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

Abstract

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

Description

Technical Field

[0001] The present invention relates to a polynucleotide delivery particle comprising:

[0002] a) at least one poly(lactic-co-glycolide);

[0003] b) at least one cationic surfactant;

[0004] c) at least one polynucleotide; and

[0005] d) optionally at least one additive; wherein:

[0006] The poly(lactic acid-co-glycolide) has a weight average molecular weight Mw of 1000 to 9500 g / mol, measured by gel permeation chromatography using polystyrene standards and chloroform; In addition, the present invention relates to a method for forming a polynucleotide delivery particle according to the present invention, wherein the particle is formed by nanoprecipitation or nanoemulsion. In addition, the present invention relates to an oral drug delivery composition or a parenteral drug delivery composition comprising at least one polynucleotide delivery particle according to the present invention and their use as a medicament. Background Art

[0007] Polynucleotide-based drugs are a new class of therapeutics that have emerged over the past 10 to 20 years. They hold great promise for cancer treatment and vaccination, as well as offering new treatment options for previously undruggable diseases.

[0008] The major obstacle to the successful application of polynucleotide therapy is to deliver them to the site of action, i.e., to the cytoplasm or nucleus. Polynucleotides are large biomolecules that are generally susceptible to chemical and enzymatic degradation and are not easy to enter cells. Therefore, suitable formulations must provide protection against any hazardous environments that polynucleotide drugs will inevitably encounter when applied topically, systemically or orally. In addition, the formulation should mediate the uptake of the polynucleotide drug into the target cell and ultimately promote its release from the endosomal compartment into the cytoplasm.

[0009] Among many non-viral formulations known in the art, the so-called lipid nanoparticles (LNPs) are currently the most advanced platform. Therefore, LNPs constitute the first generation of commercially approved delivery vehicles for drugs based on siRNA and mRNA. LNPs are composed of up to four different types of surfactants (lipids) (cationic / ionizable surfactants, PEG surfactants, cholesterol and phospholipids), which makes the formulations quite complex and expensive, particularly in terms of the supply of raw materials. In addition, the mandatory use of PEG surfactants has raised concerns about possible immunogenic reactions, which may be triggered by the presence of anti-PEG antibodies in population subgroups.

[0010] Nanoparticles and microparticles formed from poly(lactic-co-glycolic acid) (PLGA) are another widely used drug delivery platform due to their excellent biocompatibility. However, due to its charge neutrality and hydrophobic characteristics, PLGA itself is a rather unsuitable material for the encapsulation of hydrophilic charged macromolecules, such as in the case of polynucleotides. In order to increase the association with polynucleotides, the combination of PLGA with positively charged excipients such as calcium phosphate has been proposed as a workaround. However, these emulsion-based strategies suffer from complex multi-step protocols, poor encapsulation efficiency, and large particle size.

[0011] Auxiliary surfactants (lipids) (cholesterol, phospholipids, PEG lipids) are essential requirements for forming LNPs, because cationic / ionizable lipids and polynucleotides themselves cannot be co-assembled into effective nanoparticles. Therefore, the object of the present invention is to provide PLGA-based polynucleotide delivery particles that can overcome one or more of the above-mentioned disadvantages.

[0012] In this regard, the inventors of the present invention surprisingly found that when a cationic lipid is used as a positively charged excipient, a simple mixing scheme can be used to embed polynucleotides into PLGA particles without the need for additional surfactants or auxiliary lipids. In addition, when the poly (lactic acid-co-glycolide) has a weight average molecular weight Mw of 1000 to 9500 g / mol, improved cell transfection can be obtained.

[0013] Furthermore, the required absence of PEG in LNPs allows for coating of lipid / PLGA particles with other materials, such as cell penetrating peptides (eg, human lactoferrin or fragments thereof), to modulate particle surface properties and increase functionality. Summary of the invention

[0014] In a first aspect, the present invention relates to a polynucleotide delivery particle comprising or consisting of:

[0015] a) at least one poly(lactic acid-co-glycolide);

[0016] b) at least one cationic surfactant;

[0017] c) at least one polynucleotide; and

[0018] d) optionally at least one additive; wherein:

[0019] The poly(lactic acid-co-glycolide) has a weight average molecular weight Mw of 1000 to 9500 g / mol, preferably a weight average molecular weight Mw of 2000 to 6800 g / mol, more preferably a weight average molecular weight Mw of 4000 to 6800 g / mol, and most preferably a weight average molecular weight Mw of 6000 to 6800 g / mol, as measured by gel permeation chromatography using polystyrene standards and chloroform.

[0020] In a second aspect, the invention relates to a method of forming a polynucleotide delivery particle according to the invention, wherein the particle is formed by nanoprecipitation or nanoemulsion method.

[0021] In a third aspect, the present invention relates to an oral drug delivery composition comprising at least one polynucleotide delivery particle of the present invention.

[0022] In a fourth aspect, the present invention relates to a parenteral drug delivery composition comprising at least one polynucleotide delivery particle of the present invention.

[0023] In a fifth aspect, the present invention relates to an oral drug delivery composition of the present invention or a parenteral drug delivery composition of the present invention for use as a medicament. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Agarose gel electrophoresis of free mRNA and different DODMA:PLGA based particle samples. 1 μg of mRNA or an equivalent amount of particles was applied to each well.

[0025] Figure 2 : Agarose gel electrophoresis of free mRNA and different DOTMA:PLGA based particle samples. 1 μg mRNA or an equivalent amount of particles was applied per well.

[0026] Figure 3 : 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.

[0027] Figure 4: 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.

[0028] Figure 5 : Transfection efficiency of different DOTMA:PLGA-based particle samples in HeLa cells after different incubation times. 100 ng of mRNA was applied per well under each condition.

[0029] Figure 6 : Transfection efficiency of DOTMA:PLGA particles coated with different amounts of hLFF in Caco-2 cells after 24 h of incubation. 100 ng of mRNA was applied per well for each condition.

[0030] Figure 7 : Release kinetics of DOTMA:PLGA particles after dissolution assay in 0.1 N HCl (0 to 120 min) and pH 6.8 phosphate buffer (120 to 180 min) as determined by Ribogreen (representative from n=2).

[0031] Figure 8 : Transfection efficiency of DOTMA:PLGA particle samples in HeLa cells after different pretreatments and 24 hours of incubation. 100 ng of mRNA was applied per well under each condition. DETAILED DESCRIPTION

[0032] In one aspect, the present invention refers to a polynucleotide delivery particle comprising or consisting of:

[0033] a) at least one poly(lactic acid-co-glycolide) (also known as PLGA);

[0034] b) at least one cationic surfactant;

[0035] c) at least one polynucleotide; and

[0036] d) optionally at least one additive; wherein:

[0037] The poly(lactic acid-co-glycolide) has a weight average molecular weight Mw of 1000 to 9500 g / mol, preferably a weight average molecular weight Mw of 2000 to 6800 g / mol, more preferably a weight average molecular weight Mw of 4000 to 6800 g / mol, and most preferably a weight average molecular weight Mw of 6000 to 6800 g / mol, as measured by gel permeation chromatography using polystyrene standards and chloroform.

