DOTMA:PLGA particles with human lactoferrin fragments to improve transfection of mRNA into intestinal epithelial cells (example of the present invention). In this example, to further improve uptake and transfection efficiency in intestinal epithelial cells, DOTMA:RG501H particles are coated with the cell-penetrating peptide human lactoferrin fragment (hLFF).

[0116] Preparation of DOTMA:RG501H:hLFF particles [Table 7]

[0117] 264 μL of ribonuclease-free water, 44 μL of 100 mM acetate pH 4 buffer, and 44 μL of FLuc mRNA (1 g / L) collectively formed the aqueous phase, which was added to a sterile 1.5 mL safe-lock tube. The aqueous phase was vortexed and spun down. 31.65 μL of DMSO with 20 g / L DOTMA stock solution, 25.32 μL of 50 g / L RG501H stock solution, and 23.03 μL of DMSO collectively formed the organic phase, which was added to a second 1.5 mL safe-lock tube. The organic phase was vortexed and spun down. 320 μL of the aqueous phase was removed from the first tube and added to the second tube in one vigorous movement with vigorous pipetting. The two phases were further mixed by frequent pipetting. The resulting mRNA-loaded DOTMA:RG501H particles were stored in solution at 4° C. until further use. In separate 1.5 mL tubes, 2 μL, 6 μL, or 12 μL of 2.5 g / L hLFF stock solution in water was mixed with 48 μL, 44 μL, or 38 μL of ribonuclease-free water, respectively, so that each tube contained a dilute hLFF solution with a final volume of 50 μL. An additional tube contained 50 μL of pure water as a negative control. 50 μL of preformed DOTMA:RG501H particle solution was added to each of the prepared tubes, respectively, by vigorous pipetting in one go followed by frequent pipetting. The coated particles were stored in solution at 4° C. until further use.

[0118] Characterization of DOTMA:PLGA:hLFF particles Particle size was measured in a Malvern Zetasizer Nano ZS using water as the dispersant at an mRNA concentration of 10 ng / μL.

[0119] Luciferase assay was performed with human colon adenocarcinoma cells (Caco-2). One day before transfection, 10,000 cells per well were seeded in 96-well plates in DMEM medium and cultured for 24 hours at 37°C and 5% CO2. On the second day, old medium was removed and 90 μL of fresh medium was added to the cells. Samples were adjusted to an mRNA concentration of 10 ng / μL using RNAse-free water for dilution. 10 μL of each diluted sample was added to the cells equivalent to an amount of 100 ng of mRNA per well in a total volume of 100 μL. The cells were further incubated for 24 hours at 37°C and 5% CO2. On the third day, transfection efficiency was determined using a luciferase kit system according to the manufacturer's protocol (Promega GmbH). Luminescence signals were quantified by a multiplate reader (Plate Reader Infinite® 200 PRO, Tecan).

[0120] result [Table 8]

[0121] A mixture of DOTMA and RG501H forms defined nanoparticles when the organic phase is mixed with the aqueous phase containing the mRNA. Coating these particles with hLFF results in a slight reduction in particle size.

[0122] In the case of transfection efficiency in Caco-2 cells (FIG. 6), a significantly better performance is observed for hLFF-coated particles when compared to uncoated particles. The maximum transfection efficiency is achieved at a DOTMA:hLFF ratio of 1:0.948. Without being bound by any theory, the positive effect of hLFF is believed to be due to the improved cellular uptake mediated by hLFF, an effect also known from other cell-penetrating peptides.

[0123] Therefore, this experiment clearly demonstrates that DOTMA:PLGA particles can be further improved by coating with a cell-penetrating peptide such as hLFF.

[0124] Example 5: Loading of RNA-containing DOTMA:PLGA particles into enteric-coated capsules and pH-dependent release of the particles In this example, mRNA containing DOTMA:RG501H particles are applied as a relevant model drug product for combination with enteric coated capsules.

[0125] Preparation and characterization of DOTMA:PLGA particles [Table 9]

[0126] 0.5 mL of DMSO solution containing 9.5 g / L DOTMA and 19.0 g / L RG501H (organic phase) was mixed with 2.5 mL of RNAse-free 12 mM HEPES pH 7 solution containing 0.12 g / L FLuc mRNA (aqueous phase) using the Nanoassemblr® benchtop (PNI) platform. The resulting particle solution was dialyzed against 10 mM HEPES pH 7 buffer for 3 hours (Slide-A-Lyzer™, 10K MWCO) (3 buffer exchanges). After dialysis, RNAse-free trehalose solution (20 wt%) was added to the particle solution to achieve a final trehalose concentration of 10 wt%. The particles were lyophilized for 48 hours and stored at 4°C until further use.

