Method of manufacturing a pharmaceutical composition

Abstract

A method of manufacturing a pharmaceutical composition comprises incorporating a deep eutectic solvent into the pharmaceutical composition. The pharmaceutical composition comprises an active ingredient encapsulated in nanoparticles. Treating the pharmaceutical composition with the deep eutectic solvent may stabilise the active ingredient; increase the potency of the composition; and / or modify the pharmacokinetics of the composition. Also provided is a pharmaceutical composition obtainable by the method; medical uses of the composition; and the use of a deep eutectic solvent to stabilise an active ingredient of a pharmaceutical composition, wherein the active ingredient is encapsulated in nanoparticles.

Description

[0001] Method of manufacturing a pharmaceutical composition

[0002] Technical Field

[0003] The present invention generally relates to the manufacture of a pharmaceutical composition comprising an active ingredient encapsulated in nanoparticles. In particular, the active ingredient may be a biomolecule, such as a nucleic acid, and the nanoparticles may be lipid- based nanoparticles.

[0004] Further aspects of the invention provide a pharmaceutical composition obtainable by the method, medical uses of the composition, and the use of a deep eutectic solvent in the manufacture of a pharmaceutical composition.

[0005] Background

[0006] Various types of nanoparticles, such as lipid nanoparticles and liposomes, are used as vehicles to deliver active pharmaceutical ingredients ("APIs") into cells. Examples of such nanoparticles include liposomes and lipid nanoparticles.

[0007] A liposome has an aqueous solution core surrounded by a hydrophobic membrane in the form of a lipid bilayer. Hydrophilic solutes may be dissolved in the core, and cannot readily pass through the bilayer, whereas hydrophobic species may associate with the bilayer. Liposomes may therefore be loaded with a wide variety of active pharmaceutical ingredients. In use, the lipid bilayer may fuse with the cell membrane, thereby delivering the contents of the liposome into the cell.

[0008] Lipid nanoparticles are similar in structure to liposomes. In a lipid nanoparticle, the API is encapsulated within a lipid monolayer, as opposed to a lipid bilayer.

[0009] Existing mRNA vaccines, such as Pfizer-BioNTech mRNA vaccine BNT162b2 / Comirnaty which played a major role in the response to the COVID-19 pandemic, use lipid nanoparticles ("LNPs") to deliver an mRNA payload into cells. A limitation of existing mRNA vaccines is that they have stringent storage needs, e.g. requiring storage at -70 °C, to limit degradation of the highly labile mRNA payload.

[0010] A further limitation of existing RNA-LNPs is that delivery of the RNA into cells is relatively inefficient. According to estimates, only about 2 to 4 % of the mRNA is translated into protein in the cell.

[0011] There is therefore a need for compositions for drug delivery which stabilise the API, and which provide more effective delivery of the API into cells.

[0012] Curreri et al. (Adv. Healthcare Mater. 2024, 2400327) discloses mRNA delivery using lipid- based nanocomposites. The nanocomposites include a liquid selected from cholinium malonate, cholinium glutarate, cholinium hexenoate, and cholinium octanoate. Curreri et al. use the term "deep eutectic solvents" to describe the compositions. However, this terminology is inaccurate because the liquids are in fact salts, i.e. ionic liquids.

[0013] WO 2019 / 122329 Al discloses a vaccine composition comprising a live enveloped virus and a pharmaceutically acceptable carrier, the pharmaceutically acceptable carrier being a natural deep eutectic solvent.

[0014] WO 2020 / 201048 Al discloses a vaccine composition comprising live Mollicutes bacteria and a natural deep eutectic solvent.

[0015] WO 2022 / 254209 Al discloses a composition comprising a payload molecule and an ionic liquid.

[0016] Summary

[0017] In one aspect, there is provided a method of manufacturing a pharmaceutical composition. The pharmaceutical composition comprises an active ingredient encapsulated in nanoparticles. The method comprises incorporating a deep eutectic solvent into the pharmaceutical composition. Using the deep eutectic solvent may improve the stability and / or potency of the pharmaceutical composition.

[0018] Incorporating the deep eutectic solvent into the composition may comprise encapsulating the deep eutectic solvent in the nanoparticles. Alternatively or additionally, the nanoparticles may be dispersed in a medium which medium includes the deep eutectic solvent. In accordance still another possibility, the nanoparticles may be transiently treated with the deep eutectic solvent.

[0019] A further aspect provides a pharmaceutical composition obtainable by the method.

[0020] For example, the pharmaceutical composition may comprise an active ingredient encapsulated in nanoparticles; a carrier; and a deep eutectic solvent. The nanoparticles are dispersed in the carrier. The deep eutectic solvent is present in the nanoparticles and / or present in the carrier.

[0021] The pharmaceutical composition may be for use in medicine. Optionally, the composition may be for use veterinary medicine.

[0022] A related aspect provides a method of treating or preventing a disease or disorder, the method comprising administering a pharmaceutical composition as defined herein to a subject in need thereof.

[0023] Still another aspect provides the use of a deep eutectic solvent to stabilise an active ingredient of a pharmaceutical composition, wherein the active ingredient is encapsulated in nanoparticles.

[0024] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Nor is the claimed subject matter limited to implementations that solve any or all of the disadvantages noted herein. Brief Description of the Drawings

[0025] To assist understanding of embodiments of the present disclosure and to show how such embodiments may be put into effect, reference is made, by way of example only, to the accompanying drawings in which:

[0026] Fig. 1 is a flow diagram outlining a first method of manufacturing a pharmaceutical composition;

[0027] Fig. 2 is a flow diagram outlining a second method of manufacturing a pharmaceutical composition;

[0028] Fig. 3 is a graph showing the potency of LNPs treated with choline chloride : L-arabitol for various time intervals, as discussed in Example 7A;

[0029] Fig. 4 is a graph showing the potency of LNPs treated with choline chloride : L-arabitol for various time intervals, as discussed in Example 7B;

[0030] Fig. 5 is a graph comparing the potency of LNPs treated with various DESs and individual DES components, as discussed in Example 7C;

[0031] Fig. 6 is a graph showing the effect of various dilutions the DES choline chloride : arabitol (1:1 mokmol) on LNP potency, as discussed in Example 7D;

[0032] Fig. 7 is a graph showing the potency of DES-treated LNPs after storage in various different diluents, as discussed in Example 7E;

[0033] Fig. 8 is a graph showing the potency of mRNA-LNPs treated transiently with various different DESs, as discussed in Example 7F;

[0034] Fig. 9 is a graph comparing the potency of treated and untreated mRNA-LNPs in DMEM / FCS at different temperatures, as discussed in Example 7G;

[0035] Fig. 10 is a photograph of an agarose gel showing the stability of Comirnaty® SARS- CoV-2 SARS-CoV-2 mRNA-LNPs stored at 100 °C in the DES choline chloride: sorbitol (1:1 mokmol) compared with PBS, as discussed in Example 8.

[0036] Fig. 11 is a photograph of an agarose gel showing the stability of Comirnaty® SARS- CoV-2 SARS-CoV-2 mRNA-LNPs in the DES choline chloride: sorbitol (1:1 mokmol) and PBS following 10 freeze-thaw cycles, as discussed in Example 8; Fig. 12 is a graph showing the encapsulation efficiency (EE%) of LNPs formulated in different DESs before and after vortexing as compared to a control, as discussed in Example 23;

[0037] Fig. 13 is a graph showing the potency of mRNA-LNPs formulated with three types of internal DES, as discussed in Example 24;

[0038] Fig. 14 shows semi-quantitative imaging of luciferase activity 24 hours after 6 mice were injected in each hind leg i.m. with one of six different Fluc-LNP formulations, as discussed in Example 27;

[0039] Fig. 15 is a quantitative representation of luciferase activity in the hind legs of the six mice injected with mRNA-LNPs discussed in Example 27;

[0040] Fig. 16 shows semi-quantitative imaging of Luciferase activity in nine organs of three mice, 24 hours post-injection of mRNA-LNPs as discussed in Example 27;

[0041] Fig. 17 is a graph showing LNP particle size and PDI stability for 6 DES-treated LNP samples and a control after storage at 22 °C for up to 3 months, as discussed in Example 28;

[0042] Fig. 18 shows the encapsulation efficiency and mRNA purity of the samples discussed in Example 28;

[0043] Fig. 19 is a graph showing LNP particle size and PDI stability for two DES LNP formulations ("External DES-2" and "External DES-3") compared with a control after storage at 37 °C for up to 3 months, as discussed in Example 28;

[0044] Fig. 20 is a graph showing the encapsulation efficiency and purity for two DES LNP formulations ("External DES-2" and "External DES-3") compared with a control after storage at 37 °C for up to 3 months, as discussed in Example 28;

[0045] Fig. 21 is a graph showing a comparison of potency of control LNPs and LNPs treated with Internal DES-1 following either (i) 4 months at 4 °C; or (ii) 3 months at 22 °C followed by 1 month at 4 °C; and

[0046] Fig. 22 shows the results of performing capillary electrophoresis on LNPs stored in various different media at 25 °C for 7 months, as discussed in Example 30. Detailed Description

[0047] In one aspect, the present invention provides a method of manufacturing a pharmaceutical composition, wherein the pharmaceutical composition comprises an active ingredient encapsulated in nanoparticles, which method comprises incorporating a deep eutectic solvent into the pharmaceutical composition.

[0048] It has surprisingly been found that using the deep eutectic solvent during the manufacture of the pharmaceutical composition may stabilise the active ingredient; increase the potency of the composition; and / or modify the pharmacokinetics of the composition.

[0049] Potency is a measure of the amount of protein that is translated from the mRNA transfected into cells. High potency indicates highly efficient delivery and translation of mRNA into protein. Potency is expressed as a percentage of a time point zero positive control such as a mRNA-LNP. Reduced potency may be indicative of mRNA degradation and / or cross-linking, for example.

[0050] Active ingredients

[0051] The pharmaceutical composition includes an active ingredient which is encapsulated in a nanoparticle. As used herein, the terms "active ingredient" and "payload" are synonymous.

[0052] The nature of the active ingredient is not particularly limited, and may be selected as appropriate. Typically, the active ingredient is to be transported across a cell membrane by the nanoparticle.

[0053] The active ingredient may comprise a biomolecule. Examples of biomolecules include nucleic acids such as RNA and DNA; peptides; proteins, such as phosphoproteins; lipids; and carbohydrates. The biomolecule may for example be selected from a nucleic acid, a peptide, a protein, and combinations thereof. Examples of proteins which may be delivered by the pharmaceutical composition include antigens, enzymes, and antibodies.

[0054] The biomolecule may be a nucleic acid, optionally an RNA, and further optionally an mRNA. The methods described herein have been found to be effective for stabilising RNA, and increasing the potency of compositions which deliver RNA to cells.

[0055] The nucleic acid may encode all or part of a peptide, polypeptide, or protein such as an antigen; an antibody; an enzyme; a structural protein; a peptide hormone such as glucagon- like peptide-1 (GLP-1); a hormone such as insulin or erythropoietin, interferon, monoclonal antibodies, growth hormone, factor VIII, tissue plasminogen activator, interleukin-2, tumour necrosis factor, granulocyte colony-stimulating factor (GCSF), glucagon, albumin, follicle- stimulating hormone (FSH), streptokinase, chymotrypsin, alpha-1 antitrypsin, coagulation factor such as IX or a growth factor.

[0056] The nucleic acid may be for use as a personalized mRNA cancer vaccine including coding for cellular neoantigens, gene therapy, a vaccine, gene or RNA editing including CRISPR-based gene editing for treating genetic, neurodegenerative, cardiovascular, metabolic, autoimmune disorders or infections.

[0057] The nucleic acid may comprise DNA or RNA. For example, the nucleic acid may be selected from single-stranded RNA, double-stranded RNA, single-stranded DNA, double-stranded DNA, mRNA, siRNA, tRNA, rRNA, RNAi, gRNA, ASO, an aptamer, an oligonucleotide (for example, single-stranded or double-stranded oligonucleotides of 6-100 nucleotides and polynucleotides; deoxy- or ribo-oligonucleotides), short-hairpin RNA (shRNA), microRNA (miRNA), self-amplifying RNA (saRNA), CRISPR RNA (crRNA), long non-coding RNA (IncRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), guide RNA (gRNA), non-coding RNA (ncRNA), antisense RNA (asRNA), piRNA, circular RNA (circRNA), circular single-stranded or double-stranded DNA or RNA (including mRNA), linear RNA, base modified RNA or DNA (e.g. Nl-Me-Pseudouridine nucleobases), 5' capped mRNA, 3' poly A tailed mRNA, conjugated nucleic acid (e.g. small molecule or protein conjugated with a nucleic acid), ribozyme, peptide nucleic acid (PNA), 2' -OH modified RNA, nucleic acid control RNA, a mixture of types of nucleic acids (e.g. DNA molecule mixed with an RNA molecule), plasmid DNA, genomic DNA or RNA, mitochondrial DNA or RNA, non-coding RNA, non-spliced or partially-spliced intron containing mRNA, single or double-stranded viral DNA or RNA sequence, chimeric DNA and RNA sequences and linear, circular or branched DNA or RNA. Examples of 2' -OH modified RNAs include 2'-F, 2'-0Me, and 2'-OAcetyl RNA. In accordance with another possibility, the nucleic acid may be a phosphate modified RNA or DNA (e.g. phosphorothioate).

[0058] The nucleic acid may be obtained using any suitable technique. For example, the nucleic acid may be produced using a synthetic organic procedure (e.g. using an oligonucleotide synthesizer) or an in vitro enzymatic reaction (e.g. an in vitro RNA transcription reaction). The nucleic acid may be from a natural biological source, and may for example be extracted from a virus or a cell.

[0059] The active ingredient may comprise small molecule with a therapeutic effect such as an antibiotic, a hormone including peptide hormones such as GLP-1 including Ozempic®, Wegovy® or other semaglutides, an inhibitor of a cellular receptor, an activator of a cellular receptor, a cellular receptor, or other type of active pharmaceutical ingredient.

[0060] The composition may comprise a mixture of two or more active ingredients, in other words a "multiple payload". For example, the composition may include mixtures of both RNA and DNA, mixtures of nucleic acid types such as siRNA and mRNA or mixtures of nucleic acids and proteins such as a mRNA and an antibody, or a mixture of a nucleic acid or protein with a small molecule such as an antibiotic. Another example of a multiple payload comprises two or more different nucleic acids, e.g. two or more different mRNAs, encoding respective different proteins. There is no particular limitation on the maximum number of active ingredients which may be present. For instance, a neotantigen cancer vaccine may include 20 or more different mRNAs, each encoding a respective different protein.

[0061] In accordance with still another possibility, the active ingredient may comprise a virus. The virus may be an inactivated or attenuated virus. For example, certain RNA viruses have diameters of less than 40 nm, e.g. 30 to 40 nm, and may be loaded into a lipid nanoparticle having a diameter of about 100 nm. Larger viruses may be delivered by adjusting the size of the nanoparticle.

[0062] Nanopartides

[0063] The nature of the nanoparticles is not particularly limited, and may be selected as appropriate depending upon the desired therapeutic application of the composition. The nanoparticles are configured to deliver the active ingredient into the cell.

[0064] The nanoparticles may be selected from: lipid-based nanoparticles, such as liposomes, emulsified lipid nanoparticles, lipid nanoparticles, a nanostructured lipid carrier, or solid lipid nanoparticles; polymeric nanoparticles, such as dendrimers, micelles, polymeric micelles, polymersomes, cubosomes, nanospheres, or charge altering release transporters; inorganic nanoparticles, such as silica nanoparticles, carbon nanotubes, or metallic nanoparticles; and protein-based nanoparticles, such as viral particles, virus-like particles, or a bacterium. It is contemplated that, in some implementations, the active ingredient may be the RNA of a virus, and the nanoparticle may be the protein coat of the virus.

[0065] Typically, the nanoparticles are lipid-based nanoparticles. For example, the nanoparticles may be lipid nanoparticles or liposomes. Most preferably, the nanoparticles are lipid nanoparticles.

[0066] The physical properties of nanoparticles may be described using various metrics.

[0067] Particle size is measured by Dynamic Light Scattering (DLS), and expressed in nanometres. The particle sizes reported herein are Z-average particle sizes, Dz. The Z-average is the harmonic intensity averaged hydrodynamic size of the particles: where Si is the scattered intensity from particle i, and Di is the diameter of particle i. Software supplied with many commercially-available DLS systems is capable of calculating the Z- average particle size.

[0068] As used herein, the term "nanoparticle" refers to a particle having a size of less than or equal to 1 pm. For example, the nanoparticles may have a particle size of less than or equal to 500 nm, and most typically a size in the range 70 to 130 nm. Sizes greater than 300 nm may reduce transfection efficiency of cells but may be tolerated in some implementations.

[0069] Polydispersity Index (PDI) is a measure of the degree of heterogeneity of particle sizes in a sample. The larger the PDI, the greater the variation in particle size. Variations in particle size may make cell transfection more difficult, and so the PDI is desirably as low as possible. PDI is measured by DLS. A PDI <0.3 is desirable, and a PDI <0.2 is considered excellent.

[0070] It is desirable for particle size and PDI to remain as constant as possible during storage of the composition. Put differently, aggregation of nanoparticles into larger particles is undesirable. Larger particles that may be fragile and less able to transfect into cells.

[0071] Zeta Potential is the net surface charge of the nanoparticle. Negative zeta potentials are desirable because a negative charge may allow better uptake of the nanoparticle by a cell. The zeta potential is typically in the range 0 to -20.

[0072] Encapsulation Efficiency (EE%) refers to the percentage of RNA located internally in the nanoparticles compared with the total amount of RNA in the sample (external and internal). EE% may be measured by determining the amount of RNA detectable using a fluorescent marker (such as Ribogreen®) before and after rupturing the LNP with a non-ionic detergent. EE% may be determined immediately after formulation of the LNP (referred to in the Examples as EE% lgnite+), or post-dialysis (referred to in the Examples as EE% Dialysis).

[0073] Payload concentration is the amount of the payload (e.g., RNA) per unit volume of nanoparticles, expressed in mg / mL. Good sources of information about determining the physical attributes of particles including LNPs, and discussion of lipid formulations and methods of making LNPs are: Evers et al., Small Methods, 2, 1700375; Schober et al., Scientific Reports, 14, 2403; Hou et al., Nature Reviews Materials, 6(12), 1078-1094; and Tenchov et al., ACS Nano, 15(11), 16982-17015.

[0074] In implementations where the nanoparticles include a cationic lipid and the active ingredient is a nucleic acid, a Nitrogen to Phosphate (N / P) ratio may be calculated. The N / P ratio of an LNP is the number ratio of nitrogen atoms in the cationic liquid and phosphorous atoms in the nucleic acid. The N / P ratio describes the charge interaction between the amino (N+) group of the ionizable cationic lipid and the anionic nucleic acid phosphate (PC -) groups. In other words, the N / P ratio provides a measure of the relative amounts of cationic liquid and nucleic acid.

[0075] External DES treatments

[0076] An example method of manufacturing a pharmaceutical composition will now be described with reference to Fig. 1. Fig. 1 is a flow diagram outlining the method.

[0077] The method of the present example may produce a composition comprising nanoparticles dispersed in a medium, the medium including the deep eutectic solvent. Such compositions are referred to herein as "external-DES" compositions. The External-DES composition may be used directly. In other implementations of the present method, the composition may be further processed to remove the nanoparticles from the DES environment yielding a "transient-DES" composition.

[0078] At block 101, a dispersion of the nanoparticles having the active ingredient encapsulated therein is prepared.

[0079] The method used to prepare the nanoparticles is not particularly limited, and may be selected as appropriate depending upon the nature of the nanoparticles and the active to be encapsulated. Methods for preparing nanoparticles encapsulating active ingredients are known in the art. Compositions comprising actives encapsulated in nanoparticles are commercially-available.

[0080] Lipid nanoparticles containing RNA ("RNA-LNPs") may be prepared by mixing an organic phase comprising dissolved lipid(s) with an aqueous solution of the RNA. The organic phase includes a water-miscible organic solvent, such as ethanol. The lipid(s) comprise a cationic lipid. Mixing the organic phase with the aqueous solution results in self-assembly of the lipids around the negatively-charged RNA. The mixing may comprise T-junction or microcapillary mixing.

[0081] A detailed experimental protocol for preparing RNA-LNPs is set out in Example 2, further below.

[0082] Subsequently, at block 102, the dispersion is mixed with an additive. The additive may reduce aggregation of the nanoparticles when the dispersion is subsequently mixed with a DES. Alternatively or additionally, the additive may improve the mixing of the dispersion with the DES.