[0038] The polynucleotide delivery particle can be a nanoparticle or a micron particle. In a specific embodiment, the particle has a D(v,0.5) value between 50 and 500 nanometers, and / or a z-average particle size of 1 to 1000 nanometers, preferably 20 to 500 nanometers, more preferably 20 to 200 nanometers.

[0039] The term "particle" as used herein preferably refers to particles with a particle size of less than 10 μm (10,000 nm), for example, a particle size range of about 1 nm to 25 nm, to 50 nm, to 100 nm, to 250 nm, to 500 nm, to 1000 nm (1 μm), to 2,500 nm (2.5 μm), to 5,000 nm (5 μm) or to 10,000 nm (10 μm). In some embodiments, dry particles can exist as aggregates with a diameter greater than 10,000 nm, but after adding an aqueous fluid and mixing using a technique such as a vortex, they are dispersed into a particle size less than 10,000 nm. In some embodiments, the particles described herein can be generally spherical. In some embodiments, the particles described herein can be irregular geometries. The particles in the compositions of the present invention typically have a size distribution in an aqueous fluid, wherein the z-average and / or D(V,0.5) value is less than 5,000 nm, for example, in the range of 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.

[0040] As used herein, "nanoparticles" refer to particles with a certain size distribution in an aqueous fluid, wherein the z-average value is in the range of 1 nm to 500 nm. As used herein, "microparticles" refer to particles with a certain size distribution in an aqueous fluid, wherein D(V,0.5) is in the range of 500 nm to 5000 nm.

[0041] Particle size can be determined (measured) using methods available in the art. For example, using photon correlation spectroscopy, dynamic light scattering or quasi-elastic light scattering, particle size can be determined. These methods are based on the correlation of particle size obtained by Brownian motion measurement with the diffusion properties of particles. Brownian motion is the random movement of particles caused by the collision of solvent molecules around particles. The larger the particle, the slower the Brownian motion. The speed is defined by the translational diffusion coefficient (D). The measured value 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.

[0042] Particle size can also be determined by 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 an angle inversely proportional to their size. Large particles produce a diffraction pattern with high intensity at low scattering angles, while small particles produce a wide-angle low-intensity signal. If the intensity of light scattered by a sample is measured as a function of angle, the particle size distribution can be calculated. The angle information is compared with a scattering model (e.g., Mie theory) to calculate the size distribution.

[0043] Typically, particle size is determined at room temperature and involves multiple analyses of the sample in question (eg, at least 3 replicate measurements of the same sample) to arrive at an average value for the particle size.

[0044] These values ​​are preferably determined by dynamic light scattering, more preferably in accordance with DIN ISO 22412:2018-09.

[0045] The polynucleotide delivery particles may have a polydispersity index of 0.01 to 0.5, preferably measured by dynamic light scattering, more preferably measured according to DIN ISO 22412:2018-09.

[0046] In one embodiment, the polynucleotide delivery particle according to the present invention has an N / P ratio of the cationic surfactant to the polynucleotide of 1:1 to 50:1, preferably 5:1 to 20:1, more preferably 8.

[0047] In one embodiment, the polynucleotide delivery particle according to the present invention has a weight ratio of poly(lactic acid-co-glycolide) to the polynucleotide of 1 to 200, or 2 to 150, or 5 to 100.

[0048] The polynucleotide delivery particle comprises at least one poly(lactic acid-co-glycolide) having a weight average molecular weight Mw of 1000 to 9500 g / mol, preferably 2000 to 6800 g / mol, more preferably 4000 to 6800 g / mol, most preferably 6000 to 6800 g / mol, as measured by gel permeation chromatography using polystyrene standards and chloroform.

[0049] In one embodiment, the at least one poly(lactic acid-co-glycolide) has a number average molecular weight Mn of 1000 to 3000 g / mol, preferably a number average molecular weight Mn of 2000 to 2800 g / mol, as measured by gel permeation chromatography using polystyrene standards and chloroform.

[0050] In one embodiment, the at least one poly(lactic acid-co-glycolide) has a molar ratio of lactide to glycolide of 40:60 to 60:40, preferably a molar ratio of lactide to glycolide of 50:50.

[0051] In one embodiment, the at least one poly(lactic acid-co-glycolide) has an intrinsic viscosity of 0.05 to 0.25 dl / g, preferably 0.08 to 0.16, as measured by viscometry.

[0052] In one embodiment, the at least one poly(lactic acid-co-glycolide) has an acid value of 20 to 30 mg KOH / g, preferably an acid value of 22.5 mg KOH / g, preferably measured according to DIN EN 14104:2021-04.

[0053] In one embodiment, the at least one poly(lactic acid-co-glycolide) is present in an amount of 0.26 to 98.5 wt % based on the total weight of the polynucleotide delivery particle.

[0054] Suitable poly(lactic acid-co-glycolide) is available, for example, under the trade name Commercially available from Evonik Industries AG, e.g. RG 501H or Condensate RG polymer.

[0055] The particles comprise at least one cationic surfactant.

[0056] The term "surfactant" is derived from the phrase "surface-active chemical agent". Surfactants accumulate at interfaces (e.g., at liquid-liquid interfaces, liquid-solid interfaces, and / or liquid-gas interfaces) and change the properties of the interface. Surfactants as used herein include detergents, dispersants, suspending agents, emulsion stabilizers, neutral lipids, ionizable lipids, cationic lipids, and anionic lipids.

[0057] A cationic surfactant is provided to impart a charge to the particles.

[0058] In one embodiment, the at least one cationic surfactant is selected from the following salts: 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]butylcarboxamido]ethyl]-3,4-di[oleyloxy]-benzamide, N 4 -cholestyl-spermine, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, O,O'-di(tetradecanoyl)-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 Acyl-sn-glycerol-3-ethylphosphorylcholine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-ethylphosphorylcholine, 1,2-dimyristoleoyl-sn-glycerol-3-ethylphosphorylcholine, 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-di(oleoyloxy)propane-1-ammonium and 3-β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol.

[0059] In one embodiment, the at least one cationic surfactant is a salt of 1,2-di-O-octadecenyl-3-trimethylammonium propane, preferably a chloride salt of 1,2-di-O-octadecenyl-3-trimethylammonium propane.

[0060] In one embodiment, the at least one cationic surfactant is present in an amount of 0.85 to 98 wt. %, based on the total weight of the polynucleotide delivery particle.

[0061] In this regard, various salt forms of the aforementioned cationic surfactants can be provided, including halide salts and hydrohalide salts, such as chlorides, bromides, iodides and hydrochlorides. Where a specific salt (e.g., chloride) is listed, it is understood that other salts (e.g., bromides, iodides, etc.) can also be used. In one embodiment, the preferred salts of the cationic surfactants are chlorides and bromides, more preferably chloride salts.

[0062] The particles of the invention comprise at least one polynucleotide.