[0127] Filling capsules with particles The lyophilized particles were filled into enteric coated capsules (types P0001 / 21 and 22274 / 27 as disclosed in European Patent Application No. 21175704.2, Examples 5 and 8) in an amount equivalent to 100 μg of mRNA per capsule. The filled capsules were sealed and stored at 4° C. until further use.

[0128] Capsule dissolution assay To mimic the fed gastric environment, the capsules were incubated in 10 mL of 0.1 N HCl containing 2 g / L pepsin at 37° C. for 2 h on a rocking shaker. Samples for release analysis were taken after 60 and 120 min. The acid medium was then replaced with 10 mL of phosphate buffer (18.8 mM phosphate, 145.4 mM NaCl, pH 6.8) and the capsules were incubated for an additional 60 min, with samples taken at 15 min intervals.

[0129] As a negative control, pure DOTMA:RG501H particles without capsule protection were incubated under the same conditions. 40 μL of particle solution (containing 50 ng / μL mRNA) was mixed with 100 μL of 0.1 N HCl containing 2 g / L pepsin and incubated for 2 hours at 37° C. and 300 rpm on an orbital shaker. Then, 60 μL of phosphate buffer was added to the mixture and incubation was continued for another 60 minutes.

[0130] After the dissolution assay, the medium containing the dissolved capsules and PLGA particles was used immediately for the cell transfection assay without any storage in between. Samples taken at regular intervals were stored at 4°C until further analysis by Ribogreen assay.

[0131] RiboGreen Assay Ribogreen assay was applied to detect and quantify RNA after release of particles from the capsules. The mRNA concentration was measured at different time intervals to establish the release kinetics.

[0132] The Quant-iT™ RiboGreen™ RNA Assay Kit was used for this assay. The RiboGreen assay is based on the measurement of fluorescence and therefore applies black 96-well assay plates with clear bottom.

[0133] The procedure was carried out according to the manufacturer's protocol with minor adjustments. In a first step, 1x TRIS / EDTA (TE) buffer was prepared by diluting the buffer stock with ribonuclease-free water. Particle samples were diluted to a theoretical concentration of 1 μg / ml using TE buffer and added to the plate in a volume of 50 μl. To measure the concentration of available mRNA, 50 μl of TE buffer was added to the samples. Calibration standards containing the corresponding Fluc mRNA and buffer were applied and added to the same plate as the samples. Standard solutions of Ribogreen dye were prepared by diluting the reagent 1:100 in TE buffer. 100 μl of the standard solution was added to each well and then mixed thoroughly by pipetting up and down. The fluorescence signal was measured using a microplate reader with excitation / emission values ​​of 480 / 520 nm. All samples and standards were measured in duplicate.

[0134] Luciferase assay One day before transfection, 10,000 cells per well were seeded in a 96-well plate and cultured at 37°C and 5% CO2 for 24 hours. On the second day, the old medium was removed and 90 μL of fresh medium was added to the cells. All samples were adjusted to an mRNA concentration of 10 ng / μL using RNAse-free water for dilution. 10 μL of each diluted sample was added to the cells equivalent to an amount of 100 ng of mRNA per well in a total volume of 100 μL. The cells were further incubated at 37°C and 5% CO2 for 24 hours. On the third day, the transfection efficiency was determined using a luciferase kit system according to the manufacturer's protocol (Promega GmbH). Addition of luciferase substrate to the cells produces a luminescent signal that can be quantified by a multiplate reader (Plate reader Infinite® 200 PRO, Tecan).

[0135] result [Table 10]

[0136] The DLS data clearly show that defined DOTMA:RG501H particles containing mRNA can be produced by microfluidic methods (Nanoassemblr® platform). The particles obtained from microfluidic mixing are significantly smaller in size compared to samples produced by pipetting. Moreover, the particle size remains constant in various processing steps and different storage conditions.

[0137] The RiboGreen assay (Figure 7) clearly demonstrates that the release of DOTMA:RG501H particles from the enteric coated capsules is pH dependent, as measured by the signal derived from available mRNA within the particles.

[0138] Within 30 min after changing the incubation medium from acidic pH to pH 6.8, the particles were completely rehydrated and released from the capsules, which proceeded along with complete capsule dissolution.

[0139] Importantly, no release of particles and mRNA was observed during 120 min of incubation in 0.1 N HCl, confirming the integrity of the capsule structure under acidic conditions.