[0083] The additive may be selected from: glycerol; a serum; a protein or peptide (optionally selected from an albumin such as bovine serum albumin; a globulin, such as an antibody, e.g. IgG; gelatin; a silk protein; or collagen); a polyalkylene glycol, such as polyethylene glycol; a buffer, such as ammonium acetate; and combinations thereof.

[0084] In particular, the additive may comprise glycerol. The glycerol may be mixed with the dispersion at a dispersion to glycerol ratio in the range 1:1 to 1:20 by volume. The addition of glycerol has been found to enhance the potency of the pharmaceutical composition by reducing particle aggregation.

[0085] Without wishing to be bound by theory, glycerol is believed to inhibit aggregation of the nanoparticles when the dispersion is mixed with a DES. Mixing a DES with a dispersion of nanoparticles removes water from between the particles, which may encourage aggregation. Glycerol, unlike water, is not subject to the osmotic force of the DES. It has been found that, while glycerol improves potency in the short term, the potency of compositions including glycerol declines over time. In implementations where glycerol is present in the composition, the composition is preferably used within 1 month of manufacture.

[0086] In accordance with another possibility, the additive may comprise a cyclic polysaccharide. The use of a cyclic polysaccharide may improve the storage stability of the composition. In implementations where the DES includes a sugar or sugar alcohol, the use of a cyclic polysaccharide may enhance potency of the composition. Compositions including a cyclic polysaccharide may have greater storage stability than compositions lacking an additive, or compositions including glycerol.

[0087] The cyclic polysaccharide may be added to the dispersion in a dry solid form.

[0088] The cyclic polysaccharide may comprise a cyclodextrin ("CD"). Cyclodextrins are starch derivatives, and comprise glucose subunits in a ring structure. For example, a cyclodextrin may comprise 6 (a-CD), 7 ( -CD) or 8 (y-CD) glucose subunits. The cyclodextrin ring defines a cavity, the dimensions and properties of which depend upon the structure of the CD. The properties of a-CD, -CD, and y-CD are described in Paduraru et al, Pharmaceutics, 14(8):1748 (doi: 10.3390 / pharmaceuticsl4081748). For further discussion of cyclodextrins as pharmaceutical excipients, see Ferreira et al, Eur. J. Biopharm, 178, 33-52.

[0089] CDs may form 'guest-host' interactions with actives. In other words, CDs may capture small molecules inside the CD ring, which is more hydrophobic than the outer hydrophilic surface of the ring. Therapeutic applications of CDs include topical treatments with fungicides, and parenteral intravenous (i.v.) delivery of chemotherapeutics. Doses can be as much as several grams per i.v. dose, so CDs are not toxic excipients. CDs are also commonly found in homeuse aerosol air-fresheners which capture VOCs, again demonstrating CDs lack of toxicity.

[0090] The cyclic polysaccharide may comprise a cyclodextrin derivative. Examples of cyclodextrin derivatives include methyl-a-cyclodextrin, dimethyl-a-cyclodextrin, trimethyl-a-cyclodextrin, methyl-p-cyclodextrin, dimethyl-|3-cyclodextrin, trimethyl-|3-cyclodextrin, methyl-y- cyclodextrin, dimethyl-y-cyclodextrin, trimethyl-y-cyclodextrin, ethyl-a-cyclodextrin, diethyl- a-cyclodextrin, triethyl-a-cyclodextrin, ethyl-|3-cyclodextrin, diethyl-|3-cyclodextrin, triethyl- P-cyclodextrin, ethyl-y-cyclodextrin, diethyl-y-cyclodextrin, triethyl-y-cyclodextrin, propyl-a- cyclodextrin, dipropyl-a-cyclodextrin, tripropyl-a-cyclodextrin, methyl-|3-cyclodextrin, dimethyl-p-cyclodextrin, trimethyl-|3-cyclodextrin, propyl-y-cyclodextrin, dipropyl-y- cyclodextrin, tri propyl-y-cyclodextrin acetyl-a-cyclodextrin, diacetyl-a-cyclodextrin, triacetyl- a-cyclodextrin, acetyl-|3-cyclodextrin, diacetyl-|3-cyclodextrin, triacetyl-|3-cyclodextrin, acetyl- y-cyclodextrin, diacetyl-y-cyclodextrin, triacetyl-y-cyclodextrin, hydroxypropyl-a- cyclodextrin, hydroxypropyl-|3-cyclodextrin, hydroxypropyl-y-cyclodextrin, mono-6-O-(p- toluensulphonyl-a-cyclodextrin, mono-6-O-(p-toluensulphonyl-|3-cyclodextrin, mono-6-O-(p- toluensulphonyl-y-cyclodextrin, mono-2-O-(p-toluensulphonyl-a-cyclodextrin, mono-2-O-(p- toluensulphonyl-p-cyclodextrin, mono-2-O-(p-toluensulphonyl-y-cyclodextrin, 3A-Amino-3A- deoxy-(2AS, 3AS)-a-cyclodextrin, 3A-Amino-3A-deoxy-(2AS, 3AS)-|3-cyclodextrin, 3A-Amino- 3A-deoxy-(2AS, 3AS)-y-cyclodextrin, Heptakis (2,6-di-O-methyl)-a-cyclodextrin, (2- Hydroxyethyl)-|3-cyclodextrin, Succinyl-|3-cyclodextrin, Carboxymethyl-|3-cyclodextrin, Sulfobutylether-P-Cyclodextrin, Hepta kis(2,6-di-O-methyl)-|3-cyclodextrin, (2-Hydroxyethyl)- P-cyclodextrin, Succinyl-|3-cyclodextrin, Carboxymethyl-|3-cyclodextrin, Sulfobutylether-|3- Cyclodextrin, Heptakis(2,6-di-O-methyl)-y-cyclodextrin, (2-Hydroxyethyl)-y-cyclodextrin, Succinyl-y-cyclodextrin, Carboxymethyl-y-cyclodextrin, Sulfobutylether-y-Cyclodextrin, 6-0- a-Maltosyl-a-cyclodextrin, 6-O-a-Maltosyl-|3-cyclodextrin, 6-O-a-Maltosyl-y-cyclodextrin and Sulfobutylether-p-cyclodextrin.

[0091] The nature of the CD or CD derivative may be selected as appropriate depending upon the nature of the active ingredient to be delivered. In implementations where the active ingredient comprises a nucleic acid such as an RNA, the cyclic polysaccharide may be selected from a-CD, -CD or y-CD. These cyclic polysaccharides were found to be effective for improving storage stability. When used with a DES comprising a choline derivative and a sugar or sugar alcohol, a-CD, -CD or y-CD also increased the potency of the composition. In particular, the CD may comprise y-CD. Of the CDs tested in the Examples, y-CD was found to have the broadest optimum concentration range.

[0092] The amount of cyclic polysaccharide may be selected to provide a final amount in the range 0.5 to 20 %, optionally 1 to 10 %, further optionally 1 to 5 % or about 2.5 % by weight based on the weight of the deep eutectic solvent and cyclic polysaccharide.

[0093] The method further includes preparing a deep eutectic solvent at block 103. The deep eutectic solvent may be prepared before, after, or in parallel to performing the operations of blocks 101 and 102.

[0094] The identity of the DES is not particularly limited. The DES may be selected from any of the DESs described herein. DES formulations and methods for preparing DESs are described in detail in WO 2014 / 131906 Al.

[0095] The DES used in the External-DES and Transient-DES formulations may comprise a hydrogen bond acceptor which is a choline derivative, paired with a suitable hydrogen bond donor.

[0096] The expression "choline derivative" encompasses choline salts and salts of choline analogues, but excludes free choline. Free choline, in other words choline base or choline hydroxide, is strongly alkaline. DESs based on choline hydroxide are therefore much less effective at stabilising biomolecules such as RNA.

[0097] The term "choline derivative" refers in particular to compounds of formula: wherein:

[0098] R1is H, OH, and -OC(O)CH3;

[0099] R2is selected from H, CH3, Cl, Br;

[0100] R3, R4, and R5are each independently selected from Cl to C3 alkyl groups; and X- is a counterion, such as a halide.

[0101] Typically, R3, R4, and R5are each methyl groups.

[0102] Typically, the counterion is not OH". The counterion is typically not a carboxylate.

[0103] The choline derivative may be a choline salt.

[0104] Examples of choline salts include choline nitrate, choline tetrafluoroborate, choline bitartrate, choline dihydrogen citrate, choline p-toluenesulfonate, choline bicarbonate, choline chloride, choline bromide, choline iodide and choline fluoride. The preferred choline salt is choline chloride.

[0105] The hydrogen bond donor may be a sugar or sugar alcohol.

[0106] A sugar alcohol is a compound of formula:

[0107] HOCH2(CHOH)nCH2OH where the value of n is not particularly limited, n may be zero. Typically, n is in the range 2 to 24, optionally 2 to 5. Preferably, the sugar alcohol has 5 or 6 carbon atoms (i.e ., n is 3 or 4).

[0108] Examples of useful sugar alcohols include arabitol (optionally D(+)arabitol or L(-)arabitol), sorbitol, xylitol, and combinations thereof. Ribitol is a further example of a useful sugar alcohol.

[0109] In implementations where the DES comprises a choline derivative and a hydrogen bond donor such as a sugar alcohol, the molar ratio of the hydrogen bond donor to the choline derivative may be in the range 1:3 to 3:1, optionally 1:2 to 2:1, further optionally 1:0.8 to 1:1.2; or is about 1:1. One useful class of DES comprises a choline salt and a sugar alcohol having 5 or 6 carbon atoms. DESs in this class include: choline salt : arabitol, optionally at a molar ratio of 1:1; choline salt : L(-)a ra bitol, optionally at a molar ratio of 1:1; choline salt : D(+)a ra bitol, optionally at a molar ratio of 1:1; choline salt : xylitol, optionally at a molar ratio of 1:1; choline salt : sorbitol, optionally at a molar ratio of 1:1; and choline chloride : ribitol, optionally at a molar ratio of 1:1 or 1:2.

[0110] DESs based on choline salts and sugar alcohols were found to be highly effective for improving potency and stability of RNA-LNPs. In addition, it was found that potency of the composition may be enhanced when such DESs are used in combination with a cyclic polysaccharide.

[0111] In implementations where the hydrogen bond donor is a sugar, the sugar may have a general formula of CeH Oe. For example, the DES may be choline chloride : mannose, optionally at a molar ratio of 1:2.

[0112] As an alternative to a sugar or sugar alcohol, a choline derivative may be paired with urea or a urea derivative having a structure of: wherein:

[0113] A is selected from O, S, and NH;

[0114] R3is selected from: H; -NH2; a Cl to C6 alkyl, alkenyl, or haloalkyl group; and -NH-(CH2)nCH3, where n is 0 or an integer from 1 to 5; and

[0115] R4is H and or a Cl to C3 alkyl group.

[0116] For example, the hydrogen bond donor may be urea. Surprisingly, even though free urea is an irritant, it has been found that an External-DES composition comprising urea and choline chloride produced no observable adverse reactions when injected intramuscularly into a mouse. When the hydrogen bond acceptor which is a choline derivative and the hydrogen bond donor is urea or a urea derivative, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor may be in the range 1:1.5 to 1:2.5, or may be about 1:2.

[0117] One example of a useful DES is choline chloride : urea at a molar ratio of 1:2.

[0118] The method then proceeds to optional block 103A, in which the deep eutectic solvent is combined with cyclic polysaccharide.

[0119] Combining the deep eutectic solvent with the cyclic polysaccharide may comprise mixing the deep eutectic solvent with a solution comprising the cyclic polysaccharide. Alternatively, the cyclic polysaccharide may be mixed with the DES in solid form.

[0120] The nature and amount of cyclic polysaccharide may be as described with reference to block 102, above. Combining the cyclic polysaccharide with the DES, rather than with the dispersion, may be more convenient.

[0121] Addition of a cyclic polysaccharide to both the DES and the dispersion of the nanoparticles is also contemplated.

[0122] After preparing the DES, the temperature of the DES may be adjusted at block 104. Adjusting the temperature of the DES may modify the mixing behaviour of the DES. The temperature may be adjusted either upwardly or downwardly.

[0123] Adjusting the temperature of the DES is one technique for adjusting viscosity of the DES. Heating reduces viscosity, and cooling increases viscosity.

[0124] Heating the DES, for example to a temperature in the range 25 to 75 °C, may reduce the viscosity of the DES thereby making the DES easier to handle. It has also been found that cooling the DES, e.g. to a temperature in the range 0 °C to 10 °C, may improve mixing efficiency. A viscous environment may protect nanoparticles from physical damage during mixing.

[0125] After preparing the dispersion of nanoparticles and the DES, the dispersion and the DES and mixed to obtain a mixture comprising the nanoparticles dispersed in a medium.

[0126] Typically, the dispersion is mixed with an excess of the deep eutectic solvent. By an "excess of deep eutectic solvent" is meant a proportion of deep eutectic solvent which is high enough that the medium is a deep eutectic solvent environment as opposed to an aqueous solution comprising the components of the DES.

[0127] For example, the deep eutectic solvent may be mixed with the dispersion at a deep eutectic solvent to dispersion ratio of >4:1, optionally >8:1, >10:1, >15:1, or >20:1.

[0128] The mixing technique used to combine the dispersion with the DES is not particularly limited. For example, the mixing may comprise mechanical mixing and / or ultrasound mixing.

[0129] The mixing conditions are preferably selected to produce a homogenous mixture without causing excessive physical damage to the nanoparticles, e.g. rupturing the nanoparticles. Inefficient mixing may result in aggregation of the nanoparticles thereby reducing potency, whereas overly aggressive mixing may break up the nanoparticles.

[0130] Adding the dispersion of nanoparticles to bulk DES, as opposed to adding the DES to the dispersion, may improve mixing efficiency.

[0131] The method may terminate after mixing the DES and the dispersion, yielding an "external- DES" composition at block 106A.

[0132] Alternatively, the method may proceed to blocks 106B to 108 to produce a "transient-DES" composition. At block 106B, the external-DES composition obtained from block 105 is incubated for a time interval.

[0133] The time interval is typically in the range 10 seconds to 24 hours, and may be for example 30 seconds to 19 hours. Optionally, the time interval may be in the range 20 seconds to 90 minutes, 30 seconds to 1 hour, or 1 to 30 minutes. Further optionally, the time interval is in the range 1 to 15 minutes, optionally wherein the time interval is about 1 minute.

[0134] Exposing the nanoparticles to the DES for a fixed time interval has been found to increase the potency of the composition. It has been empirically established that incubation times of about 1 to 30 minutes provide an optimum increase in potency.

[0135] The incubation may be performed at a temperature in the range 18 to 40 °C, optionally about 20 °C or about 37 °C.

[0136] After incubating the nanoparticles in the DES, the nanoparticles are removed from the DES environment by eliminating the deep eutectic solvent from the medium or separating the nanoparticles from the medium, at block 107.

[0137] By "eliminating the deep eutectic solvent" is meant modifying the medium such that the medium no longer behaves as a deep eutectic solvent. The DES components may remain in the medium, but no longer interact in the manner characteristic of a DES. Eliminating the deep eutectic solvent typically comprises diluting the medium with an excess of a diluent to convert the deep eutectic solvent of the medium into a solution.

[0138] By "an excess of diluent" is meant enough diluent to remove the DES character of the medium. By way of illustration, adding more than about 55 % water by volume to a typical DES converts the DES into a solution.

[0139] The diluent may be selected from: i) water; ii) a cell culture medium, such as Dulbecco's modified Eagle's medium ("DMEM"); iii) an aqueous buffer, such as phosphate buffered saline, ammonium acetate, a citrate buffer, or TRIS; iv) a serum; v) a solution of a protein or a peptide, optionally selected from an albumin such as bovine serum albumin; a globulin, such as an antibody, e.g. IgG; gelatin; a silk protein; or collagen; vi) a pharmaceutically-acceptable excipient, such as glycerol or polyethylene glycol; vii) an aqueous solution of a cyclic polysaccharide, optionally a cyclodextrin, further optionally a-cyclodextrin, p-cyclodextrin, or y-cyclodextrin; and viii) combinations thereof, such as a combination of the cell culture medium and the serum.

[0140] Nanoparticles stored in a combination of a cell culture medium (such as DMEM) and serum (e.g., 10 % serum) have been found to have improved stability in comparison with those stored in water or buffer solutions.

[0141] Providing a cyclic polysaccharide in the storage medium may improve storage stability and / or potency, as discussed with reference to block 102.

[0142] Alternatively, block 107 may comprise separating the nanoparticles from the medium such that the nanoparticles are no longer in a DES environment. The separation may be performed by ultrafiltration, ultracentrifugation, tangential flow filtration, or dialysis.

[0143] The nanoparticles may be transferred to a storage medium. The storage medium may comprise any of the diluents identified above. For example, the storage medium may be a combination of a cell culture medium (such as DMEM) and serum (e.g., 10 % serum). In accordance with another possibility, the storage medium may include a cyclic polysaccharide such as a-cyclodextrin, p-cyclodextrin, or y-cyclodextrin.

[0144] The transient treatment or "blanching" of the nanoparticles in the DES has been found to improve potency, and may in some implementations also improve stability. Potency may be higher than that of untreated nanoparticles. Even more surprisingly, the potency of transiently treated nanoparticles may be higher than that of nanoparticles which remain permanently in an external DES. The mechanism by which the transient treatment improves potency has not been definitively established.

[0145] Various modifications may be made to the described method.

[0146] As an alternative or in addition to mixing an additive with the dispersion, an additive may be mixed with the deep eutectic solvent between blocks 103 and 105. The additive to the deep eutectic solvent may be selected from any of the additives described with reference to block 102, for example glycerol or a cyclic polysaccharide.

[0147] The use of an additive is optional, and block 102 may be omitted.

[0148] Block 104 is optional and may be omitted.

[0149] In implementations where an organic solvent such as ethanol is used in the preparation of the nanoparticles, the method may further comprise separating the nanoparticles from the organic solvent before the nanoparticles are combined with the DES. The separation may be performed using any suitable technique, such as dialysis, ultrafiltration, etc.

[0150] The method may further comprise an optional drying step, in which water is removed by evaporation. The drying step may comprise exposing the composition to a low-humidity environment, e.g. an environment with a relative humidity of less than or equal to 30 %. For example, the composition may be placed in a dry inert gas environment; or humidity may be reduced using a drying agent such as silica gel. Residual water has a small negative effect on the integrity of mRNA-LNP during storage. Removing residual water may therefore improve the stability of the composition.

[0151] The method may further comprise loading the composition into a medical device suitable for delivering the composition to a patient in need thereof. For example, the composition may be loaded into or onto a transdermal patch or a microneedle array. The loading may comprise lyophilisation. The external-DES compositions obtained at block 106A may be especially suitable for delivery in this manner. Such compositions may have a high viscosity in comparison with aqueous compositions, and may stick to the device thereby improving storage, transport, and handling.

[0152] Manufacture of compositions with internal DES

[0153] As an alternative, or in addition, to providing an external DES, the DES may be encapsulated within the nanoparticles. An example method of manufacturing a pharmaceutical composition having an internal DES will now be described with reference to Fig. 2.

[0154] At block 201, a first phase comprising the active ingredient is prepared.

[0155] The first phase may be an aqueous solution comprising the active ingredient, and optionally one or more additives.

[0156] Examples of suitable additives for the first phase include salts, such as NaCI; cryoprotectants, e.g. a sugar, such as sucrose; buffers, such as Tris, PIPES, MES, HEPES, MOPSO, CAPS, CAPSO, BICI NE, BIPES, BAPTA, citrate, phosphate, imidazole, glycine, and acetate; and combinations thereof.

[0157] The additive may comprise glycerol. The additive may comprise a cyclic polysaccharide, such as a-cyclodextrin, p-cyclodextrin, or y-cyclodextrin. A cyclic polysaccharide may improve storage stability and / or potency.

[0158] In particular, the additive may comprise a buffer. The buffer may be present in the aqueous solution at a concentration of 1 to 100 mM, optionally 1 to 10 mM. The buffer may maintain the aqueous solution at a pH in the range pH in the range 1 to 9, optionally 4 to 6.5. It has been found that, in implementations where the active ingredient comprises RNA, adjusting the pH to a pH of 4 to 6.5 may improve the stability of the active ingredient.

[0159] For example, the aqueous solution may comprise a citrate buffer and may have a pH in the range 4 to 6.5. In implementations where the pharmaceutical composition is to be frozen (e.g., for storage before use), the inclusion of a cryoprotectant such as sucrose may be desirable.

[0160] Subsequently, at block 202, the first phase is mixed with a deep eutectic solvent.