[0063] The term "polynucleotide" as used herein refers to a homopolymer or heteropolymer of at least two nucleotide units (also referred to herein as "nucleotides"). "Nucleotides forming polynucleotides" as defined herein include naturally occurring nucleotides, such as ribonucleotides and deoxyribonucleotides, and equivalents, derivatives, variants and analogs of naturally occurring nucleotides.

[0064] In one embodiment, the polynucleotide consists of 10 to 15000 nucleotides, preferably consists of 20 to 5000 nucleotides, more preferably consists of 500 to 4500 nucleotides.

[0065] A polynucleotide can be in single-stranded form or multi-stranded form (e.g., double-stranded, triple-stranded, etc.). A polynucleotide can be in linear form or non-linear form (e.g., containing circular, branched, etc. elements). A polynucleotide can be natural, synthetic, or a combination of both.

[0066] When introduced into a host cell, a polynucleotide may be capable of self-replication. Thus, examples of polynucleotides include self-replicating RNA and DNA, such as selected from replicons, plasmids, cosmids, phagemids, transposons, viral vectors, artificial chromosomes (e.g., bacteria, yeast, etc.) and other self-replicating species.

[0067] Polynucleotides include those that express antigenic polypeptides in host cells (e.g., antigens containing polynucleotides). Polynucleotides include self-replicating polynucleotides into which natural or synthetic sequences derived from eukaryotic or prokaryotic organisms (e.g., genomic DNA sequences, genomic RNA sequences, cDNA sequences, etc.) have been inserted. Specific examples of self-replicating polynucleotides include RNA vector constructs and DNA vector constructs, etc. The sequences that can be expressed include native sequences and modifications, such as deletions, additions, and substitutions (usually conservative substitutions) of native sequences, etc.

[0068] These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through mutation of the host that produces the antigen.

[0069] In one embodiment, the at least one polynucleotide is selected from single-stranded polynucleotides or multi-stranded polynucleotides, preferably selected from artificial messenger RNA (mRNA), chemically modified or unmodified mRNA, which contains at least one coding sequence, self-replicating RNA, circular RNA, viral RNA and replicon RNA; selected from linear DNA, plasmid DNA (pDNA), minicircle DNA, doggybone DNA (dbDNA); selected from small interfering RNA (siRNA), micro RNA (miRNA), guide RNA, small activating RNA (saRNA), antisense oligonucleotide (ASO); or any combination thereof, most preferably mRNA.

[0070] In one embodiment, the at least one polynucleotide is present in an amount of 0.1 to 50 wt.%, preferably 0.2 to 40 wt.%, more preferably 0.3 to 35 wt.%, based on the total weight of the polynucleotide delivery particle.

[0071] The polynucleotide delivery particle according to the present invention may comprise at least one additive.

[0072] In one embodiment, the weight ratio of the at least one additive to the at least one polynucleotide is in the range of 0.01 to 50; or 0.01 to 30; or 0.01 to 10; or 0.01 to 5; or 0.01 to 2; or 0.01 to 1; or 0.01 to 0.1.

[0073] In one embodiment, the at least one additive, preferably a cell penetrating peptide, more preferably human lactoferrin or a fragment thereof, is present at 0.01 to 88 wt % based on the total weight of the polynucleotide delivery particle.

[0074] Any additive known in the art is suitable as long as it is pharmaceutically acceptable.

[0075] "Pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual without causing any undue undesirable biological effect in the individual or interacting in an unduly deleterious manner with any component of the composition in which it is contained.

[0076] For example, the term "additive" includes buffers such as phosphate, acetate, citrate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and other organic compounds; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol 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; amino acids such as glycine, glutamine, asparagine, amine, histidine, arginine or lysine; carbohydrates, including monosaccharides, disaccharides and other glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (such as zinc-protein complexes); carriers; binders; disintegrants; immunoadjuvants, such as cell penetrating peptides, such as human lactoferrin or its fragments, 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, Bac7, CTP, K5-FGF, HAP-1, 293P-1, KALA, GALA, LAH4-L1, bee venom peptide, Penetratin, EB1, MPG, CADY, Pep4, preferably including human lactoferrin or its fragments; fillers (diluents); lubricants; glidants (flow enhancers); compression aids; pigments; antifreeze agents; sweeteners; suspending / dispersing agents; film formers / coatings; flavors; printing inks; non-ionic surfactants; ionizable surfactants; lipids, such as cholesterol, phospholipids, sphingolipids, ceramides, fatty acids; lipids linked to hydrophilic polymers.

[0077] In one embodiment, the polynucleotide delivery particle comprises at least one additive selected from a buffer; an antifreeze agent; an ionizable surfactant; a non-ionic surfactant; a lipid, such as cholesterol, phospholipids, sphingolipids, ceramides, fatty acids; a lipid linked to a hydrophilic polymer.

[0078] In one embodiment, the polynucleotide delivery particle solution comprises at least one buffer, and the at least one buffer is 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-piperazineethane sulfonic acid).

[0079] In one embodiment, the polynucleotide delivery particle comprises less than 0.5 wt % of a nonionic surfactant, preferably less than 0.1 wt % of a nonionic surfactant, and more preferably contains no nonionic surfactant, based on the total weight of the polynucleotide delivery particle.

[0080] Suitable nonionic surfactants vary, and a number of examples are described below.In certain preferred embodiments, the nonionic surfactant is selected from polyvinyl alcohol, polysorbates (eg, polysorbate 20, polysorbate 80), and poloxamers.

[0081] In one embodiment, the polynucleotide delivery particle further comprises at least one ionizable surfactant, preferably at an N / P ratio of the ionizable surfactant to the polynucleotide of 1 to 50. The at least one ionizable surfactant is preferably selected from the following salts: 1,2-distearoyl-3-dimethylammonium-propane, 1,2-dipalmitoyl-3-dimethylammonium-propane, 1,2-dimyristoyl-3-dimethylammonium-propane, 1,2-dioleoyl-3-dimethylamine-propane, 1,2-dioleyloxy-3-dimethylaminopropane, (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28 ,31-tetraen-19-yl 4-(dimethylamino)butyrate, 9-heptadecyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecanyloxy)hexyl]amino}octanoate, N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethylamine (ethanamine), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)di(2-hexyldecanoate).

[0082] In certain embodiments, the particles according to the present invention may contain an immunoadjuvant. Examples of immunoadjuvants include: E. coli heat-labile toxin, alum, liposaccharide phosphate compounds, liposaccharide phosphate mimetics, monophosphoryl lipid A analogs, small molecule immunopotentiators, muramyl tripeptide phosphatidylethanolamine, and tocopherols.

[0083] For example, the immunoadjuvant can be associated with the surface of the particle (e.g., adsorbed or otherwise bound), embedded within the particle, or both. An immunoadjuvant increases or diversifies the immune response to an antigen. Thus, an immunoadjuvant is a compound that is capable of enhancing the immune response to an antigen. An immunoadjuvant can enhance humoral and / or cellular immunity.