[0140] To evaluate particle functionality after release from the capsule, a luciferase transfection assay in human epithelial (HeLa) cells was used (Figure 8). Lyophilized particles further incubated in simulated gastric and intestinal fluids in the fed state only rehydrated or without capsule protection served as positive and negative controls, respectively. The luciferase assay clearly demonstrated the functionality of the DOTMA:RG501H particles after release from the capsule. This is because HeLa cells incubated with these samples showed a clear expression of the embedded Fluc mRNA. The protection of the particles against simulated gastric and intestinal fluids in the fed state is further validated by considering the particle negative control exposed to the same medium without any capsule protection. The transfection efficiency of the capsule-protected particles is about 2 logs higher than that of the unprotected particles, confirming the clear beneficial effect of the enteric-coated capsule on particle functionality. Compared to the positive control, i.e., the lyophilized particles rehydrated and directly applied for the transfection assay, the efficiency of the released particles is about 1 log lower. Without being bound by any theory, this may be due to dissolving capsule components that may interact with the particles and compromise their integrity. The decrease in efficiency is comparable for both capsule types tested.

Claims

1. Polynucleotide delivery particles, a) at least one poly(lactic acid-coglycolide), b) At least one cationic surfactant, c) at least one polynucleotide, d) Optionally, including or comprising at least one additive, Here, the poly(lactic acid-coglycolide) is measured by gel permeation chromatography using a polystyrene standard and chloroform to have a weight-average molecular weight Mw of 1000 to 9500 g / mol. Polynucleotide delivery particles.

2. The above-mentioned at least one poly(lactic acid-coglycolide) is i) The number-average molecular weight Mn, and / or 1000–3000 g / mol, is measured by gel permeation chromatography using polystyrene standards and chloroform. ii) A molar ratio of lactide to glycoside in the range of 40:60 to 60:40, and / or iii) Measured by viscosity assay, the intrinsic viscosity is 0.05 to 0.25 dl / g, and / or iv) Having an acid value of 20-30 mg KOH / g, The polynucleotide delivery particle according to claim 1.

3. The at least one cationic surfactant is i) 1,2-di-O-octadecenyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium propane, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide, N ４ -Cholesteryl-spermine, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, O,O'-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine, 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 1,2-distearoyl-sn-glycero-3-ethylphosphocholine, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1-palmitoyl-2-oleoyl Selected from salts of -sn-glycero-3-ethylphosphocholine, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, dimethyldioctadecylammonium, 1,2-dimyristoyl-3-trimethylammonium-propane, 1,2-dipalmitoyl-3-trimethylammonium-propane, 1,2-stearoyl-3-trimethylammonium-propane, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-aminium and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, or ii) It is 1,2-di-O-octadecenyl-3-trimethylammonium propane salt, Polynucleotide delivery particles according to claim 1 or 2.

4. The polynucleotide delivery particle according to claim 1 or 2, wherein the at least one polynucleotide is selected from single-stranded or multi-stranded polynucleotides.

5. i) The N / P ratio of the cationic surfactant to the polynucleotide is in the range of 1:1 to 50:

1. and / or ii) The molar ratio of poly(lactic acid-coglycolide) to the polynucleotide is in the range of 1:1 to 200:

1. Polynucleotide delivery particles according to claim 1 or 2.

6. The polynucleotide delivery particle according to claim 1 or 2, wherein the at least one additive is selected from buffers, cryoprotectants, ionizable surfactants, nonionic surfactants, cholesterol, phospholipids, sphingolipids, ceramides, lipids such as fatty acids, and lipids linked to hydrophilic polymers.

7. The aforementioned particles i) Measured by dynamic light scattering, having a z-average particle size of 1 to 1000 nm, and / or ii) Measured by dynamic light scattering, having a polydispersity index of 0.01 to 0.5, Polynucleotide delivery particles according to claim 1 or 2.

8. The polynucleotide delivery particle according to claim 1 or 2, wherein the particle has at least one additive adsorbed on the outer coating layer or the surface of the particle.

9. The polynucleotide delivery particle according to claim 8, wherein the outer coating layer contains human lactoferrin protein or a fragment thereof, or the at least one additive adsorbed on the surface of the particle is human lactoferrin protein or a fragment thereof.

10. A method for forming polynucleotide delivery particles according to claim 1, wherein the particles are formed by a nanoprecipitation method or a nanoemulsion method.

11. An oral drug delivery composition comprising at least one polynucleotide delivery particle as described in claim 1.

12. An parenteral drug delivery composition comprising at least one polynucleotide delivery particle as described in claim 1.

13. An oral drug delivery composition according to claim 11 or a parenteral drug delivery composition according to claim 12, for use as a pharmaceutical product.

Images