[0161] The nature of the DES is not particularly limited. The DES may be selected from any of the DESs described herein, or those described in WO 2014 / 131906 Al. Many DESs are effective for stabilising biomolecules. Since in this example the DES will be formulated internally to the nanoparticles, the dose of DES to be delivered is very low and the risk of DES toxicity is likewise low.

[0162] The DES may comprise a choline derivative and urea or a urea derivative, as previously described with reference to Fig. 1.

[0163] The DES may comprise a choline derivative and a sugar or sugar alcohol, as previously described with reference to Fig. 1.

[0164] Examples of DESs which may be included in internal-DES formulations include: choline chloride : urea choline chloride : xylitol choline chloride : sorbitol choline chloride : arabitol betaine (i.e., N,N,N-trimethylglycine) : xylitol proline : sorbitol.

[0165] The DES optionally further comprises an additive. In particular betaine : xylitol and proline : sorbitol may further comprise an additive to reduce their viscosity, such as water. The amount of water is desirably minimized in implementations where the active ingredient is susceptible to hydrolysis. The additive may comprise a cyclic polysaccharide, as discussed with reference to blocks 102 and 103A of Fig. 1. The cyclic polysaccharide may improve the potency and / or storage stability of the composition.

[0166] The proportion of DES which is encapsulated in the nanoparticles is desirably at least 50 % by volume, so as to stabilise more effectively the active ingredient. To this end, the DES is mixed with the first phase at a deep eutectic solvent to first phase ratio of at least 1:1 by volume. Higher ratios of DES to first phase may be used.

[0167] The method further comprises, at block 203, preparing a second phase. The second phase comprises an encapsulant material.

[0168] The encapsulant material comprises the nanoparticles, or one or more components which form nanoparticles when combined with the active ingredient. The nature of the encapsulant material may therefore be selected as appropriate based on the type of nanoparticles desired.

[0169] For example, in implementations where the nanoparticles will be LNPs, the second phase may be an organic phase, comprising an appropriate lipid dissolved in a water-miscible organic solvent. The water-miscible organic solvent most typically comprises ethanol.

[0170] Lipids, and combinations of lipids, suitable for forming LNPs are known in the art. For example, a commercially-available lipid formulation may be used.

[0171] As will be appreciated, "first" and "second" are used for ease of discussion to distinguish between the two phases, and do not limit the order in which the phases are prepared. The second phase may be prepared before, after, or in parallel to the first phase.

[0172] After preparing the first and second phases, the two phases are combined at block 204 to form nanoparticles having the active ingredient and DES encapsulated therein.

[0173] The technique used to combine the nanoparticles and the active ingredient may be selected as appropriate based on the natures of the nanoparticles and the active ingredient. For example, LNPs may be obtained by mixing the first and second phases using T-junction mixing, microcapillary mixing, or the like. Example 2 sets out an illustrative experimental protocol for preparing LNPs.

[0174] The first and second phases may be combined at a temperature in the range 18 to 75 °C, optionally 25 to 55 °C, and further optionally 25 to 45 °C. Heating the phases during the mixing step may increase the proportion of DES in the resulting nanoparticles, thereby stabilising the active ingredient more effectively during storage.

[0175] The ratio of first phase to second phase may be selected as appropriate. For example, in implementations where the nanoparticles are LNPs, the active ingredient comprises a nucleic acid, and the nanoparticles include a cationic lipid, the mixing ratio of the two phases may be selected to obtain nanoparticles having an N / P ratio of at least 7, e.g. 10 to 20, 10 to 14, or 12 to 18.

[0176] The N / P ratio is a measure of the amount of cationic lipid relative to the amount of nucleic acid. Decreasing the relative amount of nucleic acid increases the N / P ratio. For example, Pfizer-BioNTech mRNA vaccine BNT162b2 / Comirnaty has an N / P ratio of 6. Increasing the N / P ratio has been found to improve encapsulation efficiency.

[0177] Subsequently, at block 205, excess deep eutectic solvent is removed from the composition. In this context, "excess" deep eutectic solvent means deep eutectic solvent which is not encapsulated in the nanoparticles.

[0178] The excess deep eutectic solvent may be removed using any appropriate technique, for example dialysis, tangential flow filtration, ultrafiltration, or ultracentrifugation.

[0179] Most typically, the excess deep eutectic solvent is removed by dialysis against a dialysate. In the Examples set out herein, the terms "dialysate" and "dialysis buffer" are used interchangeably. As will be appreciated, a dialysis buffer may be, but is not necessarily, a pH buffer. 1

[0180] The ratio of the composition to the dialysate is typically at least 1:200 by volume, and is optionally at least 1:1000 by volume.

[0181] The dialysate is not particularly limited, and may be selected as appropriate. It has been found that properties of the nanoparticles may be adjusted by modifying the conditions used for dialysis, such as the nature of the dialysate; the concentration of solutes present in the dialysate; the pH of the dialysate; the temperature at which dialysis is performed; and the number of dialysis steps.

[0182] Typically, the dialysate comprises water. The dialysate may further comprise one or more of: a salt, such as NaCI; a cryoprotectant, such as sucrose; and a buffer. The buffer may be selected from Tris, PIPES, MES, HEPES, MOPS, MOPSO, CAPS, CAPSO, BICINE, BIPES, BAPTA, citrate, phosphate, imidazole, glycine, acetate, and combinations thereof. In accordance with another possibility, the dialysate may comprise a cyclic polysaccharide, optionally a cyclodextrin such as a-, |3-, ory-cyclodextrin. The cyclic polysaccharide may improve potency and / or storage stability.

[0183] In implementations where the dialysate comprises a buffer, the buffer may be at a concentration in the range 1 to 100 mM, optionally 1 to 10 mM.

[0184] The dialysate may have a pH in the range 1 to 8, optionally 3 to 7.

[0185] The dialysis may be performed at a temperature in the range 4 to 37 °C, optionally 4 to 18 °C.

[0186] The dialysis may comprise performing two or more rounds of dialysis. For example, the dialysis may comprise or consist of: performing a first round of dialysis against a first dialysate, the first dialysate having a pH in the range 3.5 to 4.5 and optionally a pH of about 4; and subsequently performing a second round of dialysis against a second dialysate, the second dialysate having a pH in the range 7 to 8, and optionally a pH of about 7.4. In the two-round dialysis process, the first dialysate may comprise a buffer at a concentration of 1 to 15 mM, optionally about 10 mM. The second dialysate may comprise a buffer at a concentration of 1 to 10 mM, optionally about 10 mM. The first dialysate may be a citrate buffer; and the second dialysate may be a Tris buffer.

[0187] The two-round dialysis process has been found to maintain a high encapsulation efficiency.

[0188] Various modifications may be made to the example method.

[0189] In the example, the deep eutectic solvent is mixed with the first phase. In principle, the deep eutectic solvent may alternatively or additionally be mixed with the second phase between blocks 203 and 204.

[0190] After preparing an internal-DES composition in accordance with a method as described with reference to Fig. 2, an external DES may be added to the composition as described with reference to blocks 101 to 106A of Fig. 1 to obtain an "internal-external DES composition". In such implementations, two different DESs may be incorporated into the composition, or the internal DES and the external DES may be the same.

[0191] Depending upon the nature of the DES chosen, an internal-external DES composition may perform better or worse than an internal DES used alone.

[0192] For internal-external DES compositions, the DES preferably comprises a choline derivative paired with urea or a urea derivative. For example, choline chloride : urea has been found to have improved performance when used as both an external and internal DES, compared to choline chloride : urea used as an internal DES only.

[0193] In accordance with another possibility, the operations of block 205 may be omitted from the method of Fig. 2, particularly in implementations where the DES comprises a choline derivative paired with urea or a urea derivative. In implementations where the first and / or second phase includes an organic solvent such as ethanol, the method typically includes separating the nanoparticles from the organic solvent or diluting the organic solvent to stabilize the nanoparticles. The presence of high concentrations of organic solvent in the composition may reduce the stability of the nanoparticles.

[0194] Pharmaceutical Compositions and Medical Uses

[0195] Another aspect provides a pharmaceutical composition obtainable by a method as defined herein.

[0196] The pharmaceutical composition may comprise: an active ingredient encapsulated in nanoparticles; a carrier, wherein the nanoparticles are dispersed in the carrier; and a deep eutectic solvent.

[0197] The deep eutectic solvent may be present in the nanoparticles (termed an "internal-DES composition"); or present in the carrier (termed an "external-DES composition"), or present in both the nanoparticles and the carrier (termed an "internal-external DES composition").

[0198] Regarding the compositions which are transiently treated with the DES, the structural changes caused by the DES have not been definitively identified.

[0199] The physical form of the pharmaceutical composition is not particularly limited and may be selected as appropriate depending upon the desired therapeutic use. For example, the composition may be formulated as drops, a spray, a tablet, a capsule, a pill, an injection, a depot, an implant, a patch, a cream, an aerosol, a gel, or a suppository.

[0200] Typically, the composition includes a liquid carrier, and the nanoparticles are dispersed in the liquid carrier.

[0201] The liquid carrier may include: water; a cell culture medium, such as Dulbecco's modified Eagle's medium; an aqueous buffer, such as phosphate buffered saline, ammonium acetate, a citrate buffer, and / or TRIS; a serum; a solution of a protein or a peptide, optionally selected from an albumin such as bovine serum albumin; a globulin, such as an antibody, e.g. IgG; gelatin; a silk protein; or collagen; a pharmaceutically-acceptable excipient, such as glycerol, polyethylene glycol or a cyclic polysaccharide (e.g., a cyclodextrin, optionally a-, |3- or y- cyclodextrin); a deep eutectic solvent; and combinations thereof, such as a combination of the cell culture medium and the serum.

[0202] For example, the liquid carrier may comprise a combination of a cell culture medium and serum. The use of such carriers has been found to improve further the stability of the active ingredient.

[0203] In accordance with another possibility, the composition may be loaded into a medical device for delivery. In particular, the composition may be loaded into a microneedle array or transdermal patch. External-DES compositions may be especially suitable for delivery in this manner. An external-DES composition may have a high viscosity compared to e.g. an aqueous composition, and this may improve handling, transport, and / or delivery.

[0204] Medical Uses

[0205] The pharmaceutical composition may be for use in medicine. As will be appreciated, medicine encompasses veterinary medicine as well as human medicine.

[0206] Equivalently, there is provided a method of treating or preventing a disease or disorder in a subject, which method comprises administering to the subject an effective amount of a composition as defined herein.

[0207] The medical indication is not particularly limited. The methods and compositions provided herein are compatible with a very wide range of active ingredients. Any active ingredient which is suitable for delivery, e.g. delivery into a cell, using nanoparticle encapsulation may be used. The compositions provided herein are particularly suitable for the delivery of nucleic acids such as RNA. The composition may be for use in gene therapy or as a genetic vaccine (e.g., an mRNA vaccine).

[0208] For example, there is provided a method of preventing an infectious disease, which method comprises administering to a subject a composition as defined herein, wherein the active ingredient is a nucleic acid, optionally an mRNA, encoding an antigen.

[0209] There is no particular limitation for the route of administration of the composition. Examples include; intraocular, intravitreal, intracerebral, oral, sublingual, buccal, nasal, intracardiac, pulmonary, epidural, intrathecal, intraosseous, intraarticular, cutaneous (including microneedles), subdermal, subcutaneous, intradermal, intramuscular, intraarterial, perivascular, intracavernous, intrauterine, rectal, intra-peritoneal, and intravenous.

[0210] In particular, the composition may be delivered using a microneedle or an array of microneedles, or using transdermal patch. A microneedle is a sharp projection configured to penetrate the stratum corneum of human skin when in use. A microneedle may have a length in the range 25 to 2000 pm and a diameter in the range 1 to 100 pm. A microneedle may be hollow, and may have a cavity which is loaded with the composition. Alternatively, a microneedle may be solid and the composition may be coated on the surface of a microneedle. A microneedle array comprises a plurality of microneedles arranged on a support, e.g. a patch.

[0211] In accordance with another possibility, the composition may be formulated for administration by injection, optionally intramuscular injection.

[0212] The composition may deliver the active ingredient to the spleen and / or a ganglion selectively over the liver.

[0213] It has been found that treatment with a DES may modify the pharmacokinetics of the composition in comparison with an untreated composition. When administered by injection, the composition may be better localised to the injection site. The composition may act selectively on the spleen and ganglions. Delivery to the spleen is desirable. Delivery to the liver is undesirable, because the liver is known to inactive active ingredients.

[0214] The deep eutectic solvent increases potency of the active ingredient. By "increasing the potency" is meant increasing potency in comparison with a comparative composition differing only in that the comparative composition is not treated with a DES.

[0215] The pharmaceutical composition may administered at a temperature of 30 to 40 °C, optionally about 37 °C. It has been found that warming the pharmaceutical composition before administration may increase the potency of the composition, particularly in implementations where the composition includes a combination of a cell culture medium and serum.

[0216] Uses

[0217] Provided is the use of a deep eutectic solvent to inhibit the degradation and increase the potency of nucleic acid therapeutics, as well as maintaining or enhancing the potency of delivery vehicles including Lipid NanoParticles (LNPs). It has also surprisingly been discovered that DES treatment of the nanoparticle may modify the biodistribution of the therapeutic compound, as one example by reducing uptake by the liver and / or increase the uptake in the spleen and ganglions.

[0218] One aspect provides the use of a deep eutectic solvent to stabilise an active ingredient of a pharmaceutical composition, wherein the active ingredient is encapsulated in nanoparticles.

[0219] The use may be in the context of any of the methods and compositions provided herein. As will be appreciated, any of the features described with reference to the methods and compositions may equally be implemented in the context of the use.

[0220] For example, the nanoparticles are lipid-based nanoparticles, optionally lipid nanoparticles or liposomes. The active ingredient may comprise a biomolecule, optionally a nucleic acid, further optionally an RNA. The use may comprise transiently contacting the deep eutectic solvent with the pharmaceutical composition. Surprisingly, it has been found that transient treatment may increase the stability and / or potency of the composition.

[0221] Example Deep Eutectic Solvents

[0222] The abbreviation "DES" refers to a deep eutectic solvent.

[0223] Deep eutectic solvents are massively hydrogen bonded non-aqueous non-volatile liquids which replace water molecules surrounding biomolecules including protein, DNA and RNA as well as large particles, viruses and cells. Following the replacement of water with DES, the structure and integrity of biomolecules and particles is extended by orders of magnitude, notably at elevated temperatures such as 37-100 °C. DESs are non-toxic, long shelf-life thermostabilizing liquids with unusually broad properties including; (i) protecting any type / size RNA as naked RNA or in particles, viruses and cells, (ii) conserving protein structure including enzyme activity and antigenicity.

[0224] DESs are mixtures of two or more components that when combined together have a eutectic point, which is the temperature of solidification or freezing (Fp). The eutectic point of the combined components is generally much lower than either of the components individually and occurs at a single temperature without separation of the individual components on solidification.

[0225] There is no particular maximum number of components. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more components may be mixed to produce a DES.

[0226] The properties of DESs have been described in Abbott et al., Chem. Commun. 7 (2003) 70-71; Abbott et al., J. Am. Chem. Soc. 126 (2004) 9142-9147; Imperato et al., Chem. Commun. 9 (2005) 1170-1172; Gore et al., Green Chem. 13 (2011) 1009-1013; Gorke et al., Chem. Commun. 10 (2008) 1235-1237; Abbott et al., Aust. J. Chem. 62 (2009) 341-347; Choi et al. Plant Physiol. 156 (2011) 1701-1705; and reviewed by Zhang et al., Chem Soc Rev. 2012 Nov 7;41(21):7108-46. Industrially, DESs have been used for electrochemical plating, mining applications and drill lubricants (US 2009 / 0247432 Al), industrial enzyme applications (US 2009 / 0117628 Al), preparation of inorganic compounds (Freudenmann et al., Angew. Chem., Int. Ed., (2011), 50, 11050-11060) or organic compounds (Gore et al., Green Chem., (2011), 13, 1009-1013), biological extractions (WO 2011 / 155829 Al), in electrochemistry as electrolytes for dye- sensitized solar cells and metal electropolishing (Jhong et al. Electrochem. Commun., (2009), 11, 209-211), for electrodeposition (Gomez et al., ]. Electroanalytical Chem., (2011), 658, 18- 24), for purification of biodiesel (Shahbaz et al., Energy Fuels, (2011), 25, 2671-2678), for solubilisation of drugs (Morrison et al., Int. J. Pharm., (2009), 378, 136-139), solubilisation of metal oxides (Abbott et al, J. Am. Chem. Soc., (2004), 126, 9142-9147) and solubilisation of CO2(Li et al., J. Chem. Eng. Data, (2008), 53, 548-550).

[0227] DESs are not considered to be ionic liquids because: (i) they are not entirely composed of ionic species and (ii) they can also be obtained from non-ionic species, and (iii) they are mixtures and not compounds. As compared to the traditional ionic liquids, DESs have several advantages such as (1) low cost, (2) chemically inert to water, (3) easy to prepare by simply mixing two or more components, (4) most are biodegradable and non-toxic, (5) low volatility even when heated and (6) non-flammable. All DESs are liquids below 150 °C and many are liquid between room-temperature and 70 °C, with a few notable examples that are liquid below 0 °C.

[0228] In contrast to DESs, ionic liquids consist of ions (Lei et al., Introduction: Ionic Liquids. Chem. Rev. 117, 10, 6633-663 (2017)), and have been described as "molten salts" (Ksenia et al., Biological Activity of Ionic Liquids and Their Application in Pharmaceutics and Medicine. Chemical Reviews 117 (10), 7132-7189 (2017)). An example ionic liquid consists of choline and lactic acid (Ukidve, Cu, Goetz, Angsantikul, Curreri, Tanner, Lahann, Mitragotri, "lonic-Liquid-Based Safe Adjuvants", Adv Mater. 2020; 32(46): e2002990).