[0084] In one embodiment, the particles have an outer coating layer or at least one additive adsorbed to the surface of the particles, preferably a cell penetrating peptide, more preferably human lactoferrin or a fragment thereof. Human lactoferrin or a fragment thereof and a method for producing it according to the present invention are described, for example, in WO 2007076904 A1, and the patent is incorporated herein by reference.

[0085] In one embodiment, the weight ratio of the cell penetrating peptide, preferably human lactoferrin or a fragment thereof, to the at least one polynucleotide is 0.01 to 50, preferably 0.1 to 30.

[0086] "Carbohydrates" as defined herein include monosaccharides, oligosaccharides, and polysaccharides, as well as substances derived from monosaccharides, for example, by reduction (e.g., sugar alcohols), by oxidation of one or more terminal groups to carboxylic acids (e.g., glucuronic acid), or by replacement of one or more hydroxyl groups by a hydrogen atom or an amino group (e.g., β-D-glucosamine and β-D-galactosamine).

[0087] As defined herein, a "monosaccharide" is a polyol, i.e., an alcohol further comprising an aldehyde group (in which case the monosaccharide is an aldose) or a ketone group (in which case the monosaccharide is a ketose). Monosaccharides generally contain 3 to 10 carbons. Furthermore, monosaccharides generally have the empirical formula C n H 2n O n , wherein n is an integer of 3 or greater, typically 3 to 10. Examples of aldoses of 3 to 6 carbons include glyceraldehyde, erythrose, threose, ribose, 2-deoxyribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, and talose.

[0088] Examples of ketoses of 3 to 6 carbons include dihydroxyacetone, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, and tagatose. Naturally occurring monosaccharides usually exist in the D-isomer form rather than the L-form.

[0089] "Oligosaccharide" refers to a relatively short monosaccharide polymer, i.e., a polymer containing 2 to 30 monosaccharide units. "Polysaccharide" is a monosaccharide polymer (i.e., a polymer containing more than 30 monosaccharide units) that exceeds the length of an oligosaccharide. In addition, the term "polysaccharide" as used herein also refers to a monosaccharide polymer containing two or more connected monosaccharides. To avoid ambiguity, the second limitation should always apply unless there is a clear indication to the contrary. The term "polysaccharide" also includes polysaccharide derivatives, such as amino-functionalized polysaccharide derivatives and carboxyl-functionalized polysaccharide derivatives, etc. Monosaccharides are usually connected by glycosidic bonds. Specific examples include disaccharides (e.g., sucrose, lactose, trehalose, maltose, gentiobiose and cellobiose), trisaccharides (e.g., raffinose), tetrasaccharides (e.g., stachyose) and pentasaccharides (e.g., verbascose).

[0090] 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 carbohydrate. Examples include carbohydrate antifreeze, carbohydrate antigens, antigens comprising a carbohydrate conjugated to a carrier peptide, etc. A "polysaccharide-containing species" is a molecule, at least a portion of which is a polysaccharide.

[0091] As used herein, "antifreeze" is a chemical agent that protects the composition from adverse effects during freezing and thawing. For example, in the present invention, an antifreeze, such as a polyol and / or a carbohydrate, etc., can be added to prevent a large amount of particle aggregation from occurring when the freeze-dried composition of the present invention is resuspended.

[0092] Various methods can be used to produce the particles of the present invention. For example, nanoprecipitation can be used, i.e., mixing an aqueous phase containing the polynucleotide with a water-miscible organic phase containing excipients and additives, or nanoemulsion can be used, i.e., mixing an aqueous phase containing the polynucleotide with a water-immiscible organic phase containing excipients and additives.

[0093] In some embodiments, the particles can be formed using an oil-in-water (o / w) or water-in-oil-in-water (w / o / w) solvent evaporation process or using a nanoemulsion method.

[0094] The w / o / w solvent evaporation process is described in, for example, O'Hagan et al, Vaccine (1993) 11: 965-969, Jeffery et al, Pharm. Res. (1993) 10: 362 and WO 00 / 06123A1. PLGA and a cationic surfactant (e.g., selected from those listed above) are dissolved in one or more organic solvents to form an organic solution. The solvent or solvent mixture may include one or more organic solvents, such as selected from 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. Preferred solvents or solvent mixtures may include EtOAc, DCM, EtOAc and DMSO or DCM and DMSO. Then, the organic solution is 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, physiological saline, a buffer solution, such as phosphate buffered saline (PBS) or sodium citrate / ethylenediaminetetraacetic acid (sodium citrate / ETDA) buffer solution, etc. Typically, the volume ratio of the organic solution to the aqueous solution is in the range of about 2:1 to about 20:1, more typically about 10:1. Emulsification is performed using any equipment suitable for this task. The most common methods include simple mechanical stirring, ultrasonic treatment, high shear mixing (HSM), high pressure homogenization (HPH), and microfluids or millifluids such as T or Y mixing.

[0095] A volume of the water-in-oil emulsion is then combined with a second, larger volume of an aqueous solution, which may contain an emulsion stabilizer, such as an uncharged surfactant (e.g., PVA (polyvinyl alcohol), povidone (also known as polyvinyl pyrrolidone or PVP), sorbitan esters, polysorbates, or poloxamers, etc.), or an anionic surfactant or a cationic surfactant (e.g., selected from those listed above, etc.). The volume ratio of the aqueous solution to the 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 produce particles.

[0096] Nanoprecipitation, also known 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", U.S. Pat. No. 5,049,322 of the same title to Devissaguet et al., U.S. Pat. No. 5,118,528 to Fessi et al., entitled "Process for the preparation of dispersible colloidal systems of a substance in the form of microparticles", and WO 2008 / 051245 A1 to Wendorf et al., 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, etc.) can be dissolved in one or more organic solvents (e.g., hydrophilic organic solvents such as acetone, ethanol, DMSO, etc., or any mixture thereof). Then, the resulting organic solution can be combined with another solvent, which is miscible with the organic solvent and is a non-solvent for the polymer, typically an aqueous solution. The aqueous solution can be, for example, deionized water, physiological 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. Then, the organic solution and the aqueous solution can be combined with suitable relative volumes, typically in a 1:9 to 9:1 combination. For example, while stirring or homogenizing or shaking, the organic solution can be poured, injected or dripped into the non-solvent, and vice versa. The mixing of the two solutions can also be achieved by standard T / Y-tube mixing techniques, microfluid mixing, millifluid mixing, turbulent mixing, grinding mixing, or a combination thereof. By selecting a system in which the polymer is soluble in the organic solvent and is significantly less soluble in a miscible blend of the organic solvent and the non-solvent, a suspension of particles can be formed almost instantaneously. Subsequently, the organic solvent can be removed from the suspension, for example by evaporation, dialysis or diafiltration.

[0097] As previously mentioned, in certain embodiments, it is desirable to provide one or more additives (in addition to PLGA) that may be associated with the interior (e.g., embedded) and / or surface (e.g., by adsorption, covalent attachment, co-lyophilization, etc.) of the particle, or may be unassociated with the particle. These additional species may include, for example, chemicals that adjust tonicity or pH, cryoprotectants, immune adjuvants, antigens, etc.