[0229] Examples of DES components include: chlorocholine chloride, bromocholine bromide, iodocholine iodide, acetylcholine hydroxide, acetylcholine bitartrate, acetylcholine dihydrogen citrate, acetylcholine p-toluenesulfonate, acetylcholine bicarbonate, acetylcholine chloride, acetylcholine bromide, acetylcholine iodide, acetylcholine fluoride, chloroacetylcholine chloride, bromoacetylcholine bromide, iodoacetylcholine iodide, butyrylcholine hydroxide, butyrylcholine bitartrate, butyrylcholine dihydrogen citrate, butyrylcholine p-toluenesulfonate, butyrylcholine bicarbonate, butyrylcholine chloride, butyrylcholine bromide, butyrylcholine iodide, butyrylcholine fluoride, chlorobutyrylcholine chloride, bromobutyrylcholine bromide, iodobutyrylcholine iodide, acetylthiocholine chloride, l-carnitine, d-carnitine, betaine, sarcosine, trimethylamine N-oxide, betaine hcl, cetyl betaine, cetyltrimethylammonium fluoride, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, lauryl betaine, N,N-dimethylenethanolammonium chloride, N,N-diethyl ethanol ammonium chloride, beta-methylcholine chloride, phosphocholine chloride, choline citrate, benzoylcholine chloride, lauryl sulphobetaine, benzyltrimethylammonium chloride, methyltriphenylphosphonium chloride, methyltriphenylphosphonium bromide, methyltriphenylphosphonium iodide, methyltriphenylphosphonium fluoride, N,N-diethylenethanol ammonium chloride, ethylammonium chloride, tetramethylammonium chloride, tetramethylammonium bromide, tetramethylammonium iodide, tetramethylammonium fluoride, tetraethylammonium chloride, tetraethylammonium bromide, tetraethylammonium iodide, tetraethylammonium fluoride, tetrabutylammonium chloride, tetrabutylammonium bromide, tetrabutylammonium iodide, tetrabutylammonium fluoride, (2-chloroethyl) trimethylammonium chloride, terbium (III) chloride, zinc (II) chloride, zinc (II) bromide, zirconium (III) chloride, iron (III) chloride, tin (II) chloride, copper (II) chloride, magnesium (II) chloride; with one or more other component(s) that can also form a DES, including for example, but without limitation, one or more of the following: urea, formamide, thiourea, 1- methylurea, 1,1-dimethylurea, 1,3-dimethylurea, carbohydrazide, tetramethylurea, 1,3- bis(hydroxymethyl)urea, benzamide, Girards Reagent T, lactamide, acetamide, fluoroacetamide, difluoroacetamide, trifluoroacetamide, chlorofluoroacetamide, chlorodifluoroacetamide, chloroacetamide, dichloroacetamide, dichlorofluoroacetamide, trichloroacetamide, bromoacetamide, dibromoacetamide, tribromoacetamide, bromofluoroacetamide, bromodifluoroacetamide, bromochlorofluoroacetamide, iodoacetamide, diiodoacetamide, triiodoacetamide, 2-methyl-2,2-difluoroacetamide, 2- methyl-2-fluoroacetamide, 2,2-dimethyl-2-fluoroacetamide, 2-ethyl-2,2-difluoroacetamide, 2-ethyl-2-fluoroacetamide, 2,2-diethyl-2-fluoroacetamide, 2-propyl-2,2-difluoroacetamide, 2-propyl-2-fluoroacetamide, 2,2-propyl-2-fluoroacetamide, 2-fluoropropionamide, 3- fluoropropionamide, 2,2-difluoropropionamide, 2,3-difluoropropionamide, 3,3- difluoropropionamide, 3,3,3-trifluoropropionamide, 2-fluoro-3,3,3-trifluoropropionamide, 2- chloro-3,3,3-trifluoropropionamide, 2,2-chloro-3,3,3-trifluoropropionamide, 2-bromo-3,3,3- trifluoropropionamide, 2,2-bromo-3,3,3-trifluoropropionamide, pentafluoropropionamide, heptafluorobutyramide, trimethylacetamide, l-(trifluoroacetyl)imidazole, N,O- Bis(trifluoroacetyl)hydroxylamine, Bistrifluoroacetamide, N-Methyl-fluoroacetamide, N-

[0230] Methyl-difluoroacetamide, N-Methyl-trifluoroacetamide, N-Methyl-chlorofluoroacetamide,

[0231] N-Methyl-chlorodifluoroacetamide, N-Methyl-chloroacetamide, N-Methyl- dichloroacetamide, D N-Methyl-dichlorofluoroacetamide, N-Methyl-trichloroacetamide, N-

[0232] Methyl-bromoacetamide, N-Methyl-dibromoacetamide, N-Methyl-tribromoacetamide, N-

[0233] Methyl-bromofluoroacetamide, N-Methyl-bromodifluoroacetamide, N-Methyl- bromochlorofluoroacetamide, N-Methyl-iodoacetamide, N-methyl-diiodoacetamide, N- methyl-triiodoacetamide, N-methyl-2-methyl-2,2-difluoroacetamide, N-methyl-2-methyl-2- fluoroacetamide, N-methyl-2,2-dimethyl-2-fluoroacetamide, N-methyl-2-ethyl-2,2- difluoroacetamide, N-methyl-2-ethyl-2-fluoroacetamide, N-methyl-2,2-diethyl-2- fluoroacetamide, N-methyl-2-propyl-2,2-difluoroacetamide, N-methyl-2-propyl-2- fluoroacetamide, N-Methyl-2,2-propyl-2-fluoroacetamide, N-Methyl-2-fluoropropionamide,

[0234] N-methyl-3-fluoropropionamide, N-methyl-2,2-difluoropropionamide, N-methyl-2,3- difluoropropionamide, N-methyl-3,3-difluoropropionamide, N-methyl-3,3,3- trifluoropropionamide, N-Methyl-2-fluoro-3,3,3-trifluoropropionamide, N-methyl-2-chloro- 3,3,3-trifluoropropionamide, N-methyl-2,2-chloro-3,3,3-trifluoropropionamide, N-methyl-2- bromo-3,3,3-trifluoropropionamide, N-methyl-2,2-bromo-3,3,3-trifluoropropionamide, N- methyl-pentafluoropropionamide, N-methyl-heptafluorobutyramide, N,N-dimethyl-2,2,2- trifluoroacetamide, N-ethyl-2,2,2-trifluoroacetamide, N,N-diethyl-2,2,2-trifluoroacetamide, N-(hydroxymethyl)trifluoroacetamide, ethyltrifluoroacetate, dithiothreitol, dithioerythritol, beta-mercaptoethanol, penicillamine, tiopronin, acrylamide, methanol, ethanol, propanol, butanol, formaldehyde, glutaraldehyde, taurine, aconitic acid, adipic acid, benzoic acid, citric acid, malonic acid, malic acid, DL-maleic acid, oxalic acid, phenylacetic acid, phenylpropionic acid, succinic acid, levulinic acid, tartaric acid, gallic acid, p-toluenesulphonic acid, glycine, alanine, valine, leucine, isoleucine, serine, threonine, tyrosine, cysteine, methionine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, lysine, histidine, phenylalanine, tryptophan, proline, ethylene glycol, triethyleneglycol, glycerol, resorcinol, phenol, 1,2- propanediol, 1,3— propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1,8- octanediol, 1,12-dodecanediol, m-cresol, imidazole, 1-methylimidazole, 4-methylimidazole, N-methylpyrrolidone, N-ethylpyrrolidone, N-benzylpyrrolidone, 2-imidazolindone, tetrahydro-2-pyrimidione, guanidine, guanidine HCI, guanidine isothiocyanate, guanidine sulphate, ammonium acetate, ammonium bicarbonate, ammonium chloride, ammonium citrate dibasic, ammonium formate, ammonium iodide, ammonium nitrate, ammonium phosphate monobasic, ammonium phosphate dibasic, ammonium sulfamate, ammonium sulfate, ammonium tartrate dibasic, ammonium isothiocyanate, ammonium benzoate, ammonium bromide, ammonium fluoride, ammonium hydrogensulphate, ammonium trifluoroacetate, ammonium thiosulphate, adonitol, ribitol, rhamnose, trehalose, d-sorbitol, l-sorbitol, sorbose, xylitol, glucose, sucrose, lactose, fructose, maltose, mannose, mannitol, arabinose, galactose, raffinose, inositol, erythritol or xylose.

[0235] The deep eutectic solvent may comprise a first component and a second component, the first component being a compound of Formula 1 or a salt thereof:

[0236] Formula I wherein:

[0237] R6is H or OH; and

[0238] R7is selected from H, CH3, Cl, Br, a carbonyl oxygen, and -Z-C(O)R8where Z is selected from -CH2-, O and S; and R8is R11or OH; and the second component comprises a compound of Formula II or a salt thereof:

[0239] Formula II wherein:

[0240] A is selected from O, S, and NH; R1is selected from H, an alkene group having 1 to 6 carbon atoms, R9, -NH2, - NH-(CH2)aCH3, and -C(R3)(R4)(R5); wherein a is 0 or an integer from 1 to 5;

[0241] R2is selected from H and linear alkyl group having 1 to 3 carbon atoms;

[0242] R3is an optionally substituted 5- or 6- membered aliphatic or aromatic ring, wherein the substituent is R10;

[0243] R4and R5are each independently H or F; and wherein R9, R10, and R11are each independently selected from alkyl groups having one to three carbon atoms, monochloroalkyl groups having one to three carbon atoms, and mono-, di- or tri-fluoroalkyl groups having one to three carbon atoms.

[0244] A may be selected from O, S, and NH. Ri may be selected from H, -CH=CH2, R9, -NH2, -NHCH3, and -C(R3)(R4)(R5). R2may be selected from H and -CH3. R9, R10, and R11may each independently be selected from alkyl groups having one to three carbon atoms, monochloroalkyl groups having one to three carbon atoms, and mono-, di- or tri-fluoroalkyl groups having one to three carbon atoms.

[0245] R7may be selected from H, Br, a carbonyl oxygen, and -ZC(O)R8.

[0246] A may be O or S.

[0247] R2may be H.

[0248] Ri may be selected from H, R9, -CH=CH2, and C(R3)(R4)(R5). In this arrangement, R1may be R9, and R9may have one carbon atom.

[0249] The second component may be acetamide or 2-chloroacetamide.

[0250] In another arrangement, R9may be a mono-, di- or tri-fluoromethyl group. For example, the second component may be trifluoroacetamide, trifluorothioacetamide or N- methyltrifluoroacetamide. In another arrangement, R9may have two carbon atoms. R9may be a mono-, di- or trifluoroethyl group. The second component may be 2,2-difluoropropanamide or 3,3,3- trifluoropropanamide.

[0251] In another arrangement, the second component may be formamide or acrylamide.

[0252] In a further arrangement, R1is C(R3)(R4)(R5). R4and R5may each be F.

[0253] R3may be an optionally substituted 6-membered aromatic ring, such as an optionally substituted phenyl group. Preferably, the first component is 2,2-difluoro-2-phenylacetamide. Alternatively, R3 comprises a substituent, preferably at the 2-position of the phenyl group.

[0254] The substituent is preferably a mono-, di- or tri-fluoromethyl group. Thus, in one arrangement the second component is 2-(trifluoromethyl)phenyl acetamide.

[0255] In a further arrangement R1is selected from -NH2 and -NHCH3. Preferably, the second component is urea, thiourea or 1,3-dimethylurea.

[0256] In a further arrangement, A is NH. In this arrangement the second component may be guanidine, optionally wherein the guanidine is present in the form of a hydroisothiocyanate salt.

[0257] In a further arrangement, R7is H. In this arrangement the first component may be choline.

[0258] In another arrangement, the first component comprises bromocholine.

[0259] In another arrangement, the first component is N,N,N-trimethylglycine, optionally wherein the second component is selected from trifluoroacetamide and urea.

[0260] In another arrangement R7is -ZC(O)R8. Z may be O or S and Rs may be Rn. Rn may have one carbon atom. The first component may comprise acetylcholine or acetylthiocholine. Alternatively, R11may have three carbon atoms. For example, the first component may be butyrylcholine.

[0261] In a further arrangement Z may be CH2. In this arrangement, R8may be OH. In this arrangement, the second component is preferably carnitine.

[0262] The first component may comprise a counterion, which counterion is typically a halide anion but can be another anion such as nitrate (NO3 ) or tetrafluoroborate (BF4 ). The halide anion may be selected from fluoride, chloride, bromide, and iodide. The first component may be choline chloride, and the second component may be selected from trifluoroacetamide, trifluorothioacetamide; 3,3,3-trifluoropropanamide; 2,2-difluoro-2-phenylacetamide; thiourea and urea.

[0263] The molar ratio of the first component to the second component may be in the range 1:3 to 2:1, optionally 1:1.5 to 1:2.5, further optionally 1:2.

[0264] Another example class of DES comprises a choline salt and an optionally-substituted imidazole. The or each substituent may be an alkyl group having 1 to 3 carbon atoms.

[0265] The molar ratio of the choline salt to the optionally-substituted imidazole may be in the range 2.8:1 to 2:1.

[0266] The choline salt may be choline chloride. The substituted imidazole may be a methyl imidazole, such as N-methylimidazole or 4-methylimidazole. The use of benzyl-substituted imidazoles such as 1-benzylimidazole is also contemplated. Alternatively, the imidazole is unsubstituted. In a further arrangement, the deep eutectic solvent comprises a first component and a second component, wherein the first component comprises a compound of Formula III:

[0267] Formula III wherein:

[0268] R6is H or OH;

[0269] R7is selected from H, CH3, Cl, Br, a carbonyl oxygen, and -ZC(O)R8;

[0270] Z is selected from -CH2-, O and S; and

[0271] R8is selected from OH, an alkyl group having one to three carbon atoms, a monochloroalkyl group having one to three carbon atoms, and a mono-, di- or tri-fl uoroa Ikyl group having one to three carbon atoms; and the second component is a sugar or a sugar alcohol having at least 3 carbon atoms.

[0272] R7is optionally selected from H, Br, a carbonyl oxygen, and -ZC(O)R8.

[0273] The sugar alcohol may be selected from glycerol, thrietol, xylitol, sorbitol and volemitol. Optionally, the sugar alcohol may be selected from glycerol, xylitol and sorbitol. Further optionally, the sugar alcohol may be sorbitol.

[0274] The sugar may be trehalose.

[0275] In this arrangement, the second component may be a choline salt, such as choline chloride. The molar ratio of the first component to the second component may be in the range 1:2 to 2:1, optionally in the range 1:0.8 to 1:1.2.

[0276] In accordance with another possibility, the deep eutectic solvent comprises a choline salt and an alkanediol having 5 to 7 carbon atoms. The alkanediol may be hexanediol. Another class of DES comprises a choline salt and an N-alkyl pyrrolidone, wherein the N-alkyl group has 1 to 5 carbon atoms. The N-alkyl pyrrolidone may be N-methylpyrrolidone. The choline salt may be choline chloride. The molar ratio of the choline salt to the N-alkyl pyrrolidone may be 1:2.

[0277] Still another class of DES comprises a first component of Formula III:

[0278] Formula III wherein:

[0279] R6is H or OH;

[0280] R7is selected from H, CH3, Cl, Br, a carbonyl oxygen, and -ZC(O)R8;

[0281] Z is selected from -CH2-, O and S; and

[0282] R8is selected from OH, an alkyl group having one to three carbon atoms, a monochloroalkyl group having one to three carbon atoms, and a mono-, di- or tri-fl uoroa Ikyl group having one to three carbon atoms; and a second component which is a sugar or a sugar alcohol having at least 3 carbon atoms.

[0283] R7is optionally selected from H, Br, a carbonyl oxygen, and -ZC(O)R8.

[0284] The sugar alcohol may be selected from glycerol, thrietol, xylitol, sorbitol and volemitol. Optionally, the sugar alcohol may be selected from glycerol, xylitol and sorbitol. Further optionally, the sugar alcohol may be sorbitol.

[0285] The sugar may be trehalose.

[0286] The first component may be a choline salt, such as choline chloride. The molar ratio of the first component to the second component may be in the range 1:2 to 2:1, optionally in the range 1:0.8 to 1:1.2. In a further arrangement the deep eutectic solvent comprises a first component and a second component, wherein the first component comprises choline and wherein the second component is an alkanediol having 5 to 7 carbon atoms. The alkanediol is preferably hexanediol.

[0287] In a still further arrangement the deep eutectic solvent comprises a choline salt and an N-alkyl pyrrolidone, wherein the N-alkyl group has 1 to 5 carbon atoms. The N-alkyl pyrrolidone may be N-methylpyrrolidone. The choline salt may be choline chloride. In this arrangement, the molar ratio of the first component to the second component may be 1:2.

[0288] Examples of two-component DESs include:

[0289]

[0290] Other examples of DESs include choline chloride:trifluoroacetamide (1:2 mokmol), choline chloride:glycerol (1:2 mokmol), choline chloride:sorbitol (1:2 mokmol), choline chloride:galactose (1:2 mokmol), choline chloride:xylitol (1:2 mokmol), trimethylglycine:urea (1:2 mokmol), trimethylglycine:glycerol (1:2 mokmol), trimethylglycine:sorbitol (1:2 mokmol), trimethylglycine:galactose (1:2 mokmol), trimethylglycine:xylitol (1:2 mokmol) and / or trimethylglycine:trifluoroacetamide (1:2 mokmol).

[0291] The DES may further include an additive to modify one or more properties of the DES. Examples of additives include: i) a detergent, quaternary ammonium salt or saponin to improve penetration of the cell plasma membrane, such as SDS, sodium lauryl sulphate, cetyltetrabutylammonium bromide, tetrabutylammonium bromide, sodium deoxycholate, Brij-35, Brij-58, NP-40, Triton X-100, Triton X-114, Tween-20, Tween-80, octyl beta glucoside, CHAPS, and solanine; ii) an anti-microbial such as an antibiotic or antiseptic, for example streptomycin, penicillin, sodium nitrate, sodium nitrite, or sodium benzoate; iii) an anti-oxidant to reduce damaging oxidative effects during storage, for example vitamin C or glutathionine; iv) a ribonuclease inhibitor, an anti-lactoferrin antibody or lactoferring inhibitor, or a protease inhibitor such as phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, or aprotinin; v) a buffer such as Tris-HCI, PIPES, MES, HEPES, MOPS, MOPSO, CAPS, CAPSO, BIPES, phosphate, or imidazole, optionally at a concentration in the range 0.5 to 20 mM; vi) a chelator to remove divalent metal cations, such as BAPTA, EDTA, EGTA, citric acid, or D-Penicillamine, optionally at a concentration in the range 0.5 to 20 mM; vii) a dissolved gas, such as oxygen, CO2, or argon; viii) a nutrient, such as glucose or an amino acid, to enhance cell, tissue and organism viability; ix) water or an alcohol such as ethanol to reduce DES viscosity; and x) combinations thereof.

[0292] As previously discussed, cyclic polysaccharides such as cyclodextrins are also useful as additives, and may improve the storage stability and / or potency of the composition.

[0293] Typically, no more than one component of the deep eutectic solvent has a net charge. The deep eutectic solvent is not an ionic liquid.

[0294] Typically, the deep eutectic solvent includes at least one component that is uncharged.

[0295] Typically, no more than one component of the deep eutectic solvent is a salt. The deep eutectic solvent may comprise one salt and at least one non-ionic or zwitterionic component.

[0296] The deep eutectic solvent may be free of carboxylic acids. It has been found that DESs including carboxylic acids tend to be less effective for stabilising biomolecules.

[0297] The deep eutectic solvent may comprise a hydrogen bond acceptor and a hydrogen bond donor, wherein the hydrogen bond acceptor is choline or a choline derivative, and the hydrogen bond donor is selected from urea, a urea derivative, a sugar, and a sugar alcohol.

[0298] The choline derivative may have a structure of: wherein:

[0299] R1is selected from H, a halogen, a Cl to C3 alkyl group (e.g. -CH3), and an oxo group;

[0300] R2is selected from H, -OH, a halogen (e.g. -Cl, -Br, I), -OP(O)(OH)2, and -OC(O)R6, where

[0301] R6is a Cl to C3 alkyl group; and

[0302] R3, R4, and R5are each independently selected from Cl to C3 alkyl groups.

[0303] Optionally, R3, R4, and R5are each methyl groups.

[0304] The choline derivative may comprise N,N,N-trimethylglycine (referred to as "betaine" in the Examples):

[0305] However, in implementations where the composition is an external-DES composition or a transient-DES composition, the hydrogen bond acceptor is typically not N,N,N- trimethylglycine. More generally, there is an optional proviso that when R1is an oxo group, R2is not OH.

[0306] The hydrogen bond donor is most typically a salt, further comprising a counterion. The counterion may be a halide (e.g. chloride, bromide, or iodide). Other counterions may be used, such as bicarbonate or tosylate. The counterion may in some implementations be a carboxylate (e.g., citrate, or bitartrate), however the DES is typically free of carboxylates and carboxylic acids. The counterion is typically not OH-.

[0307] Examples of choline derivates include chlorocholine chloride, bromocholine bromide, iodocholine iodide, acetylcholine hydroxide, acetylcholine bitartrate, Acetylcholine dihydrogen citrate, acetylcholine p-toluenesulfonate, acetylcholine bicarbonate, acetylcholine chloride, acetylcholine bromide, acetylcholine iodide, acetylcholine fluoride, chloroacetylcholine chloride, bromoacetylcholine bromide, iodoacetylcholine iodide, butyrylcholine hydroxide, butyrylcholine bitartrate, butyrylcholine dihydrogen citrate, butyrylcholine p-toluenesulfonate, butyrylcholine bicarbonate, butyrylcholine chloride, butyrylcholine bromide, butyrylcholine iodide, butyrylcholine fluoride, chlorobutyrylcholine chloride, bromobutyrylcholine bromide, and iodobutyrylcholine iodide.

[0308] The hydrogen bond donor may in particular have a structure of: wherein:

[0309] R1is H or OH;

[0310] R2is selected from H, CH3, Cl, Br; and

[0311] X- is a counterion, such as a halide.

[0312] The preferred hydrogen bond acceptors in this class are choline salts, such as choline chloride.

[0313] The hydrogen bond donor may be a sugar or a sugar alcohol.

[0314] The sugar may be any monosaccharide or disaccharide, and is typically a monosaccharide. Examples of sugars useful as hydrogen bond donors include mannose, glucose, sucrose, lactose, fructose, maltose, mannose, arabinose, galactose, raffinose, inositol, and xylose. For example, the sugar may be mannose.

[0315] The sugar alcohol is not particularly limited. The sugar alcohol may have 5 or 6 carbon atoms. The sugar alcohol may be selected from arabitol, optionally D(+)arabitol or L(-)arabitol; sorbitol; xylitol; ribitol; and combinations thereof.

[0316] When the hydrogen bond acceptor is choline or a choline derivative, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor may be in the range 1:3 to 3:1, optionally 1:2 to 2:1, further optionally 1:0.8 to 1:1.2; or is about 1:1. As an alternative to a sugar or sugar alcohol, the hydrogen bond donor may be urea or a urea derivative. For example, the hydrogen bond donor may have a structure of: wherein:

[0317] A is selected from O, S, and NH;

[0318] R3is selected from: H; -NH2; a Cl to C6 alkyl, alkenyl, or haloalkyl group; and -NH-(CH2)nCH3, where n is 0 or an integer from 1 to 5; and

[0319] R4is H and or a Cl to C3 alkyl group.

[0320] Optionally, the hydrogen bond donor is urea.

[0321] When the hydrogen bond donor is urea or a urea derivative, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is in the range 1:1.5 to 1:2.5, or is about 1:2.

[0322] Alternatively, the deep eutectic solvent may comprise an amino acid and a sugar or sugar alcohol.

[0323] The amino acid may be selected from glycine, alanine, valine, leucine, isoleucine, serine, threonine, tyrosine, cysteine, methionine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, lysine, histidine, phenylalanine, tryptophan, and proline. In particular, the amino acid may be proline.

[0324] The sugar or sugar alcohol may be any of the sugars and sugar alcohols discussed hereinabove.

[0325] In accordance with still another possibility, the deep eutectic solvent may comprise a hydrogen bond acceptor selected from ectoine, hydroxyectoine, homoectoine, and hydroxyhomoectoine, and salts thereof; and a hydrogen bond donor which is a sugar or sugar alcohol, such as sorbitol, xylitol, or arabitol. For instance, the DES may be ectoine:sorbitol (1:1, 1:2 or 1:3 mokmol). It has been found that DESs in this class may prolong the viability of viruses and other microbes, even after storage at temperatures of 60°C or more. A DES comprising ectoine or a derivative thereof may be especially suitable in implementations where the composition is a vaccine comprising a live or attenuated virus.

[0326] Specific examples of DES useful in the methods and compositions provided herein are: choline chloride : urea; choline chloride : xylitol; choline chloride : sorbitol; choline chloride : arabitol;

[0327] N,N,N-trimethylglycine : xylitol;

[0328] N,N,N-trimethylglycine : arabitol;

[0329] N,N,N-trimethylglycine : urea; proline : sorbitol; ectoine : sorbitol; choline chloride : mannose; and choline chloride : ribitol.

[0330] In implementations using an internal DES, the DES may in particular be choline chloride : urea.

[0331] General Definitions

[0332] Unless otherwise stated, all percentages are by volume, based on the volume of the relevant mixture. Volumes are measured at a temperature of 20 °C and at atmospheric pressure (1 atm).

[0333] The term "about" where used in connection with a numeral contemplates a variance of ± 10 %.

[0334] As used herein, "betaine" refers to N,N,N-trimethylglycine.

[0335] RNA % Purity is the integrity of the full-length complete RNA, expressed as a percentage. RNA including mRNA with a purity of >90 % is considered substantially intact. Purity is measured by Capillary Electrophoresis (CE) and is expressed as a percentage of full-length RNA compared to RNA degradation products and any contaminating double-stranded RNA generated during in vitro transcription. Stability of mRNA vaccines is discussed in detail in; Schoenmaker L, Witzigmann D, Kulkarni JA, Verbeke R, Kersten G, Jiskoot W, Crommelin DJA. Int J Pharm. 2021 May 15;601:120586. doi: 10.1016 / j.ijpharm.2021.120586.

[0336] Examples

[0337] Example 1: preparation of DESs

[0338] Six different DESs were prepared as set out in the table below. Three different Hydrogen

[0339] Bond Acceptors ("HBA") were used: Choline chloride, Betaine and Proline. Four different

[0340] Hydrogen Bond Donors ("HBD") were used (Urea, Xylitol, Sorbitol, L(-) or D(+) Arabitol). DES

[0341] 5 and DES 6 included water to reduce the viscosity of the DES.

[0342] Methods for preparing DESs as well as further DES formulations are set out in WO 2014 / 131906 Al.

[0343] As one example, in a glass bottle a molar stoichiometric amount of a hydrogen bond acceptor such as choline chloride was mixed with either one or two molar amounts of a hydrogen bond donor such as sorbitol, shaken to crudely mix the 2 components and was then subjected to 25 kHz ultrasound in a water bath heated at 80 °C until a clear viscous liquid DES such as Choline chloride:Sorbitol (1:1 or 1:2 mokmol) was obtained with no visible particles. The DES can be purified by passing through a 0.1 or 1 pm filter and stored at ambient temperature. Typically, 80 °C is the maximum temperature necessary to reliably make a DES. The temperature chosen may depend on the DES formulation. There is a risk of sugars caramelising if prolonged heating at 100°C is carried out. DESs are typically hygroscopic and so should be stored in an airtight container.

[0344] Example 2: Preparation of Control LNPs using the NanoAssemblr® lqnite+™

[0345] Typically, lipid nanoparticles ("LNPs") are formed by rapid mixing of two miscible phases: an organic phase containing dissolved lipids, and an aqueous phase comprising a dissolved payload, such as RNA.

[0346] The two phases may be mixed using a variety of methods, such as T-junction or microcapillary mixing. One example method using NxGen™ cartridges, a commercially-available product for preparing LNPs, is described here.

[0347] Two different organic phases were used:

[0348] Organic Phase A comprising the lipids ALC-0315 : DSPC : Cholesterol : ALC-0159 in a molar ratio of 46.5 : 9.4 : 42.7 : 1.6, respectively, dissolved in ethanol to a total lipid concentration of 16 mg / ml.

[0349] Organic Phase B comprising SM-102 (heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate), PEG2000-DMG (1- monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000), DSPC (l,2-Distearoyl-sn-glycero-3 phosphocholine) and Cholesterol in a 50 : 10 : 38.5 : 1.5 % molar ratio, dissolved in ethanol.

[0350] The aqueous phase contained the 1913 nucleotide long 5' Capped Nl-Me-Pseudouridine Fluc- mRNA (Cat. No. CT-072 CATUG, China) dissolved in an acidic buffer (Buffer A, 10 mM Citrate buffer pH 4) to a final concentration of 104 pg / mL with an N / P ratio (Cationic lipid ucleic acid Phosphate molar ratio) of 6-24, preferably 12; a Flow Rate Ratio (FRR) of aqueous:organic of between 1:1 to 5:1, preferably 3:1; and a Total Flow Rate (TFR) of 1-12 mL / min, preferably 8 mL / min.

[0351] To facilitate the self-assembly of RNA and lipids, the organic phase and aqueous phase were combined using a microfluidic mixer system (NanoAssemblr® lgnite+™ system from Precision NanoSystems Inc., Canada) and standard NxGen™ cartridges, to form RNA-Lipid nanoparticles (RNA-LNPs). The NanoAssemblr® lgnite+™ equipment was operated in accordance with the manufacturer's instructions.

[0352] After encapsulation, the RNA-LNPs were diluted at a volumetric ratio of 1:1 (LNP product : dialysis buffer) using acidic buffer to stabilise the RNA-LNPs.

[0353] Dialysis was performed to purify the RNA-LNPs using a Float-A-Lyzer® device (with molecular weight cut-off 100 kDa). RNA-LNPs were dialysed against 10 mM citrate buffer pH 4 for 4 hours, followed by 10 mM Tris buffered saline (TBS) at pH 7.4 overnight. Dialysed samples were then retrieved from the device and measured by DLS to ascertain size and PDL LNPs were subsequently stored at 2-8 °C.

[0354] Various modifications may be made to the process described in this example.

[0355] For larger-scale manufacturing of mRNA-LNPs, NxGen500 cartridges were used instead of standard NxGen cartridges, and a total flow rate of 60 mL / min used for microfluidic mixing. Following manufacture of the RNA-LNPs, Tangential Flow Filtration ("TFF") was used instead of dialysis to purify the RNA-LNPs. This was performed using KrosFlo® KR2i TFF system (from Repligen, UK). A hollow fibre filter (pore size 100 kDa), reservoirs, pumps, pressure sensors and scales were set up according to manufacturer's instructions. The RNA-LNPs were diafiltrated against 10 mM TBS and then concentrated to achieve a desired RNA concentration.

[0356] Experiments were also conducted using a commercially-available mRNA-LNP composition (0.3 mg / mL Flue mRNA, Pfizer lipid formulation, (Cat. No. CT0072-LNP, CATUG, China)). Example 3: Preparation of External DES Stabilised LNPs

[0357] 5 pl of mRNA-LNP was added to 95 pL of a DES in a 2 mL screw-cap tube and stirred with the tip of a 100 pL plastic pipette in a figure-8 stirring movement 20 times or until the mixture appeared homogenous.

[0358] In order to improve particle mixing, it is preferred that the mRNA-LNP is added to the DES and mixed rather than the DES added to mRNA-LNP already present in the tube and then mixed. Inefficient mixing can result in particle aggregation or fusion.

[0359] The mixing ratio of LNP to DES may be selected as appropriate based on the initial concentration of LNP, because the addition of DES results in dilution. For example, the volume:volume ratio of LNP to DES may be in the range of 1:1 to 1:1000, 1:3 to 1:500, 1:5 to 1:200, 1:10 to 1:100, or 1:20 to 1:50.

[0360] Mixing of the LNP in the DES may be improved by first premixing by stirring 1 volume LNP with 1 to 20 volumes of glycerol, before mixing by stirring with at least 19 volumes of DES. Glycerol reduces potential particle aggregation, in the DES thereby enhancing the potency of the LNP formulation.

[0361] The viscosity of the DES may be reduced by adding water to the DES. Reducing the viscosity of the DES may simplify handling of the DES.

[0362] However, LNPs in higher viscosity DESs are better protected from vibration during, for example, transport which can lead to LNP damage in low viscosity aqueous DES. Higher viscosity DESs are therefore advantageous for storage of LNPs during transport or other environments where there is substantial mechanical movement which would otherwise lead to LNP damage. It has been found that both ultrasound (25 kHz) and mechanical mixing can lead to extensive particle damage in aqueous solutions, but the viscous properties of DES can provide protection from such damage (Example 6). Furthermore, RNA is susceptible to hydrolysis, and the presence of excessive water may compromise potency if the composition is to be stored for an extended period and / or at elevated temperatures.

[0363] The table below shows a comparison of the effect of 4 different external DESs on LNPs. In this example, DES-1 maintained a high EE% and diameter. LNPs treated with external DES-2, DES-3 and DES-4 had a reduced EE% of 63, 66 and 23 % respectively and slightly increased particle size.

[0364] Example 4: Mixing LNPs with DES

[0365] In a 2 mL tube, a portion of the DES stock was prewarmed to 70 °C to reduce liquid viscosity, and then mixed by vortexing at maximum speed for about 10 seconds. The tube was then transferred to a 70 °C heating block for 5 minutes.

[0366] The DES was allowed to cool for five minutes and then the aqueous LNP sample was added by pipetting. The pipette tip was placed immediately under the meniscus of the DES to a depth of 2 to 5 mm and the aqueous LNP was ejected slowly over 2 to 4 seconds.

[0367] The LNP was then mixed into the DES by stirring, before sealing the tube to exclude further contact with the air.

[0368] Additional methods to improve mixing and reduce LNP aggregation include premixing the mRNA-LNP with one or more of the following: blood serum such as FCS or FBS; a purified protein such as Bovine Serum Albumin (BSA), gelatin, an antibody such as IgG, silk protein or collagen in the range of 0.01 to 10 % final weight:volume; glycerol 1-95 % final volume; 5-80 kDa PEG at 0.01-10 % final weight:volume; or ammonium acetate pH 4-7.5 or tetraethylammonium chloride 10 mM to 1 M final concentration. In particular, premixing the LNPs with glycerol prior to the addition of the external DES was found to maintain high potency.

[0369] Mixing the mRNA-LNP into chilled DES in the range of 0 to 10 °C can also improve mixing efficiency as determined by DLS. In order to avoid mRNA-LNP sample loss on plastic surfaces such as polypropylene it has been found that silicone solid surfaces are effective solid alternatives for handling and mixing.

[0370] Example 5: Transient DES treatment of LNPs

[0371] It was found that a brief treatment of LNPs with a DES markedly increases their potency as measured by cell transfection. It was also found that neither a high concentration of aqueous solutions of the individual DES components such as 5 M choline chloride or 5 M sorbitol, or the same amount of DES but prediluted before addition of the LNP have this potency enhancing effect.

[0372] The mechanism(s) by which the transient DES treatment increase the potency of the LNP have not been definitively established.

[0373] Without wishing to be bound by theory, the DES may change one or more of: the structure or arrangement of the Lipids in the LNP; the size or morphology of the LNP; the organization of the lipids and / orthe RNA in the LNP; or the Zeta potential of the LNP.

[0374] Alternatively or additionally, the DES components may enter or attach to the mRNA-LNP thereby enhancing one or more of the following attributes of the mRNA-LNP:

[0375] (i) stability;

[0376] (ii) attachment to the cell; (iii) uptake by the cell;

[0377] (iv) intracellular release of the mRNA from the LNP;

[0378] (v) intracellular release of the mRNA from the cationic lipid;

[0379] (vi) intracellular release of the mRNA from the endosome;

[0380] (vii) availability and binding of the mRNA to the ribosome; and

[0381] (viii) translation template.

[0382] It is also possible that the DES components may be transferred inside the cell at the same time as the LNP thus improving the viability of the cell and / or the rate of translation of the mRNA; and / or may increase the stability of the mRNA by altering its molecular structure so that the mRNA is translated into protein for longer than an untreated mRNA-LNP.

[0383] It has been found that the optimum time for the DES treatment of the mRNA-LNP is less than 1 hour at 20 °C, preferably less than 30 minutes and more than 10 seconds, or approximately in the range of 1-15 minutes. The DES is then removed from the treated mRNA-LNP by one of several methods including dilution in tissue culture media, a buffer such as PBS or a protein solution such as 10 % FCS, or by dialysis or Molecular Weight Cutoff (MWCO) spin ultrafiltration.

[0384] Detailed protocol: In a polypropylene 8-tube PCR tube containing 10 pL of DES such as Choline chloride:sorbitol (1:1 mokmol) or choline chloride:urea (1:2 mokmol) was added 1 pL (0.3 mg / ml) of mRNA-LNP such as 5' Capped Nl-Me-Pseudouridine Flue mRNA-LNP, CATUG (China) Cat. CT072-LNP and mixed with a pipette tip 10 times in a figure-8 movement.

[0385] Optionally, the LNPs may be pre-mixed with glycerol at an amount in the range of 0.2 to 50 volumes, optionally 1 to 10 volumes of glycerol to LNP, prior to the addition of the external DES. Adding glycerol was found to provide high potency. The mixture of DES and mRNA-LNP was incubated for 15 minutes at 20-24 °C before removal of the DES from the mRNA-LNP by either:

[0386] (i) adding twenty volumes of DMEM / bovine serum (FCS) and gently pipetting up and down at least ten times to dilute the DES to less than 5 % concentration final volume; or

[0387] (ii) ultrafiltration with a 50-300 kDa MWCO membrane device.

[0388] The volumes of DES and mRNA-LNP that are mixed together are not particularly limited. Typically, the ratio of DES to mRNA-LNP is at least 4:1, preferably 8:1, more preferably 10:1, more preferably 15:1 and most preferably greater than 20:1 (vokvol) to provide a final DES concentration equal to or greater than 80 %, optionally equal to or greater than 95 %.

[0389] The transiently DES treated mRNA-LNP may be stored in Dulbecco's Modified Eagle's Medium (DMEM) / 10 % serum such as FCS or FBS, or other commonly used serum growth medium at 4 °C for up to 1 month.

[0390] Alternatively, blood serum, or a purified protein such as Bovine Serum Albumin (BSA), Gelatin, an antibody such as IgG, silk protein or collagen in the range of 0.01 to 10 % final weight:volume; glycerol in the range 1 to 95 % final volume; 5 to 80 kDa PEG at 0.01 to 10 % final weight:volume; ammonium acetate pH 4 to 7.5 can be diluted in water, 1 mM Tris-HCI pH 6.5, 1 mM citrate buffer pH 6 or PBS pH 7 before mixing, dialyzing or freezing with the transiently DES treated LNPs to give a final protein concentration in the range of 0.1 to 10 % and a LNP concentration in the range of 0.01 to 0.5 mg / mL.

[0391] Example 6: in vitro Cell Assay Potency Tests

[0392] Plate preparation: HeLa cells were trypsinized and immediately counted using a BioRad Cell Counter. Then, using an 8-channel pipette, 100 pl of DMEM / 10 % FCS / lx Pen / Strep with 10,000 cells per well were added to black or white 96-well plates (preferably with an opaque bottom). The cells were allowed to attach to the plate for 1-2 hours. Sample preparation: 10 pl of DES was added using a wide-bore 10 pl pipette tip to a 250 pl PCR tube (8-strip). If necessary to reduce viscosity during pipetting, the DES can be prewarmed to 50 to 80 °C. The amount of DES added was verified using weight rather than volume, the density of DES is approximately 1.3 g / ml.

[0393] Approximately 0.5 pl of mRNA-LNP (150 ng / well) was added to 10 pl (approximately 13 mg DES) of 100 % DES in a PCR tube. For optimal RNA stability, the final amount of aqueous should be 5 % by volume or less. The mixture was mixed by stirring with a pipette tip for 5 to 10 seconds in a figure 8 movement. If necessary, the DES / LNP mixture was incubated.

[0394] Cell transfection: When the DES / LNP sample was ready for cell transfection, 200 pl of diluent (DMEM / 10 % FCS / lx Pen / Strep) was added to each of the PCR tubes containing the DES / LNP. The solution was allowed to dissolve over ten minutes without pipetting or vortexing. To ensure proper dissolution, the PCR tubes were inverted four times, allowing the denser DES to flow into the diluent. Care was taken to avoid air bubbles so that all the DES in the PCR tube was in contact with the diluent.

[0395] Using an 8-channel pipettor set at 20 pl, the diluted DES in DMEM / 10 % FCS / lx Pen / Strep was gently pipetted up and down four times to fully mix it into the diluent. Next, 20 pl of the diluted DES in diluent was gently removed from the PCR tube and added to the 100 pl of tissue culture medium already present on the cells in the 96-well plate. The mixture was gently pipetted up and down once. The 96-well plate was returned to the incubator and left overnight for at least 14 and no more than 48 hours to allow for cell transfection and mRNA translation into luciferase. The remaining unused 180 pL of diluted DES was stored at 4°C.

[0396] Cell Viability and Luciferase Assay: 5x CellTitre-Fluor cell viability assay reagent was prepared as set out in the Promega protocol Promega ONE-Glo™ + Tox Luciferase & Cell Viability. Promega USA Cat. E7120), forexample 5 pLGF-AFC was added to 1 mL Assay Buffer and mixed by vortexing. The white-walled 96-well plate was removed from incubator and 20 pL 5x CellTitre-Fluor cell viability assay reagent added to each well, then pipetted up and down once to mix. The 96-well plate was then left in a 37 °C incubator for 30 min and fluorescence read on a Fluorimeter using 390 nm excitation and 505 nm emission settings. Then 100 pL ONE- Glo reagent (Cat. No. E7120 Promega, UK) was added per well of the 96-well plate and pipetted up and down once. Luciferase activity was then measured within 10 minutes using a Luminometer (Tecan, France) using standard Luminometer settings. Generally, cell viability differed by 10 % or less between sample wells.

[0397] Potency tests are useful for determining the effects of different DES on LNPs and other RNA delivery vehicles, such as liposomes. This is because potency requires that all the following are true:

[0398] (i) the mRNA including the 5' CAP and poly A tail are intact, not cross-linked or oxidized and can be translated;

[0399] (ii) the delivery vehicle such as a LNP is of a suitable size and compatibility for cell attachment and uptake; and

[0400] (iii) the mRNA can be correctly released from the LNP and Lipids within the cell and then transported to the ribosome for translation into protein.

[0401] If any one of these factors is sub-optimal or absent there will be a reduction or absence of potency.

[0402] The luciferase activity results herein are reported in arbitrary units. Results reported in the same example or table are comparable with one another. However, results reported in different examples or tables are not necessarily comparable.

[0403] Example 7: in vitro Tissue Culture Luciferase mRNA Transfection Assay Using Transiently DES Treated LNPs

[0404] In a polypropylene 1.5 mL tube containing 5-10 pL of a DES such as choline chloride:urea (1:2 mokmol), choline chloride:xylitol (1:1 or 1:2 mokmol), choline chloride:sorbitol (1:1 or 1:2 mokmol), choline chloride:arabitol (1:1 or 1:2 mokmol) or betaine:xylitol:water (1:1:1 mokmokmol) was added 0.5 pl (0.3 mg / ml) of mRNA-LNP (such as 5' Capped Nl-Me- Pseudouridine Flue mRNA-LNP, CATUG (China) Cat. CT072-LNP) and mixed gently with a pipette tip 10 times in a figure-8 movement. Alternatively, the 0.5 pl LNP may be premixed with blood serum (FBS or FCS), a purified protein such as Bovine Serum Albumin (BSA), gelatin, an antibody such as IgG, silk protein or collagen in the range of 0.01 to 10 % final weight:volume; 5 to 80 kDa PEG at 0.01 to 10 % final weight:volume; 10 mM ammonium acetate pH 4 to 7.5; or preferably glycerol at 1 to 95 % final volume in the LNP before the addition of 1-100 volumes of DES. Such pretreatment of the LNP markedly improves potency, potentially by reducing LNP aggregation when DES is added.