[0098] The additional species may be provided during the particle formation process. 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 employed may therefore further contain various additives as desired. For example, these additives (a) may be added to the organic solution if they are in oil-soluble or oil-dispersible form, or (b) may be added to the aqueous solution if they are in water-soluble or water-dispersible form.

[0099] In some embodiments, one or more additives may be added after particle formation (typically after removal of the organic solvent, and after a washing step or a step in which the particles are dialyzed against water, if any). These additives are often added to the particles in the form of an aqueous solution or dispersion. For example, these additives may be in solution and / or accumulate at the particle-solution interface, such as adsorbed on the particle surface.

[0100] Once a suitable composition is formed (eg, using the techniques described above or others), it may be lyophilized for future use.

[0101] The polynucleotide delivery particles of the present invention may be contained in an oral drug delivery composition, or in a parenteral drug delivery composition, preferably in an injectable drug delivery composition.

[0102] Once formulated (and resuspended if necessary), the polynucleotide delivery particles of the present invention can be administered parenterally, for example, by injection (which can be needle-free injection), as well as by other routes of administration. In this regard, the particle composition is typically supplied in a lyophilized form in a vial or other container, which is supplied with a septum or other suitable device for supplying a resuspension medium (e.g., water for injection) and for extracting the resulting suspension. A suitable syringe can also be supplied for injection. For example, the composition can be injected subcutaneously, intradermally, intramuscularly, intravenously, intraarterially, or intraperitoneally.

[0103] Other routes of administration include nasal, mucosal, intraoccular, rectal, vaginal, oral and pulmonary administration, as well as transdermal or percutaneous application.

[0104] For oral administration, the polynucleotide delivery particles can be contained in a capsule, preferred capsules are described, for example, in WO 2019096833 A1, WO 2020229178 A1, WO 2020229192 A1 and EP Application No. 21175704.2.

[0105] In some embodiments, the compositions of the present invention can be used for site-specific targeted delivery. For example, intravenous administration of the compositions can be used for targeting the lungs, liver, spleen, blood circulation or bone marrow. In addition, oral administration of the compositions can be used for targeted gastrointestinal tract delivery.

[0106] Treatment may be carried out according to a single dose regimen or a multiple dose regimen. A multiple dose regimen refers to a regimen in which a primary course of administration may be given, e.g., with 1 to 10 separate doses, followed by other doses at subsequent time intervals, selected to maintain and / or enhance the therapeutic response, e.g., a second dose at 1 to 4 months, and, if necessary, subsequent doses or doses several months later. The dosage course will also be determined, at least in part, based on the needs of the subject and will depend on the practitioner's discretion.

[0107] In addition, if prevention of disease is desired, the composition is generally administered prior to the primary occurrence of the infection or condition of interest. If other forms of treatment are desired, such as reduction or elimination of symptoms or recurrence, the composition is generally administered after the primary occurrence of the infection or condition of interest.

[0108] Example

[0109] Example 1: Testing DODMA:PLGA for mRNA encapsulation and transfection of mRNA into cells (Comparative Example)

[0110] In this example, the ability of the ionizable lipid DODMA to function as an ionizable surfactant in combination with low molecular weight PLGA was explored.Particle formation, encapsulation of mRNA, and transfection of mRNA into cells were evaluated.

[0111] Preparation of DODMA:PLGA particles

[0112] Table 1: Materials used for particle preparation

[0113]

[0114] 99 μL of RNase-free water, 16.5 μL of 100 mM acetate pH 4 buffer, and 16.5 μL of firefly luciferase encoding mRNA (FLuc mRNA, 1 g / L) were added to a sterile 1.5 mL safe-lock tube, which together formed the aqueous phase. The aqueous phase was vortexed and spun down. 10.98 μL of 20 g / L DODMA stock solution in DMSO, 4.39 μL of 50 g / L RG 501H stock solution in DMSO, and 14.63 μL of DMSO were added to a second 1.5 mL safe-lock tube, which together formed the organic phase. In the case of DODMA particles only, the corresponding volume of RG 501H solution. The organic phase was vortexed (Scientific Industries SI TM Vortex-Genie TM 2) and decelerate the rotation. 120 μL of the aqueous phase was taken out from the first tube and added to the second tube at once with a strong pipette burst. The two phases were further mixed by frequent pipetting. The obtained DODMA:PLGA particles loaded with mRNA were stored in a solution at 4°C until further use.

[0115] Characterization of DODMA:PLGA Particles

[0116] Particle size was measured on a Malvern Zetasizer Nano ZS at an mRNA concentration of 10 ng / μL using water as the dispersant.

[0117] Use Invitrogen TM E-Gel TM Gel electrophoresis was performed using the Power Snap electrophoresis system with 1 μg of mRNA per well.

[0118] Luciferase assays were performed with human epithelial cells (HeLa). One day before transfection, 10,000 cells per well were seeded into 96-well plates and cultured for 24 hours at 37°C and 5% CO2. On the second day, the old culture medium was removed and 90 μL of fresh culture medium was added to the cells. RNase-free water was used for dilution and the samples were adjusted to an mRNA concentration of 10 ng / μL. 10 μL of the corresponding 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). The luminescent signal was measured by a multiplate reader (well plate reader 200PRO, Tecan) for quantification.

[0119] result

[0120] Table 2: Dynamic light scattering data of DODMA:PLGA particles

[0121] particle z-mean value [nm] PDI DODMA 134 0.120 DODMA:RG 501H 200 0.130

[0122] DODMA and a mixture of DODMA and RG 501H formed defined nanoparticles when the organic phase was mixed with the aqueous phase containing mRNA. The addition of RG 501H resulted in a significant increase in particle size.

[0123] However, according to agarose gel electrophoresis ( Figure 1 ), the mRNA was not encapsulated into the formed particles, either in the case of DODMA or in the mixture with RG 501H. Accordingly, when incubated with HeLa cells, neither of the two tested compositions achieved substantial transfection ( Figure 3 ).

[0124] Therefore, this experiment clearly demonstrated that the combination of ionizable surfactants alone with PLGA is not sufficient for mRNA encapsulation and transfection.

[0125] Example 2: Testing DOTMA:PLGA for mRNA encapsulation and transfection of mRNA into cells (Inventive Example)

[0126] In this example, the ability of the ionizable lipid DOTMA to function as an ionizable surfactant in combination with low molecular weight PLGA was explored.Particle formation, encapsulation of mRNA, and transfection of mRNA into cells were evaluated.