[0405] The mixture of DES and mRNA-LNP was incubated for a time in the range of 1 to 60 minutes, more preferably 1 to 10 minutes and most preferably 10 minutes at 20 to 24°C before removal or dilution of the DES from the mRNA-LNP using one of the following techniques:

[0406] (i) adding 10 to 20 volumes of tissue culture media such as DMEM containing 10 % bovine serum (FCS) and gently pipetting up and down at least 10 times to dilute the DES to less than 5 % concentration final volume,

[0407] (ii) adding the DES / LNP mixture to 450 pL of TBS (Tris Buffered saline pH 8.5) and gently pipetting up and down to mix before adding to a 50, 100 or 300 kD MWCO ultrafiltration device such as Vivaspin® (Sartorius Cat. No. VS0141), centrifuging at 9000 rpm for 1-5 minutes, discarding the flow-through, adding 400 TBS wash buffer to the device and repeating the centrifugation before recuperating the LNPs from the dead-stop pocket in an approximate volume of 40 pL.

[0408] The volumes of DES and mRNA-LNP that are mixed together is not particularly limited. Typically, the ratio of DES to mRNA-LNP is at least 4:1, preferably 8:1, more preferably 10:1, more preferably 15:1 and most preferably greater than 20:1 (vokvol).

[0409] Example 7 A: Effect of Treatment Time

[0410] 0.5 pl (150 ng) samples of Flue mRNA-LNP, (CATUG Cat. CT072-LNP) were pretreated with 12 pl choline chloride:L-arabitol (1:1 mokmol) for 1 min to 19 hours at 37 °C, before dilution in 200 pL DMEM / 10 % FCS. 20 pL of the diluted LNPs was added to 100 pL cells in a 96-well plate and a Luciferase assay carried out as set out in Example 4. The results of the luciferase assay are shown in Fig. 3. All DES treatments from 1 minute to 19 hours increased the potency of the LNP compared with an untreated control. The 1 minute treatment gave the strongest effect, increasing potency by approximately 4-fold compared with the control.

[0411] Example 7B: Further Optimization of Treatment Time

[0412] The experiment of Example 7A was repeated using pretreatment at 22 °C for times in the range 30 seconds to 25 minutes. A positive control, comprising the same amount (150 ng) of mRNA-LNP not treated with the DES was also tested.

[0413] All DES treatments from 30 seconds to 25 minutes increased the potency of the LNP compared with the non-DES treated control.

[0414] A test was also carried out using a diluted DES. The diluted DES was 12 pL choline chloride:L- arabitol (1:1 mokmol) prediluted in 200 pL DMEM / 10 % FCS before addition of the mRNA- LNP. No improvement in potency was observed for the diluted DES.

[0415] The results of Example 7B are shown in Fig. 4.

[0416] Example 7C: Effects of individual DES components

[0417] The compositions listed in the table below were used to treat 0.5 pL (150 ng) of Flue mRNA- LNP (CATUG Cat. CT072-LNP) for 30 minutes at 22°C. After treatment, the sample was diluted in 200 pL of DMEM / 10% FCS, and 20 pL of the diluted LNPs was added to 100 pL of cells in a 96-well plate. A luciferase assay was then conducted as described in Example 6. The positive control represents the same amount (150 ng) of untreated mRNA-LNP.

[0418] The results of the experiment are shown in Fig. 5.

[0419] L-arabitol or choline chloride used alone were found not to enhance potency. However, two different DES combinations, choline chloride:L-arabitol (1:1 mokmol) and choline chloride:xylitol (1:1 mokmol) did enhance potency. The choline chloride-based DESs were found to be much more effective than the betaine-based DES betaine : L-arabitol.

[0420] Example 7D: Effects of DES concentration

[0421] 0.5 pL (150 ng) samples of Flue mRNA-LNP, (CATUG Cat. CT072-LNP) were treated with 6 pL of various dilutions of choline chloride:D-arabitol (1:1 mokmol) for 30 minutes at 22 °C, before further dilution in 200 pL DMEM / 10 % FCS. Then, 20 pL of the diluted LNPs was added to 100 pL cells in a 96-well plate and a luciferase assay carried out as set out in Example 4. As a positive control, an untreated sample comprising the same amount (150 ng) of mRNA-LNP was also tested.

[0422] The results are shown in Fig. 6.

[0423] It was found that adding water to the DES reduced the enhancement of LNP potency when performing a transient treatment. To improve potency through transient treatment of LNPs, DESs with a water content of less than 20 % by volume should be used. Example 7E: Storage of transiently-treated mRNA LNPs

[0424] Treatment of 0.5 pL (150 ng) samples of Flue mRNA-LNP, (CATUG Cat. CT072-LNP) were treated with 6 pL of 0-100 % choline chloride:D+arabitol (1:1 mokmol) for 30 minutes at 22 °C before dilution in 200 pL of:

[0425] (i) DMEM / 10 % FCS,

[0426] (ii) water,

[0427] (iii) PBS, or

[0428] (iv) 10 mM Tris-HCI at pH 7.4.

[0429] The samples were then incubated for 12 days at 4 to 20 °C before adding 20 pL of the diluted LNPs to 100 pL cells in a 96-well plate and a Luciferase assay carried out as set out in Example 4.

[0430] The results of the luciferase assay are shown in Fig. 7. The positive control (POS) represents the same amount (150 ng) of mRNA-LNP also incubated in DMEM / FCS for 12 days at 4 to 20 °C.

[0431] It was found that transiently DES treated LNPs stored in DMEM tissue culture medium supplemented with 10 % serum (FCS) were more stable than LNPs stored in water, PBS buffer, or Tris-HCI buffer.

[0432] Example 7F: Effects of different DESs for transient treatment

[0433] 0.5 pL (150 ng) samples of Flue mRNA-LNP (CATUG Cat. CT072-LNP) were treated with 6 pL of choline chloride:D+arabitol (1:1 mokmol) ("CCA D+"), choline chloride:xylitol (1:1 mokmol) ("CCX 1:1"), choline chloride:sorbitol (1:1 mokmol) ("CCS 1:1"), betaine:xylitol (1:1 mokmol) ("BX"), betaine:arabitol (1:1 mokmol) ("BA"), ES (ectoine:sorbitol (1:1 mokmol) ("ES"), choline chloride:urea (1:2 mokmol) ("CCU") or proline:sorbitol:water (2:1:18 mokmokmol) ("PSH") for 30 minutes at 22 °C. The samples were each subsequently diluted in 200 pL DMEM / 10 % FCS, and then incubated for 12 days at 4 to 20 °C before addition of 20 pL of the diluted LNPs to 100 pL cells in a 96-well plate and a Luciferase assay carried out as set out in Example 6. The positive control (POS) represents the same amount (150 ng) of mRNA-LNP also incubated in DM EM / FCS for 12 days at 4 to 20 °C.

[0434] The results of the luciferase assays are shown in Fig. 8.

[0435] Transient treatment of LNPs with choline chloride:D+arabitol (1:1 mokmol), choline chloride:xylitol (1:1 mokmol), choline chloride:sorbitol (1:1 mokmol) and to a lesser extent choline chloride:urea (1:2 mokmol) were effective at enhancing LNP potency.

[0436] Example 7G: Improving potency by warming

[0437] 0.5 pL (150 ng) samples of Flue mRNA-LNP (CATUG Cat. CT072-LNP) were treated with 6 pL of choline chloride:urea (1:2 mokmol) (CCU) for 30 minutes at 22 °C before dilution in 200 pL DMEM / 10 % FCS, and then incubated at either 4 °C or 37 °C before addition of 20 pL of the diluted LNPs to 100 pL cells in a 96-well plate and a Luciferase assay carried out as set out in Example 6.

[0438] The results of the luciferase assays are shown in Fig. 9. The black columns correspond to samples incubated in the DMEM / FCS for 90 minutes at 37 °C, and the white columns correspond to samples incubated in the DMEM / FCS for 90 minutes at 4 °C. As may be seen, LNP potency may be improved by warming the LNPs to 37 °C for 90 minutes in DMEM / FCS for 90 minutes before carrying out a Luciferase potency assay on the LNP transfected cells.

[0439] Example 8: Agarose Gel Electrophoresis

[0440] 1 % weight / volume agarose gels were prepared in 0.5X final concentration of TAE pH 7.6 buffer (0.5X working solution was 20 mM Tris-acetate / 0.5 mM EDTA pH 7.6). Combs were used to make wells of at least 8 mm width to improve resolution and avoid RNA smearing during electrophoresis. For the load buffer, 0.01 % bromophenol blue, 135 mM trifluoroacetamide dissolved in 100 % formamide was used. Preferably, at least 50 % Load buffer by total volume per 300 ng RNA sample was used per lane. Agarose gels were electrophoresed at a maximum of 135 V, and the 0.5X TAE running buffer was chilled at 4 °C before use to prevent the gel from overheating. The gel was run until the blue Bromophenol marker had migrated a maximum of 3 to 4 cm.

[0441] Fig. 10 illustrates the stability of Comirnaty® SARS-CoV-2 mRNA-LNP at 100 °C for 2, 4, 15 or 30 min in choline chloride:sorbitol (1:1 mokmol) compared with PBS. mRNA was extracted from the LNPs before loading 300 ng / lane and analysis in a 0.5x TAE / l % agarose gel stained with ethidium bromide. The mRNA was significantly more thermostable in the DES than in an aqueous buffer.

[0442] Fig. 11 illustrates the stability of Comirnaty® SARS-CoV-2 mRNA-LNP in choline chloride:sorbitol (1:1 mokmol) compared with PBS following 10 freeze-thaw (F / Thaw) cycles, 25kHz sonication or Ing RNase A / CA-630 non-ionic detergent treatment. mRNA was extracted from the LNPs before loading 300 ng RNA / lane and analysis in a 0.5x TAE / 1 % agarose gel stained with ethidium bromide. It was found that the approximately 4000 nucleotide mRNA was significantly more stable in DES than aqueous buffer when sonicated or treated with RNase A.

[0443] RNA integrity was determined by capillary gel electrophoresis (Sciex PA-800 & CESI-8000 system).

[0444] RNA-LNPs were ruptured, and RNA isolated using a RNeasy™ Mini RNA Purification kit (Cat. No. 74104 Qiagen, UK) prior to capillary gel electrophoresis. Isolated RNA was diluted to the same concentration (e.g., 10 pg / ml) with nuclease-free water and then further diluted to a final concentration of 5 pg / ml with formamide (50 % final volume). 500 ng of each RNA sample was then heated at 70 °C for 5 minutes. A ThermoFisher Millenium™ RNA Marker (ThermoFisher, UK) ranging from 0.5 to 9 kB in size was run. The 32 Karat software was used to analyse data and determine the %Purity of the RNA as a percentage of the non-degraded time point zero mRNA (assumed to have a %Purity of 100 %), however it was found that the time-point zero control was never more than 85 to 90 %. It was found that the %Purity dropped from the Time point zero value in a non-linear manner relative to the time and temperature of storage. RNA degradation could be observed as sample detected between the intact mRNA found at approximately 16 to 17 minutes and 0 minutes of the CE run. Frequently such RNA appeared as a 'smear' of various overlapping sizes compared with the single peak of the intact RNA. An additional higher molecular weight RNA peak could be observed.

[0445] The table below shows the purity of RNA extracted from various mRNA-LNP samples after storage for 3 months at 22 °C, as measured by capillary electrophoresis.

[0446] RNA from the non-stabilised control at time point zero had a % Purity of 90 %. After 3 months storage at 22°C, the % RNA Purity descended to 41 %. DES-stabilised samples were found to maintain much higher purity after incubation under the same conditions. This demonstrates that RNA in both internal DES LNPs as well as externally DES treated LNPs is protected from degradation compared with the control.

[0447] Example 10: Dynamic Light Scattering (PLS)

[0448] RNA-LNP particle size and polydispersity was measured using Dynamic Light Scattering (DLS).

[0449] To 200 pL of 10 mM Tris buffered saline (TBS pH 8.5 / 0.7 % NaCI) in a plastic disposable microcuvette (Cat. No. ZEN0040, Brand, Germany) was added 4 pL of DES / LNP mix and gently pipetted up and down three times to mix. The sample size and PDI was measured in triplicate by DLS at 25 °C using the standard water settings (ZetaSizer ZS, Malvern Panalytical, UK).

[0450] Hydrodynamic diameter was measured in back scatter mode at an angle of 173°.

[0451] Example 11: Riboqreen RNA Encapsulation Efficiency

[0452] RNA concentration and encapsulation efficiency were measured using an Invitrogen™ Quant- iT™ RiboGreen RNA assay kit (from ThermoFisher). The assay was performed according to the manufacturer's protocol in a black 96-well plate, using a calibration curve with and without Triton X-100 (an LNP-rupturing reagent). The plate was analysed using a Spark® plate reader (from Tecan), using an excitation of 485 nm and an emission of 535 nm. The background fluorescence was subtracted from each sample. The encapsulation efficiency was determined by comparing the signal of the RNA-binding fluorescent dye RiboGreen in the absence (free mRNA) and presence (total mRNA) of Triton.

[0453] Example 12: Preparing Internal DES LNPs

[0454] It has been surprisingly found that it is possible to incorporate large amounts of DES within the RNA and lipid filled internal structure of LNPs whilst maintaining the LNP size, PDI, EE%, and RNA purity as well as potency.

[0455] The inventor's observations suggest that there is no significant reduction in the cationic lipid / anionic RNA charge interaction as well as no significant negative effect on lipid selfassembly, or at macro level no impact on the size and structure of the LNP. It is fundamental for LNP formation that during rapid mixing the RNA is incorporated into the LNP and serves as a molecular scaffold around which the lipid components congregate.

[0456] At least five DES formulations (choline chloride:urea (1:2 mokmol), choline chloride:xylitol (1:1 mokmol), choline chloride:sorbitol (1:1 mokmol), choline chloride:arabitol (1:1 mokmol), betaine:xylitol:water (1:1:1 mokmol) and proline:sorbitol:water (1:1:18 mokmokmol) have been shown to be incorporated into LNPs at a concentration of at least 50 % DES volume in the RNA phase (i.e. before mixing with lipids and formation of the LNPs). The standard temperature for encapsulation is 18 to 20 °C, however it was found that higher temperatures such as 42 or 50 °C were advantageous to reduce DES viscosity and increase incorporation into the LNP when the DES was choline chloride:urea and was present at a concentration of at least 60 % by volume.

[0457] It is advantageous to have DES present inside the LNP in order to replace the water within the LNP that can be associated with RNA hydrolysis with a consequent reduction of RNA purity and potency.

[0458] An internal DES may have further benefits in addition to reducing the rate of RNA hydrolysis. For example, the DES may also protect lipids from oxidative damage, reduce cross-linking of RNA with the cationic lipid (Packer M, et al., Nat Commun. (2021). 12(1):6777), maintain the correct structure of Lipids and mRNA within and / or on the surface of the LNP, maintain LNP surface charge, and / or provide other protection to the LNP thereby maintaining potency.

[0459] The organic phase contained the lipids ALC-0315 : DSPC : Cholesterol : ALC-0159 (CATUG, China) at a molar ratio of 46.5 : 9.4 : 42.7 : 1.6, respectively, dissolved in ethanol to a total lipid concentration of 16 mg / ml.

[0460] Alternatively, the organic phase contained: SM-102 (heptadecan-9-yl 8-((2-hydroxyethyl) (6- oxo-6-(undecyloxy) hexyl) amino) octanoate, PEG2000-DMG (1- monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000), DSPC (l,2-Distearoyl-sn-glycero-3 phosphocholine) and Cholesterol in a 50 : 10 : 38.5 : 1.5 % molar ratio, dissolved in ethanol.

[0461] The aqueous phase contained a DES in the range of 10 to 95 % by volume, as well as the 5' Capped Nl-Me-Pseudouridine Fluc-mRNA (Cat. No. CT-072 CATUG, China) to a final concentration of 104 pg / mL RNA in an encapsulation buffer selected from the list below.

[0462] To facilitate the self-assembly of RNA and lipids in the presence of DES, the organic phase containing the lipids and aqueous phase containing the DES and RNA were combined using a microfluidic mixer system (NanoAssemblr® lgnite+™ system from Precision NanoSystems Inc. PNI) and standard NxGen™ cartridges, to form RNA-lipid nanoparticles (RNA-LNPs). The NanoAssemblr® lgnite+™ equipment was operated in accordance with the manufacturer's instructions. Further details are set out above in Example 2.

[0463] There are no particular limitations on the workflow for encapsulating RNA, DES and lipids into LNPs. For example, other rapid mixing methods may be used. Examples of rapid mixing methods include the thin-lipid-film evaporation method and the T-junction method, as well as microcapillary mixing as set out in Example 2.

[0464] It was surprisingly found that DES can be mixed directly with the buffered RNA (e.g. Flue mRNA) in the aqueous phase (aqueous encapsulation mixture) prior to mixing with the ethanol phase containing the lipids. It was also found that, depending on the desired concentration of the internal DES, that one or more of the following parameters during LNP formulation with DES was beneficial to obtain LNPs with, preferably; at least 50 % internal DES concentration, an EE% of the RNA of at least 50 %, LNPs with sizes less than 400 nm, preferably less than 300 nm and a PDI of 0.3 or preferably less than 0.2.

[0465] The LNP formulation may be adjusted based on the nature and concentration of the DES. Parameters that may be adjusted include:

[0466] (i) the nature of the aqueous encapsulation medium. The medium may be water or a buffer solution. The buffer may be selected from e.g. Tris-HCI, PIPES, MES, HEPES, MOPSO, CAPS, CAPSO, BICINE, BIPES, Citrate, Phosphate, Phosphate Buffered Saline, Imidazole, BAPTA, Glycine, or Acetate.

[0467] (ii) The concentration of the encapsulation buffer, which may for be example be in the range of 0 to 100 mM, optionally 1 to lOmM.

[0468] (iii) The pH of the encapsulation medium, which may be in the range of pH 1 to 9, optionally pH 4 to 6. The pH is typically adjusted before mixing the medium with the RNA or the DES.

[0469] (iv) The temperature at which the aqueous and ethanol phases were combined. For example, the temperature may be in the range 18 to 75 °C, optionally 25 to 55 °C, and further 25 to 42 °C). After preparing the LNPs, the LNPs may be separated from the ethanol used to dissolve the lipids, as well as any excess deep eutectic solvent by dialysis. It was found the dialysis buffer ("dialyte") may affect the size, PDI and Encapsulation efficiency of the LNP.

[0470] The amount of dialysis buffer used is typically at least 1000 times the volume of the LNP sample.

[0471] Dialysis buffers may be selected from: Tris-HCI, Tris-saline, Tris-10 % sucrose, PIPES, MES, HEPES, HEPES Buffered Saline, MOPS, MOPSO, CAPS, CAPSO, BICINE, BIPES, citrate, phosphate, Phosphate Buffered Saline (PBS), imidazole, BAPTA, glycine, acetate, and water.

[0472] The concentration of the dialysis buffer may be in the range 0.1 to 100 mM, optionally 1 to 10 mM.

[0473] The pH of the dialysis buffer may be in the range pH 1 to 8, optionally pH 3 to 7.

[0474] The dialysis may be at a temperature in the range of 4 to 37 °C, optionally 4 to 18°C.

[0475] Two or more dialysis steps may be performed.

[0476] As one example of a 2-step dialysis, an initial dialysis of 1 volume LNP with at least 500 volumes of 1 mM sodium acetate pH 5.5 for 4 hours at 4 °C was performed, and then the buffer was exchanged with 1 mM Tris-HCI pH 7 and dialysed for a further 16 hours at 4 °C before the DES LNPs were recovered and measured or stored at 4 °C.

[0477] Sucrose or other cryoprotectant can be added to the dialysis buffer if LNP freezing is necessary.

[0478] It was found that the % volume and type of DES that in the encapsulated buffer may vary the properties of the LNP, such as diameter, PDI, Encapsulation Efficiency (EE%), RNA stability ('RNA Purity'), LNP particle stability, in vitro cell potency, and animal organ (bio)distribution and potency in vivo. There are no particular limitations with respect to the formulation of the DES which is added to the aqueous phase. Process parameters may be optimized for the chosen DES. Examples of process parameters include the concentration of the DES; the encapsulation buffer type, concentration and pH; the temperature of mixing with the organic phase; and the method for post-formulation removal of ethanol (e.g., dialysis, ultracentrifugation with a molecular weight cutoff membrane in the range of 50 to 300 kD, or TFF).

[0479] In particular, it has been found that DESs comprising:

[0480] (a) a hydrogen bond acceptor selected from choline chloride, betaine and proline; and

[0481] (b) a hydrogen bond donor selected from urea, xylitol, arabitol and sorbitol are compatible with encapsulation into LNPs.

[0482] Example 13: Preparing Internal LNPs with DES-1 (Choline chloride:Urea (1:2 mol:mol))

[0483] Choline chloride:Urea (1:2 mokmol) (DES-1) was encapsulated into LNPs as set out in Example 12 at 20 °C using a final citrate buffer concentration of 10 mM, pH 4, and a 2-step 4 °C dialysis using citrate buffer 10 mM, pH 4 as the first buffer (4 hours) and Tris-HCI saline pH 7.0 as the secondary dialysis buffer (16 hours) which also served as the LNP storage buffer. It was found that these conditions provided an %EE of 98 %.

[0484] We prepared a 50:50 volume:volume mixture of pure DES-1 and 20 mM citrate buffer pH 4. After mixing, this resulted in a final 10 mM citrate buffer concentration in the aqueous phase and the pH of this mixture was adjusted to pH 4 using HCI. RNA (2 mg / mL) was then added to the pre-mixed DES-buffer pH 4 solution to achieve a final RNA concentration of 104 pg / mL. The DES-buffer aqueous phase was then used for microfluidic mixing (NanoAssemblr® lgnite+™) with the organic phase (N / P = 12).

[0485] Using instead a 10 mM citrate encapsulation buffer at pH 6 and a dialysis buffer of 10 mM Tris-HCI at pH 7.4 provided an EE% of 37 %. In order to increase the DES content in the LNP formulation to a total of 75 % volume, the ethanol / lipid and aqueous DES / mRNA phases were combined at 42 °C instead of 20 °C to reduce DES viscosity. As shown in the table below, the effect of 75 % compared with 50 % DES volume was a reduction in the EE% as well as an increase in the LNP diameter. It is expected that such 75 % DES filled LNPs will have suitable potency and potentially increased stability during storage, however for certain use cases the large LNP diameter may not be desirable. In further testing, 50 % DES filled LNPs were found to have very good stability.

[0486] The above table demonstrates the influence of the pH of the citrate encapsulation buffer as well as the post-formulation 2-step dialysis steps on the EE%, particle size, and PDL A pH 4 encapsulation buffer and 2-step dialysis are preferred for formulating DES-1 into LNPs.

[0487] Example 14: Preparing Internal DES-2 (Choline chloride:Xylitol (1:1 mol:mol) and DES-4

[0488] (Choline chloride:Arabitol (1:1 mol:mol) LNPs

[0489] Using the method as set out in Example 13 but with the DES choline chloride:xylitol (1:1 mokmol), it was found that a 1 mM was superior to 10 mM Citrate encapsulation buffer. The EE% improved whilst the LNP diameter reduced significantly. The dialysis buffer type also had a strong effect on EE%, increasing from 12 % to 66 % and the LNP diameter reducing from 521 nm to 405 nm when the dialysis buffer is changed from 10 mM Tris-HCL pH 7.4 to a 2-step dialysis using 10 mM Citrate at pH 4 followed by 10 mM Tris-Saline.

[0490] Example 15: Preparing Internal LNPs with Internal Betaine:Xylitol:Water (1:1:1 mol:mol:mol)

[0491] (DESS)

[0492] The effect of heating on LNP size during encapsulation was tested. It was found that the preferred smaller LNPs were prepared when 75 % betaine:xylitol:water (1:1:1 mol:mol:mol) was heated at 42 °C, but preferably 50 °C compared with 20 °C during encapsulation. Excellent EE% of 98 % and 96 % and LNP diameter size of 92 and 115 nm was observed with 50 % and 60 % betaine:xylitol:water (1:1:1 mol:mol:mol).

[0493] The above table demonstrates that it is possible to prepare LNPs with up to 75 % volume DES-

[0494] 5 in the encapsulation buffer with very good EE%, size and PDL Example 16: Preparing Internal LNPs with Proline:Sorbitol:Water

[0495] Using the method described in Example 12, RNA-LNPs may be formulated with up to 75 % volume internal proline:sorbitol:water (2:1:6 mol:mol:mol). The table below illustrates effects of the encapsulation buffer pH on the encapsulation efficiency (EE%) and the LNP diameter.

[0496] It was found that pH of 5.8 in water resulted in LNPs with higher EE and reduced size compared to pH 4 when using 75 % proline:sorbitol:water. Additionally, with 50 % proline:sorbitol:water, a 1 mM citrate buffer at pH 6 was compatible with LNPs, resulting in a very high EE% of 97 % and a small diameter of 126 nm.

[0497] Table 7 demonstrates that both 50 % and 75 % volume compositions with the hydrogen bond acceptor Proline and the hydrogen bond donor Sorbitol are compatible with the formation of RNA LNPs, achieving the desired physical properties of high RNA content (EE%) and small size.

[0498] Example 17: Preparing Internal LNPs with Various N / P Ratios

[0499] The N / P (nitrogen / phosphate) ratio of LNPs formulated with internal betaine:xylitol:water (1:1:1 mol:mol:mol) (DES-5) was varied.

[0500] The N / P ratio provides a measure of the amount of RNA (which contains P) relative to the amount of cationic lipid (which contains N). For example, the commercially available FDA- approved Pfizer-BioNTech mRNA vaccine BNT162b2 / Comirnaty has an N / P ratio of 6.

[0501] It was found that when mRNA Flue was combined with 75 % DES-5 and then mixed with lipids including the cationic lipid ALC-0315, increasing the N / P ratio from 6 to 18 improved the EE%, and modified the size and PDI of the LNPs. Therefore, adjusting the N / P ratio is useful modifying the characteristics of LNPs when formulated with a DES.

[0502] Example 18: Preparing Internal DES-5 LNPs with Various Aqueous Encapsulation Buffers The encapsulation buffer type and pH may vary the amount of positive charge on the cationic lipid, thereby influencing binding with the negatively charged phosphate of the RNA. Many DES have an intrinsic buffering effect. It was surprisingly found that even very low concentrations of Citrate buffer such as 1 mM led to increased LNP diameters compared with non-buffered Water. Whilst buffering the RNA with Citrate prior to mixing with Lipids is considered standard practice for LNP formulation, it was found that usefully, internal DES LNPs formulated with mRNA mixed with 50 % betaine:xylitol:H2O (1:1:1 mol:mol:mol) (DES-

[0503] 5) in water pH 5.5 rather than ImM citrate buffer had favourable EE (98 %), particle size (92 nm) and PDI (0.205) following purification by dialysis. These results indicate that LNP metrics EE%, size and PDI may be improved by careful choice of the encapsulation buffer. Example 19: Preparing Internal DES LNPs with Various Types of DES Various DESs were mixed with the aqueous mRNA phase and then incorporated internally into LNPs by addition to the ethanol dissolved lipid phase (see Example 12 for lipid components and use of NanoAssemblr® lgnite+™ for mixing).

[0504] As set out in the table above, it was found that DES-1 to 6 could be all incorporated into LNPs as internal components.

[0505] Heating when combining the aqueous and ethanolic phases was found to be helpful for increasing the amount of DES incorporated into the LNPs.

[0506] For example, DES-4 could be reliably incorporated at percent volumes greater than 60 % volume in the aqueous phase when the aqueous and ethanolic phases were mixed at 42 °C and a 2-step dialysis with 10 mM citrate pH 4 followed by pH 7.4, 10 mM Tris 0.9 % saline buffer.

[0507] LNPs incorporating 60 % DES-2 by volume were obtained by heating at 42 °C.

[0508] DES-5 and DES-6 was found to be notable for smaller particle size whilst, following dialysis, encapsulating high amounts of mRNA (EE%) of 91 and 97 % respectively.

[0509] It was advantageously found that for all DES, amounts of 50 % volume or more could be included in the aqueous phase containing the mRNA. This demonstrated that at least three Hydrogen Bond Acceptors (choline chloride, betaine and proline), as well as four Hydrogen Bond Donors (urea, xylitol, sorbitol and arabitol) can be formulated into DES which are compatible with inclusion in mRNA-LNPs. Example 20: Preparing Internal LNPs at Various Mixing Temperatures

[0510] Some DESs are significantly more viscous than water and may have viscosities of several hundred or even thousands of Centipoises. High viscosity may make mixing the DES with the ethanolic lipid phase inefficient. Inefficient mixing may lead to increased particle size, for example.

[0511] Increasing the temperature and / or amount ofwater or ethanol present in the DES may reduce viscosity by orders of magnitude. By way of illustration, the viscosity of betaine:trifluoroacetamide (1:2 mokmol) is halved by the addition of 5 % water or ethanol, and reduced by a factor of 250 when the temperature is increased from 0 to 37 °C.

[0512] It was advantageously found that when mRNA is mixed with 75 % volume betaine:xylitol:water (1:1:1 mol:mol:mol) in the aqueous phase and then combined with the lipid ethanolic phase in the NanoAssemblr® lgnite+™ set at 42 °C or 50 °C produced LNPs with improved metrics compared to those prepared at 22°C. EE% increased with temperature from 59 % to 82 % to 89 %, the size decreased from 215 nm to 154 nm to 140 nm, and the PDI decreased from 0.368 to 0.368 to 0.175 following dialysis with increasing mixing temperature. Increasing the mixing temperature during mixing is therefore a straightforward way to improve the metrics of LNPs containing formulated with high concentrations of DES. Example 21: Dialysing Internal LNPs with Various Buffers

[0513] Examples of dialysis buffers that can be used with a dialyzing device (e.g., Slide A lyser™ or Float A Lyser™ (Sigma-Aldrich, UK)) or a dialyzing membrane (e.g., having a MWCO in the range of 3,000 to 500,000 Daltons) to remove ethanol from a freshly prepared LNP include: sodium citrate buffer (at e.g. pH 7), sodium acetate buffer (at e.g. pH 7), PBS (at e.g. pH 7.4), Tris Buffer (at e.g. pH 4.5-8.4),

[0514] PIPES Buffer (at e.g. pH 4.5-8.4), HEPES Buffer (at e.g. pH 4.5-8.4), MOPS Buffer (at e.g. pH 5.5-8.4), BICI NE Buffer (at e.g. pH 4.5-8.4) or MES Buffer (at e.g. pH 4.5-8.4).

[0515] The buffer may be at a concentration of 10 mM. The buffer may optionally include NaCI in an amount of 0.1-1.0 % (w / v) (e.g., about 0.9 %); and / or sucrose, e.g. in an amount of 10 % (w / v). The use of 10 % sucrose may protect the LNP during freezing and thawing.

[0516] The buffer may have a pH range in the range pH 6.5-7.5.

[0517] A 2-step dialysis can be carried out. For example the sample can be dialysed against 10 mM citrate (pH 7.4) as a first step for 4 hours followed by Tris buffered saline (pH 7.4) for 18 hours.

[0518] Example 22: Preparation of Internal DES LNPs in an External DES Environment.

[0519] The effect of adding external DES-1, DES-2 and DES-4 to the corresponding internal DES-1, DES-2 and DES-4 LNPs was determined.

[0520] The combination of internal and external DES-1 maintained a very high EE% of 100 %. The internal / external DES-2 combination and the internal / external DES-4 combination lost RNA from the LNP and had EE% of 42 and 34 % respectively.

[0521] Example 23: EE% Stability of Various DES Formulations

[0522] The effect on LNP integrity of high shear forces associated with vortexing at 2200 rpm for 5 minutes was studied using a control LNP with a formulation as described in Example 2, compared with four different DES LNP formulations:

[0523] (1) Internal DES-1 (choline chloride:urea, 1:2 mokmol, 50 % by volume)

[0524] (2) Internal DES-5 (75 % betaine:xylitol:water, 1:1:1 mol:mol:mol)

[0525] (3) External DES-3 (95 % choline chloride:sorbitol, 1:1 mokmol); and (4) External DES-4 (95 % choline chloride:arabitol, 1:1 mokmol).

[0526] All LNPs had identical lipid formulations and mRNA as described in Example 2.

[0527] The encapsulation efficiency (EE%) was measured at both time point zero and after vortexing for 5 minutes. The results are shown in Fig. 12.

[0528] It was found that the EE% at time point zero was slightly higher for Internal DES-1, but significantly higher after vortexing compared with control (EE% of 16 % vs 7 % respectively).

[0529] Both External DES-3 and External DES-4 were significantly higher EE% than the control (EE% of 26 %, 22 % and 7 % respectively)

[0530] Internal DES-5 was slightly higher than the control.

[0531] The results demonstrate that all of the DES LNP formulations were more stable, retaining a greater proportion of their encapsulated RNA within the LNP than the control. External DES LNPs were more stable during vortexing than internal DES LNPs.

[0532] Example 24: Potency Assay using Internal DES mRNA-LNPs stored for 2 months at 4 °C mRNA-LNPs were formulated with three types of internal DES: choline chloride:urea (1:2 mokmol) (CCU), choline chloride:D+arabitol (1:1 mokmol) (CCA), and betaine:xylitol:water (1:1:1 mokmokmol) (BX).

[0533] The LNPs were stored for 2 months at 4 °C and transported for 24 hours on ice before diluting 215 ng of each formulation into 200 pL DMEM / 10 % FCS. Then, 20 pL of the diluted LNP was added to 100 pL cells in a 96-well plate and a Luciferase assay carried out as set out in Example 6. The positive control (POS) represents the same amount (215 ng) of mRNA-LNP. Fig. 13 shows the results of the luciferase assay. Choline chloride:Urea and Betaine:Xylitol:Water led to a stronger luciferase signal than the positive control (POS), even after 2 months storage at 4 °C.

[0534] Example 25: Stabilising Liposomes Using an External DES

[0535] 10 pL of (66 mg / ml) ARIKAYCE™ liposomal nebulizer dispersion (Insmed UK) was added into 90 pL choline chloride:sorbitol (1:1 mokmol), stirred and incubated for 1 hour at 22 °C before dilution in 1 mL water. Subsequently, 40 pL of the diluted Liposome mixture was added to a DLS cuvette and the size determined.

[0536] It was found that there were no significant changes in liposome size compared with the non- DES treated control sample. Therefore, despite the liquid filled core of the liposome and the high osmotic pressure of the DES, the DES treatment does not reduce the nanoparticle the volume of liposomes. Liposomes are therefore compatible with DES treatment and stabilization.

[0537] Example 25A: Stabilising Liposomes Using an Internal DES

[0538] Liposomes may be formulated with internal DES.

[0539] Step 1: 0.3 pL of Lipofectamine™ MessengerMAX™ Transfection Reagent (ThermoFisher UK Cat. No. LMRNA001) was added to 5 pL of Opti-MEM™ (Cat. No. 31985062, ThermoFisher UK) containing up to 50 % volume of a DES, mixed gently and then incubated for 10 minutes at room-temperature.

[0540] Step 2: The mixture was then added to 5 pL of Opti-MEM™ containing premixed 100 ng Flue mRNA (Cat. No. CT-072, CATUG China), and incubated for 5 minutes at room-temperature. Then, 10 pL of the mixture was added to a single well containing tissue culture cells (such as HeLa cells) and 100 pL DMEM and 10 % FCS in a 96-well plate, incubated at 37 °C in 5 % CO2 for 24 hours. Luciferase activity was then measured in accordance with Example 6. Alternatively, instead of the DES being added during step 1, the DES may instead be added to the Opti-MEM™ in Step 2, in an amount of up to 50 % by volume.

[0541] It was demonstrated that choline chloride:urea (1:2 mokmol), betaine:xylitol (1:1 mokmol) or choline chloride:sorbitol (1:1 mokmol) can be added at up to 50 % volume in Opti-MEM™ during either step 1 (before addition to Lipofectamine™), or alternatively, during step 2 (to the Opti-MEM™ before or after addition of the mRNA but before addition to the Lipofectamine™).

[0542] Addition of DES to the individual components of the Liposomes provides a convenient means to encapsulate the mRNA and / or other payload including antibiotics, along with the DES inside the Liposome providing increased particle and mRNA stability during storage.

[0543] 10 pL volume of one or more of the following vehicles: Nanostructured Lipid carrier, Solid Lipid Nanoparticles, Polymeric nanoparticles including dendrimers, polymeric micelles, polymersome, cubosomes, nanospheres, inorganic nanoparticles including silica nanoparticles, carbon nanotubes and metallic nanoparticles, charge altering release transporters (CARTs) and cationic peptides, whole cells, micelles, liposomes or adeno- associated viruses (AAV) particles were added individually to 90 pL of a DES such as choline chloride:sorbitol (1:1 mokmol) or betaine:xylitol (1:1 mokmol), mixed and stored for 1 month at 22 °C prior to analysis.

[0544] It was found that these particles formed a homogenous solution in the DES, and particle integrity was maintained during the storage period.

[0545] In order to determine in vivo potency, 1 pg of each Flue mRNA-LNP formulation in 10 pL total volume was injected into each rear leg (i.m.) of female B6 / Rj -Tyr c / c albino mice. 24 hours later Luciferin was injected, and after 8 minutes, detection and quantification of whole animal bioluminescence was carried out. For mice 3-4, organs were dissected, and individual bioluminescence quantified.

[0546] The compositions delivered to each mouse are set out in the table below.

[0547] Fig. 14 shows semi-quantitative imaging of luciferase activity 24 hours after 6 mice were injected in each hind leg i.m. with one of six different Fluc-LNP formulations. Areas with the highest luciferase activity are shown in red, and those with the lowest activity are shown in blue. Prominent tissues with Luciferase activity were the leg muscle, liver, ganglions, and spleen.

[0548] Fig. 15 is a quantitative representation of the luciferase activity in the hind legs of the six mice. It was observed that Mouse 4, injected with a control non-DES mRNA-LNP formulation stored for 14 weeks at 4 °C had Luciferase activity that was substantially lower than either Mouse 2 or Mouse 5. Mouse 3 and Mouse 6 demonstrate that mRNA-LNPs transiently treated with choline chloride:sorbitol (1:1 mokmol) choline ch loride :xylitol (1:1 mokmol) respectively are active in vivo. Fig. 16 shows semi-quantitative imaging of Luciferase activity in nine organs of Mouse 4, Mouse 5, and Mouse 6, 24 hours post-injection of the mRNA-LNPs. 1. Intestine, 2. Stomach, 3. Liver, 4. Hind leg muscle, 5. Kidneys, 6. Spleen (indicated by arrow) + Pancreas 7. Lungs, and 8. Heart.

[0549] The internal DES LNPs and the two external DES treated LNPs are compatible with in vivo mRNA-LNP potency with equal or greater Luciferase activity than the controls.

[0550] None of the intramuscularly injected DES-LNP formulations led to observable inflammation, muscle atrophy or toxicity. The treated mice appeared fully healthy and showed normal behaviour. This is especially surprising for Mouse 2: intramuscular injection of urea alone would be expected to cause inflammation.

[0551] There was greater potency for the internal choline chloride:urea (14 week storage) and betaine:xylitol (22 week storage) internal DES LNPs (Mice 2 and 5) compared with the control mRNA-LNP (14 week storage) (Mouse 4) demonstrating the stabilizing effect of encapsulating DES into the LNP.

[0552] It was observed that luciferase activity in the liver was much lower in Mouse 6 than control Mouse 4. The effect of the DES-treated LNP was better localised to the injection site.

[0553] It was also noted that Luciferase activity in the spleen was significantly higher in the mouse treated with internal DES-5 mRNA-LNPs (Mouse 5) compared to the mouse treated with the control LNP (Mouse 4). Such changes to the biodistribution of mRNA-Luciferase are useful for delivering mRNA to specific targets such as a viral antigen to ganglions and the spleen. The liver is known to remove mRNA-LNPs from the blood thereby reducing activity in potentially target organs. Low LNP uptake by the liver and high LNP uptake by the spleen may be beneficial for vaccine or RNA therapeutic applications. Example 28: Stability of DES Formulated mRNA-LNPs During 3-month Storage at 20 °C or

[0554] 37 °C

[0555] The particle size, PDI, EE% and %Purity was determined for six different DES-LNP formulations at Time point zero, 2 weeks, 1 month and 3 months at either 22°C or 37°C. The DES formulations tested for their stabilisation effect were:

[0556] 95 % External DES-1 (choline chloride:urea (1:2 mokmol));

[0557] 95 % External DES-2 (choline chloride :xylitol (1:1 mokmol));

[0558] 95 % External DES-3 (choline chloride:sorbitol (1:1 mokmol)); 50 % volume Internal DES-1 (choline chloride:urea (1:2 mokmol));

[0559] 60 % volume Internal DES-2 (choline ch loride :xylitol (1:1 mokmol)); and

[0560] 50 % volume Internal DES-3 (choline chloride:sorbitol (1:1 mokmol)).