[0127] Preparation of DOTMA:PLGA particles

[0128] Table 3: Materials used for particle preparation

[0129]

[0130] 66 μL of RNase-free water, 11 μL of 100 mM acetate pH 4 buffer, and 11 μL of FLuc mRNA (1 g / L) were added to a sterile 1.5 mL safe-lock tube to form the aqueous phase. The aqueous phase was vortexed and rotated at a reduced speed. 7.91 μL of 20 g / L DOTMA stock solution in DMSO, 6.33 μL of 50 g / L RG501H, and 5.76 μL of DMSO, which formed the organic phase, were added to a second 1.5 mL safe-lock tube. In the case of DOTMA particles only, the corresponding volume of PLGA solution was replaced with DMSO. The organic phase was vortexed and rotated at a reduced speed. 80 μL of the aqueous phase was taken out from the first tube and added to the second tube at one time with a strong pipette burst. The two phases were further mixed by frequent pipetting. The obtained DOTMA:PLGA particles loaded with mRNA were stored in a solution at 4°C until further use.

[0131] Characterization of DOTMA:PLGA particles

[0132] Particle size was measured on a Malvern Zetasizer Nano ZS at an mRNA concentration of 10 ng / μL using water as the dispersant.

[0133] Use Invitrogen TM E-Gel TM Gel electrophoresis was performed using the Power Snap electrophoresis system with 1 μg of mRNA per well.

[0134] Luciferase assays were performed with human epithelial cells (HeLa). One day before transfection, 10,000 cells per well were seeded into 96-well plates in DMEM medium and cultured for 24 hours at 37°C and 5% CO2. On the second day, the old medium was removed and 90 μL of fresh medium was added to the cells. The samples were adjusted to an mRNA concentration of 10 ng / μL using RNAse-free water for dilution. 10 μL of the corresponding 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). The luminescent signal was measured by a multiplate reader (well plate reader 200PRO, Tecan) for quantification.

[0135] result

[0136] Table 4: Dynamic light scattering data of DOTMA:PLGA particles

[0137] particle z-mean value [nm] PDI DOTMA 104 0.340 DOTMA:RG 501H 125 0.120

[0138] DOTMA and mixtures of DOTMA and RG 501H formed defined nanoparticles when the organic phase was mixed with the aqueous phase containing mRNA. In contrast to DODMA, in the case of DOTMA, the particle size was within the same range for all tested mixtures, and the addition of RG 501H did not result in a significant increase in particle size. Without being bound by any theory, it is hypothesized that this can be attributed to the permanent cationic charge of DOTMA, which provides better colloidal stability of the particles compared to DODMA.

[0139] According to agarose gel electrophoresis ( Figure 2 ), for all tested compositions, the mRNA was completely encapsulated into the formed particles. Without being bound by any theory, it is hypothesized that this can again be attributed to the cationic charge provided to the particles by the use of DOTMA.

[0140] In terms of transfection efficiency ( Figure 4 ), significantly better performance of the DOTMA:RG 501H hybrid particles was observed compared to DOTMA alone and compared to all previously tested DODMA-containing compositions.

[0141] Therefore, this experiment clearly shows that the combination of cationic surfactants and the specific PLGA of the present invention is very suitable for mRNA encapsulation and transfection. In addition, the results show that the specific PLGA of the present invention has a beneficial effect on the efficiency of the particles described in the present invention when it is combined with a cationic surfactant.

[0142] Example 3: Comparison of low and medium molecular weight PLGA for mRNA encapsulation and transfection into cells (inventive example)

[0143] In this example, two PLGA polymers of low and medium molecular weight were used in combination with DOTMA to encapsulate FLuc mRNA and form particles. The transfection efficiency and kinetics of the particles depending on the PLGA used were evaluated.

[0144] Preparation of DOTMA:PLGA particles

[0145] Table 5: Materials used for particle preparation

[0146]

[0147] 75.17 μL of RNase-free water, 5.5 μL of 200 mM HEPES pH 7 buffer, and 11 μL of FLuc mRNA (1 g / L) were added to a sterile 1.5 mL safe-lock tube to form the aqueous phase. The solution was vortexed and rotated at a reduced speed. 7.91 μL of 20 g / L DOTMA stock solution in DMSO, 6.33 μL of 50 g / L RG 501H or 50 g / L RG 503H stock solution in DMSO, and 2.42 μL of DMSO were added to a second 1.5 mL safe-lock tube to form the organic phase. The solution was vortexed and rotated at a reduced speed. 83.33 μL of the aqueous phase was taken out from the first tube and added to the second tube at one time with a strong pipette burst. The two phases were further mixed by frequent pipetting. The obtained mRNA-loaded DOTMA:PLGA particles were stored in solution at 4 °C until further use.

[0148] Characterization of DOTMA:PLGA particles

[0149] Particle size was measured on a Malvern Zetasizer Nano ZS at an mRNA concentration of 10 ng / μL using water as the dispersant.

[0150] Luciferase assays were performed with human epithelial cells (HeLa). One day before transfection, 10,000 cells per well were seeded into 96-well plates, seeded in DMEM medium, and cultured for 24 hours at 37°C and 5% CO2. On the second day, the old culture medium was removed and 90 μL of fresh culture medium was added to the cells. Using RNase-free water for dilution, the sample was adjusted to an mRNA concentration of 10 ng / μL. 10 μL of the corresponding diluted sample was added to the cells, equivalent to an amount of 100 ng of mRNA per well, with 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). The luminescent signal was measured by a multiplate reader (well plate reader 200PRO, Tecan) for quantification.

[0151] result

[0152] Table 6: Dynamic light scattering data of DOTMA:PLGA particles

[0153]

[0154] DOTMA and low molecular weight Mixture of RG 501H and DOTMA with medium molecular weight The mixture of RG 503H formed defined nanoparticles when the organic phase was mixed with the aqueous phase containing the mRNA. However, the particles obtained with RG 503H were larger than those obtained with RG 501H.

[0155] At different time points of cell incubation, the in vitro transfection efficiency of particles containing RG 501H or RG 503H was assessed by luciferase assay ( Figure 5 ). The luciferase assay confirmed the functionality of the DOTMA:PLGA particles in transfecting the cells with encapsulated FLuc mRNA. Importantly, at all time points tested, particles containing RG 501H performed better than particles with RG 503H. Without being bound by any theory, it is hypothesized that this can be attributed to the lower molecular weight of RG 501H compared to RG 503H, which promotes particle disassembly and release of the encapsulated mRNA. Ultimately, the promoted mRNA release leads to faster transfection kinetics, as well as improved overall transfection efficiency of the corresponding particles. Therefore, the efficiency of the particles described in the present invention is directly related to the PLGA used in the composition.

[0156] Example 4: Coating DOTMA:PLGA particles with human lactoferrin fragments to improve mRNA transfection into intestinal epithelial cells (inventive example)

[0157] In this example, DOTMA:RG 501H particles were coated with the cell penetrating peptide human lactoferrin fragment (hLFF) to further improve uptake and transfection efficiency in intestinal epithelial cells.

[0158] Table 7: Materials used for particle preparation

[0159]

[0160] 264 μL of RNase-free water, 44 μL of 100 mM acetate pH 4 buffer, and 44 μL of FLuc mRNA (1 g / L) were added to a sterile 1.5 mL safe-lock tube, which together formed the aqueous phase. The aqueous phase was vortexed and rotated at a reduced speed. 31.65 μL of 20 g / L DOTMA stock solution in DMSO, 25.32 μL of 50 g / L RG501H stock solution, and 23.03 μL of DMSO, which together formed the organic phase, were added to a second 1.5 mL safe-lock tube. The organic phase was vortexed and rotated at a reduced speed. 320 μL of the aqueous phase was taken out from the first tube and added to the second tube at one time with a strong pipette burst. The two phases were further mixed by frequent pipetting. The obtained DOTMA:RG 501H particles loaded with mRNA were stored in a solution at 4°C until further use. In separate 1.5 mL tubes, 2 μL, 6 μL, or 12 μL of a 2.5 g / L stock solution of hLFF in water was mixed with 48 μL, 44 μL, or 38 μL of RNase-free water, respectively, so that each tube contained a final volume of 50 μL of the diluted hLFF solution. An additional tube contained 50 μL of tap water as a negative control. 50 μL of the pre-prepared DOTMA:RG 501H particle solution was added to each prepared tube, with a single strong pipette burst followed by frequent pipetting. The coated particles were stored in solution at 4°C until further use.

[0161] Characterization of DOTMA:PLGA:hLFF particles

[0162] Particle size was measured on a Malvern Zetasizer Nano ZS at an mRNA concentration of 10 ng / μL using water as the dispersant.

[0163] Luciferase assays were performed with human colorectal adenocarcinoma cells (Caco-2). One day before transfection, 10,000 cells per well were seeded into 96-well plates in DMEM medium and cultured for 24 hours at 37°C and 5% CO2. On the second day, the old medium was removed and 90 μL of fresh medium was added to the cells. The samples were adjusted to an mRNA concentration of 10 ng / μL using RNAse-free water for dilution. 10 μL of the corresponding 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). The luminescent signal was measured by a multiplate reader (well plate reader 200PRO, Tecan) for quantification.

[0164] result

[0165] Table 8: Dynamic light scattering data of DOTMA:RG 501H:hLFF particles

[0166] particle z-mean value [nm] PDI DOTMA:RG 501H, 1:2 125 0.270 DOTMA:RG 501H:hLFF,1:2:0.316 114 0.250 DOTMA:RG 501H:hLFF,1:2:0.948 102 0.160 DOTMA:RG 501H:hLFF,1:2:1.896 103 0.150

[0167] The mixture of DOTMA and RG 501H formed defined nanoparticles when the organic phase was mixed with the aqueous phase containing mRNA. Coating of these particles with hLFF resulted in a slight reduction in particle size.

[0168] In the case of transfection efficiency in Caco-2 cells ( Figure 6 ), significantly better performance of the hLFF coated particles was observed when compared to the uncoated particles. The maximum value of transfection efficiency was reached at a DOTMA:hLFF ratio of 1:0.948. Without being bound by any theory, it is hypothesized that the positive effect of hLFF stems from the improved cellular uptake mediated by hLFF, an effect also known from other cell penetrating peptides.

[0169] Therefore, this experiment clearly shows that the DOTMA:PLGA particles can be further improved by coating with cell penetrating peptides such as hLFF.

[0170] Example 5: Enteric coated capsules filled with RNA-containing DOTMA:PLGA particles and pH-dependent release of particles

[0171] In this example, DOTMA:RG 501H particles containing mRNA were used as a relevant model drug product in combination with an enteric coated capsule.

[0172] Preparation and characterization of DOTMA:PLGA particles

[0173] Table 9: Materials used for particle preparation

[0174]

[0175] use Benchtop (PNI) platform, 0.5 mL of DMSO solution containing 9.5 g / L DOTMA and 19.0 g / L RG 501H (organic phase) was mixed with 2.5 mL of 12 mM HEPES pH 7 solution containing 0.12 g / L FLuc mRNA without RNAse (aqueous phase). The resulting particle solution was dialyzed against 10 mM HEPES pH 7 buffer (Slide-A-Lyzer TM, 10K MWCO) for 3 hours (3 buffer changes). After dialysis, RNAse-free trehalose solution (20 wt %) was added to the particle solution to reach a final trehalose concentration of 10 wt %. The particles were lyophilized for 48 hours and stored at 4° C. until further use.

[0176] Filling the capsule with particles

[0177] The lyophilized particles were filled into enteric coated capsules (types P0001 / 21 and 22274 / 27 disclosed in Example 5 and Example 8 in EP application No. 21175704.2) in an amount equal to 100 μg of mRNA per capsule. The filled capsules were sealed and stored at 4°C until further use.

[0178] Capsule dissolution assay

[0179] To simulate the gastric environment in the fed state, the capsules were incubated for 2 hours at 37°C on a shaker in 10 mL of 0.1 N HCl containing 2 g / L of pepsin. Samples were collected after 60 and 120 minutes for release analysis. Subsequently, the acidic medium was exchanged against 10 mL of phosphate buffer (18.8 mM phosphate, 145.4 mM NaCl, pH 6.8), and the capsules were incubated for another 60 minutes, with samples collected every 15 minutes.

[0180] As a negative control, pure DOTMA:RG 501H particles without capsule protection were incubated under the same conditions: 40 μL of particle solution (containing 50 ng / μL of mRNA) was mixed with 100 μL of 0.1 N HCl containing 2 g / L of pepsin and incubated for 2 hours at 37° C. and 300 rpm on an orbital shaker. Subsequently, 60 μL of phosphate buffer was added to the mixture and incubation continued for an additional 60 minutes.

[0181] After the dissolution assay, the culture medium containing the dissolved capsules and PLGA particles was used immediately in the cell transfection assay without intermediate storage. The samples collected at fixed time intervals were stored at 4°C until further analysis in the Ribogreen assay.

[0182] Ribogreen assay

[0183] A Ribogreen assay was applied to facilitate detection and quantification of RNA after release of particles from capsules. mRNA concentrations were measured at different time intervals to establish release kinetics.

[0184] Quant-iT TM RiboGreenTM RNA assay kit was used for this assay. As the Ribogreen assay is based on measuring fluorescence, a black 96-well assay plate with a clear bottom was applied.

[0185] The procedure was performed according to the manufacturer's protocol with slight adjustments. In the first step, 1x TRIS / EDTA (TE) buffer was prepared by diluting the buffer stock solution with RNase-free water. Using TE buffer, the particle sample was diluted to a theoretical concentration of 1 μg / ml and added to the well plate in a volume of 50 μl. 50 μl of TE buffer was added to the sample to facilitate the measurement of the concentration of the usable mRNA. Calibration standards with corresponding Fluc mRNA and buffer were applied and added to the same well plate as the sample. The working solution of Ribogreen dye was prepared by diluting the reagent 1:100 with TE buffer. 100 μl of working solution was added to each well and then mixed thoroughly by pipetting up and down. The fluorescence signal was measured at the excitation / emission value of 480 / 520 nm with a microplate reader. All samples and standards were measured in duplicate.

[0186] Luciferase assay

[0187] One day before transfection, 10,000 cells per well were seeded into a 96-well plate and cultured for 24 hours at 37°C and 5% CO2. On the second day, the old culture medium was removed and 90 μL of fresh culture 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 the corresponding diluted sample was added to the cells, equivalent to an amount of 100 ng of mRNA per well, with a total volume of 100 μL. The cells were further incubated for 24 hours at 37°C and 5% CO2. On the third day, the transfection efficiency was determined using a luciferase kit system according to the manufacturer's protocol (Promega GmbH). By adding luciferase substrate to the cells, a luminescent signal is generated, which can be read by a multiplate reader (well plate reader 200PRO, Tecan) for quantification.

[0188] result

[0189] Table 10: DLS data of DOTMA:RG 501H particles after different treatment steps.

[0190] particle z-mean value [nm] PDI DOTMA:RG 501H, after preparation 52.9 0.315 DOTMA:RG 501H, 3 weeks at 4°C 50.9 0.256 DOTMA:RG 501H, 3 weeks at -20°C 58.7 0.193 DOTMA:RG 501H, lyophilized 58.9 0.386

[0191] DLS data indicate that defined DOTMA:RG 501H particles containing mRNA can be generated by microfluidics ( The size of the particles obtained by microfluidic mixing was significantly smaller than that of samples produced by pipetting. In addition, the particle size remained unchanged during various processing steps and different storage conditions.

[0192] The Ribogreen assay ( Figure 7 ) clearly demonstrated that the release of DOTMA:RG 501H particles from enteric coated capsules was pH dependent as measured by the signal generated by the available mRNA within the particles.

[0193] Within 30 minutes after changing the incubation medium from acidic pH to pH 6.8, the particles were fully rehydrated and released from the capsules, while the capsules were completely dissolved.

[0194] Importantly, during incubation in 0.1 N HCl for 120 min, no release of particles and mRNA was observed, confirming the structural integrity of the capsule under acidic conditions.

[0195] A luciferase transfection assay was applied in human epithelial (HeLa) cells to assess particle functionality after release from the capsule ( Figure 8 ). Freeze-dried particles that were only rehydrated or additionally incubated in gastric and intestinal fluids simulating the fed state without capsule protection were used as positive and negative controls, respectively. The luciferase assay showed the functionality of the DOTMA:RG 501H particles after release from the capsule, because HeLa cells incubated with these samples showed different expressions of embedded Fluc mRNA. The protective effect of the particles against gastric and intestinal fluids simulating the fed state was further verified by considering the negative control of the particles exposed to the same culture medium without any capsule protection. The transfection efficiency of the capsule-protected particles was about 2 logs higher than that of the unprotected particles, confirming the obvious beneficial effect of the enteric-coated capsules on the functionality of the particles. Compared to the positive control, i.e., the freeze-dried particles were rehydrated and directly applied to the transfection assay, the efficiency of the released particles was about 1 log lower. Without being bound by any theory, this may be attributed to the fact that the dissolved capsule components may interact with the particles and damage their integrity. The efficiency decline of the two tested capsule types is comparable.

Claims

1. A polynucleotide delivery particle comprising or consisting of: a) at least one poly(lactic acid-co-glycolide); b) at least one cationic surfactant; c) at least one polynucleotide; and d) optionally at least one additive; wherein: The poly(lactic acid-co-glycolide) has a weight average molecular weight Mw of 1000 to 9500 g / mol, as measured by gel permeation chromatography using polystyrene standards and chloroform.

2. The polynucleotide delivery particle according to claim 1, wherein The at least one poly(lactic acid-co-glycolide) has: i) a number average molecular weight Mn of 1000 to 3000 g / mol, as measured by gel permeation chromatography using polystyrene standards and chloroform; and / or ii) a molar ratio of lactide to glycolide in the range of 40:60 to 60:40; and / or iii) an intrinsic viscosity of 0.05 to 0.25 dl / g, as measured by viscometry; and / or iv) Acid value of 20 to 30 mg KOH / g.

3. The polynucleotide delivery particle according to any one of the preceding claims, wherein: The at least one cationic surfactant is: i) a salt selected from the group consisting of 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]butylcarboxamido]ethyl]-3,4-di[oleyloxy]-benzamide, N 4 -cholestenyl-spermine, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, O,O'-di(tetradecanoyl)-N-(α-trimethylammonioacetyl)diethanolamine, 1,2-dilauroyl-sn-glycerotrioxy-3-ethylphosphocholine, 1,2-dimyristoyl-sn-glycerotrioxy-3-ethylphosphocholine, 1,2-dipalmitoyl-sn-glycerotrioxy-3-ethylphosphocholine, 1,2-distearoyl-sn-glycerotrioxy-3-ethylphosphocholine, 1,2-dioleoyl-sn-glycerotrioxy-3-ethylphosphocholine choline, 1-palmitoyl-2-oleoyl-sn-glycerotrioxy-3-ethylphosphorylcholine, 1,2-dimyristoleoyl-sn-glycerotrioxy-3-ethylphosphorylcholine, 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-di(oleoyloxy)propane-1-ammonium and 3-β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol; or ii) a salt of 1,2-di-O-octadecenyl-3-trimethylammonium propane, preferably a chloride salt of 1,2-di-O-octadecenyl-3-trimethylammonium propane.

4. The polynucleotide delivery particle according to any one of the preceding claims, wherein the at least one polynucleotide is selected from a single-stranded polynucleotide or a multi-stranded polynucleotide.

5. The polynucleotide delivery particle according to any one of the preceding claims, wherein: 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-co-glycolide) to the polynucleotide is in the range of 1:1 to 200:

1.

6. A polynucleotide delivery particle according to any one of the preceding claims, wherein the at least one additive is selected from a buffer; an antifreeze agent; an ionizable surfactant; a non-ionic surfactant; a lipid, such as cholesterol, phospholipids, sphingolipids, ceramides, fatty acids; a lipid linked to a hydrophilic polymer.

7. The polynucleotide delivery particle according to any one of the preceding claims, wherein the particle: i) has a z-average particle size of 1 to 1000 nm as measured by dynamic light scattering; and / or ii) a polydispersity index of 0.01 to 0.5 as measured by dynamic light scattering.

8. The polynucleotide delivery particle according to any one of the preceding claims, wherein the particle has an outer coating layer or at least one additive adsorbed to the surface of the particle. 9 . The polynucleotide delivery particle according to claim 8 , wherein the outer coating layer comprises human lactoferrin or a fragment thereof, or the at least one additive adsorbed to the surface of the particle is human lactoferrin or a fragment thereof.

10. A method of forming a polynucleotide delivery particle according to any one of claims 1 to 9, wherein the particle is formed by nanoprecipitation or nanoemulsion method.

11. An oral drug delivery composition comprising at least one polynucleotide delivery particle according to any one of claims 1 to 9.

12. A parenteral drug delivery composition comprising at least one polynucleotide delivery particle according to any one of claims 1 to 9.

13. The oral drug delivery composition according to claim 11 or the parenteral drug delivery composition according to claim 12 for use as a medicament.

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