[0561] The table below sets out the 6 DES formulations and control LNPs. The internal DES formulations were as described in Example 12. The 2-step dialysis method used for Internal DES 1, 2 and 3 is set out in Example 13. Fig. 17 shows the LNP particle size and PDI stability at 22 °C for up to 3 months. The columns represent particle diameter (nm) and the line shows the PDI. Fig. 18 shows the encapsulation efficiency (columns) and mRNA %Purity (lines) over the same time period.

[0562] The 3-month stability results at 22 °C demonstrate that except for External DES-1 and Internal 60 % DES-2, all of the LNP sizes were stable over time.

[0563] External DES-2, External DES-3 and 50 % Internal DES-1 retained smaller LNP diameters. PDI was stable over time.

[0564] It is notable that the PDI for the control LNPs increased over time suggesting that, despite the apparently average stable particle diameters, the particles as a population in the control were unstable.

[0565] Both External DES-2 and External DES-3 retained an excellent %Purity of over 70 % after 3 months storage at 22 °C, compared with 45 % for the control. This demonstrates that RNA was particularly well stabilised in External DES-2 and External DES-3, with both Internal DES- 1 and Internal DES-3 also demonstrating good RNA stabilisation compared with control.

[0566] Taken together, the size, PDI, EE% and %Purity results demonstrate that External DES-2 and External DES-3, as well as Internal DES-1 and Internal DES-3 were particularly stable compared with control LNPs.

[0567] Further analysis of the robust RNA stabilisation properties of External DES-2 and DES-3 was carried out compared with control LNPs. Results for the two DES LNP formulations External DES-2 and External DES-3 compared with control LNP particle size and PDI stability at 37 °C for up to 3 months. The results are shown in Fig. 19, in which the columns represent particle diameter (nm) and the line represents PDI.

[0568] The PDI was similar for all samples, including the control. The External DES-2 and External DES-3 particle diameters were larger than the control LNPs, however, the %Purity for the External DES LNPs was significantly higher (%Purity approximately 50 %) compared with control LNPs (%Pu rity 0 %) demonstrating much better RNA stabilisation in the presence of External DES.

[0569] EE% was at least 50 % for all samples although the control LNPs were over 90 % despite the RNA being completely degraded. This demonstrates that, for the control, even though the particle size, PDI and EE% were all good the most important metric - RNA stability - was very poor. It would be expected that the control LNPs stored for 3 months at 37°C would have no potency at all in vitro.

[0570] Example 29: Comparison of mRNA-LNP Potency with and without Internal DES-1 During Storage at Either 4 °C and 22 °C

[0571] To demonstrate the stability of Internal DES-1 LNP formulation, HeLa cells were transfected in duplicate with either 30 ng Control LNPs or 50 % Internal DES-1 LNPs formulated using the method and lipids as set out in Examples 12 and 28.

[0572] Fig. 20 shows a comparison of Luciferase potency of Control LNPs compared with Internal DES-1 LNP following either: (i) 4 months at 4°C or (ii) 3 months at 22°C followed by 1 month at 4°C.

[0573] As shown in the graph, potency of the Internal DES-1 LNPs were substantially greater than Control LNPs following storage. This demonstrates that Internal DES-1 is effective at maintaining the potency of the LNP during storage.

[0574] As observed in Example 28, after 3 months at 22 °C, the %Purity of the Control LNP is approximately 40 % whilst that of Internal DES-1 LNPs is approximately 60 %. However, the results in Fig. 20 show that there is 22-fold more potency for Internal DES-1 LNPs than control.

[0575] The difference between the relatively small comparative difference in %Purity (40 and 60 %) and the very large comparative difference in Potency (22-fold) may be explained by a protective effect of DES-1 on the Lipids, the LNP structure, or the degree of cross-linking between the Cationic lipid ALC-0315 and the Flue mRNA.

[0576] As described above, lipid-RNA cross-linking is not detectable by the Capillary Electrophoresis tests that were carried out here to determine %Purity but can be detected by either (i) reversed-phase ion pair high performance liquid chromatography (RP-IP HPLC) or alternatively, (ii) using protein translation assays such as in vitro cell Luciferase assays or cell- free in vitro translation assays. Cross-linked mRNA is incapable of serving as a template for protein translation as described by Packer M, et al., Nat Commun. (2021). 12(1):6777).

[0577] Without wishing to be bound by theory, the large differences in potency observed between Control LNPs and Internal DES-1 LNPs may be at least in part due to reduced cross-linking of the mRNA thereby increasing Luciferase activity.

[0578] In a polypropylene 1.5 mL tube containing 95 pL choline chloride:urea (1:2 mokmol), choline chloride:xylitol (1:1 mokmol), choline chloride:sorbitol (1:1 mokmol) or PBS was added 5 pl (0.3 mg / ml) Flue mRNA-LNP, CATUG (China) Cat. CT072-LNP) and mixed gently with a pipette tip 10 times in a figure-8 movement. The RNA was either extracted using Rneasy Plus™ (Qiagen, Germany) immediately (Time point zero), or after 7 months storage at 25°C, 30 ng each RNA heated for 70°C for 3 minutes and analysed in a RNA Nanochip (Agilent Bioanalyser 2100™). The results of the capillary electrophoresis are shown in Fig. 22, and show that although the RNA is of similar quality at time point zero, the RNA-LNPs stored in PBS was completed degraded after 7 month storage at 25°C whereas the RNA stored in DES samples remained of high quality.

[0579] 0.5 pL samples of mRNA-LNP (0.3 mg / ml) Flue mRNA, Pfizer Lipid formulation, (Cat. No.

[0580] CT0072-LNP, CATUG, China) were added to 200 pL polypropylene PCR tubes. Then, 2 pL of an additive was added to each sample. The additives tested were: (1) DMEM tissue culture media including 10 % foetal bovine serum, (2) 100 % glycerol; (3) a 10 % w / v aqueous solution of dextran, (4) a 10% w / v aqueous solution of PVP (Kollidon CL™), (5) a 10 % w / v aqueous solution of P-cyclodextrin (Kleptose™), (6) a 10 % w / v aqueous solution of chitosan, (7) a 10 % w / v aqueous solution of glycerol stearate; and (8) a 10 % w / v aqueous solution of guar gum. A control sample with no additive was also prepared.

[0581] To each sample was then added 10 pL of choline chloride:sorbitol (1:1 mokmol), before mixing in a figure-8 movement using a plastic pipette tip and incubating the tube for 20 minutes at 20°C. Each sample was then diluted with 200 pL DMEM tissue culture media / 10 % bovine serum and gently mixed by tube inversion and pipetting. 20 pL portions of the diluted LNP was transfected onto 100 pL of tissue culture cells in a 96-well plate and a Luciferase assay carried out as set out in Example 6.

[0582] Results of the LNP pre-treatment are shown in the table below:

[0583] It was found that compared with the no additive control sample, both glycerol and P-cyclodextrin pre-treated LNPs increased mRNA-LNP potency substantially by more than five-fold when subsequently mixed with choline chloride:sorbitol (1:1 mokmol). Example 32: comparison of adding different amounts of dry powdered / 3-Cydodextrin to the LNP before adding DES

[0584] In 200 pL polypropylene PCR tubes was added either: (1) control - no addition, (2) 2 pL glycerol, (3) 1 mg P-CD, (4) 2 mg P-CD, (5) 5.6 mg P-CD, (6) 10 mg P-CD, or (7) 5 mg Dextran. Then, 0.5 pL of mRNA-LNP (0.3 mg / ml) Flue mRNA, Pfizer Lipid formulation, (Cat. No. CT0072- LNP, CATUG, China) was added on top of each powder, followed by the addition of 10 pL of choline chloride:sorbitol (1:1 mokmol) before incubating at 20°C for 10 minutes. A luciferase potency assay was then carried out as set out in Example 6.

[0585] It is possible to carry out a two-step process of (i) pre-mixing the cyclodextrin with the mRNA- LNP, followed by (ii) addition of the DES. The increase in LNP potency was found to be dependent on the amount of -CD added, as shown in the table below. Glycerol (sample (2)) had a positive effect on potency. -CD had an even stronger positive effect when added at amounts in the range 1 to 5.6 mg (samples 3 to 5). 2 mg amount of P-CD (sample 4) provided a 43x increase in potency compared to the control (sample 1). The 5 mg sample of the linear polysaccharide dextran (sample (7)) had no positive effect on potency.

[0586] It was found that 2mg P-CD dry powder when added to 0.5 pL LNP followed by the addition of 10 pL of choline chloride:sorbitol (1:1 mokmol) led to the greatest increase in potency. Increasing the amount of P-CD to 6 mg reduced the magnitude of the increase. The linear polysaccharide dextrin, unlike CDs, had no positive effect on potency demonstrating the unusual properties of CDs compared with other polysaccharides. In this example it was found that adding 0-CD to the LNP followed by DES required greater w / w amounts (20 %) of 0-CD than when mixed into the DES prior to addition of the LNP (2.5 %) to obtain greatest potency.

[0587] Pre-mixing cyclodextrins into DES before the addition to the mRNA-LNP was found to be more convenient than adding dry cyclodextrin powder to the LNP followed by the addition of the DES requiring only a single mixing step. 650 mg of y-CD (2.5% (w / w) (Cat. No. C0869 TCI Belgium) was dissolved into 20 mL choline chloride:sorbitol (1:1 mokmol) by heating and sonicating the tube at 80 °C / 37 kHz in an ultrasonic water bath for 1 hour. To avoid cyclodextrin clumping and forming almost insoluble mixtures in the DES, it was added in portions of no more than 100 mg to 20 ml of DES before heating and ultrasonic mixing until the final amount of 2.5 % CD (w / w) was attained.

[0588] Once the cyclodextrin was completely dissolved in the DES, the mixture was found to be stable with no visible particle aggregation even after 3 months storage at 22 °C. In order to reduce viscosity, the stock DES-CD mixture was briefly warmed to 37 °C before pipetting and use. It was found that positive displacement pipettes (e.g., Microman E series (Gilson, France) were very helpful to handle the viscous mixtures.

[0589] Using a Microman E series pipette, 10 pL of 2.5% (w / w) y-CD / choline chloride:sorbitol (1:1 mokmol) was added to a 200 pL polypropylene PCR tube and 0.5 pL of mRNA-LNP (0.3 mg / ml) Flue mRNA, Pfizer Lipid formulation (Cat. No. CT0072-LNP, CATUG, China) was pipetted into the CD / DES and immediately mixed using a figure-8 movement of the pipette tip and incubated for 20 minutes at 20 °C before dilution by adding 200 pL of DMEM tissue culture media / 10 % bovine serum (ThermoFisher) followed by gentle mixing. 20 pL portions of the diluted LNP was then transfected onto 100 pL of tissue culture cells in a 96-well plate and a Luciferase assay carried out as set out in Example 6. It was found that the inclusion of y-CD in the DES improved both potency and stability of the mRNA-LNP compared with DES without y-CD.

[0590] Example 34: Variation of Potency with increasing amounts a-CD, / 3-CD or y-CD in choline chloride:sorbitol (1:1 mohmol) and subsequently stored at 50°C for 1H a-CD, 0-CD or y-CD powder (TCI, Belgium) were each dissolved separately in the range 0-20 % (w:w) in choline chloride:sorbitol (1:1 mohmol). Then 10 pL portions of each were added to a 200 pL polypropylene PCR tube and then 0.25 pL of mRNA-LNP (0.3 mg / ml) Flue mRNA, Pfizer Lipid formulation, (Cat. No. CT0072-LNP, CATUG, China) was added to each tube and mixed in a figure-8 movement, then incubated at 50 °C for 1 hour to test thermostability. A luciferase potency assay was then carried out as set out in Example 6. It was found that the addition of all three types of CD (a, p and y-CD) led to a significant increase in mRNA-LNP potency compared with choline chloride:sorbitol (1:1 mohmol) without CD even following heat treatment at 50°C for 1 hour. The optimum ranges of amounts of a, P and y-CD in DES varied. For a-cyclodextrin, amounts in the range 5 to 10 % improved potency. For p-cyclodextrin, amounts in the range 0.5 to 5 % were particularly effective, with about 2.5 % being optimal, y-cyclodextrin had the broadest range of effective amounts, which spanned 0.5 to 12.5 %, gave strong effects over the range of 1.25-5% (wt:wt), and a peak in efficacy at about 2.5 %. y-CD at 1.25% (w:w) leading to over 2.5-times the potency compared with the DES without added y-CD.

[0591] Example 35: LNP potency following storage in five different DES, mixed with either [3-CD, y- CD compared with controls

[0592] In order to determine the effect on LNP potency of mixing either 0-CD or y-CD with five different DES, the following experiment was carried out. To 100 pL DES was added 2.5 mg 0- CD or y-CD powder and dissolved by sonication at 37 kHz at 70 °C for 10 minutes. To the cooled mixture was added 2 pL of mRNA-LNP (0.3 mg / ml) Flue mRNA, Pfizer Lipid formulation, (Cat. No. CT0072-LNP, CATUG, China) and mixed using a pipette tip and a figure-8 movement before incubation for 45 minutes at 15 °C. A 5 pL portion was removed and added to 200 pL DMEM / 10%FCS in a 200 pL polypropylene PCR tube and then dissolved using a head-over- tails movement of the tube, 20 pL of the dissolved mixture was then added to 100 pL cells in tissue culture medium and a Luciferase assay carried out after 2 hours of transfection as described in Example 6.

[0593] Results are shown in the table below.

[0594] It was found that the potency of DES treated LNPs following short-term incubation was significantly increased when P-CD or y-CD was mixed with the sugar based DES; choline chloride:xylitol (1:1 mokmol), choline chloride:sorbitol (1:1 mokmol) and choline chloride:sorbitol (1:2 mokmol). Mixing P-CD or y-CD with betaine:xylitol (1:2 mokmol) resulted in lesser increases in potency.

[0595] Mixing P-CD ory-CD with choline chloride:urea had only a small effect on short-term potency, but provided a strong improvement in longer-term storage stability (please see Example 36, below).

[0596] For the sugar-based DESs, P-CD ory-CD both had similar effects on LNP potency increasing by up to 8-10-fold compared with DES with no CD. The DES choline chloride:sorbitol (1:1 mokmol) was considered to be the best of those tested in this example, due to its lower viscosity and larger increase in potency compared with choline chloride:sorbitol (1:2 mokmol).

[0597] Therefore, it was found that CDs may provide a strong increase in short-term potency when used with sugar-based DESs. Example 36: LNP potency following storage in aqueous buffer with and without / 3-CD, y-CD for five days at 4 °C.

[0598] In order to determine the effect on LNP potency of storing diluted in aqueous buffer five different DES with or without 0-CD or y-CD, the following experiment was carried out. To 100 pL DES was added 2.5 mg 0-CD or y-CD powder and dissolved by sonication at 37 kHz at 70 °C for 10 minutes. To the cooled mixture was added 2 pL of mRNA-LNP (0.3 mg / mL) Flue mRNA, Pfizer Lipid formulation, (Cat. No. CT0072-LNP, CATUG, China) and mixed using a pipette tip and a figure-8 movement before incubation for 45 minutes at 15 °C. A 5 pL portion was removed and added to 200 pL DMEM / 10%FCS in a 200 pL polypropylene PCR tube and then dissolved using a head-over-tails movement of the tube, followed by storage for five days at 4°C. 20 pL of the stored diluted mixture was then added to 100 pL cells in tissue culture medium and a Luciferase assay carried out after 14 hours of transfection as described in Example 6.

[0599] As set out in the Table above, it was found that the potency of the LNPs that had been stored for 5 days at 4°C in aqueous buffer varied depending upon the type of DES and the amount of P-CD or y-CD in the diluted mixture. It was found that LNPs diluted to 2.5 % in betaine:xylitol (1:2 mokmol) in DMEM / 10 % FCS all had very low potency whether CDs had been added or not. However, LNPs treated in one of the four DES types; choline chloride:xylitol (1:1 mokmol), choline chloride:urea (1:2 mokmol), choline chloride:sorbitol (1:1 mokmol) and choline chloride:sorbitol (1:2 mokmol) and then diluted to 2.5 % in DMEM / FCS and stored for five days at 4 °C demonstrated much better potency when either P-CD or y-CD was also in the diluted DES-LNP mixture at a final CD concentration of 0.0625% (w:v). Aqueous diluted y-CD improved potency when used with both choline chloride:xylitol (1:1 mokmol) and choline chloride:urea (1:2 mokmol), whilst -CD and y-CD had similar effects on potency with chloride:sorbitol (1:1 mokmol) and choline chloride:sorbitol (1:2 mokmol).

[0600] As one example, P-CD / choline chloride:urea (1:2 mokmol) mixtures when diluted into DMEM / FCS led to potency that was 1.4-times greater than without CD, whilst y-CD / choline chloride:urea (1:2 mokmol) mixtures when diluted into DMEM / FCS led to potency that was 1.9-times greater than DES without any CD. It is noteworthy the potency of the LNP treated choline chloride:urea (1:2 mokmol) mixtures were very similar at time point zero before storage. Therefore, it has been shown that CDs can have a positive effect on LNP storage stability even when the CD and DES have been diluted and subsequently stored in an aqueous buffer.

[0601] Example 37: variation of Potency with different types of CD in choline chloride:sorbitol (1:1 mohmol) stored at 60°C for 14H

[0602] To (1) 2.5 % (w:w) y-CD, (2) 2.5 % (w:w) hydroxypropyl-P-CD, (3) 2.5% (w:w) methyl-P-CD; (4) 2.5 % (w:w) di-O-methyl-P-CD, or (5) 2.5% (w:w) trimethyl-P-CD in choline chloride:sorbitol (1:1 mokmol), water or PBS was added 0.5 pL mRNA-LNP (0.3 mg / ml) Flue mRNA, Pfizer Lipid formulation (Cat. No. CT0072-LNP, CATUG, China), and mixed using a pipette tip and a figure- 8 movement before incubation for 14 hours at 60 °C to test LNP thermostability. A 5 pL portion was removed and added to 200 pL DMEM / 10%FCS in a 200 pL polypropylene PCR tube and then dissolved using a head-over-tails movement of the tube, 20 pL of the dissolved mixture was then added to 100 pL cells in tissue culture medium and a Luciferase assay carried out after 2 hours of transfection as described in Example 6. Control refers to the potency of 0.5 pL mRNA-LNP immediately thawed from frozen and added to tissue culture cells in the same proportion as the test samples.

[0603] It was observed that only 2.5 % (w:w) y-CD + choline chloride:sorbitol (1:1 mokmol) led to high mRNA-LNP potency after storage for 14 hours at 60°C. None of the four modified CDs (hydroxypropyl-p-CD, methyl-|3-CD, di-O-methyl-|3-CD or trimethyl-|3-CD) when mixed at 2.5% (w:w) with choline chloride:sorbitol (1:1 mokmol) maintained significant potency, and led to potency results equivalent to water or PBS storage. Even after 16 hour storage at 60°C, the y- CD 2.5% (w:w) choline chloride:sorbitol (1:1 mokmol) retained potency as determined by luciferase assay of 3.5x that of an equal amount of mRNA-LNP control that had not been stored at 60°C demonstrating the extraordinary protective effect associated with y-CD but not the side-chain modified CDs listed in the table. Example 38: variation of potency with different amounts of added water and potency following treatment of mRNA-LNPs for 30minutes at 80°C choline chloride:sorbitol (1:1 mohmol) with and without 2.5% (w:w) y-CD (FLUC 42) y-CD powder (TCI, Belgium) was dissolved at 2.5% (w:w) in choline chloride:sorbitol (1:1 mokmol).

[0604] To ten 200 pL polypropylene PCR tubes were added separately: (1) 10 pL of choline chloride:sorbitol (1:1 mokmol), (2) 10 pL of choline chloride:sorbitol (1:1 mokmol) plus 2 pL water, (3) 10 pL of choline chloride:sorbitol (1:1 mokmol) plus 6 pL water, (4) 10 pL of choline chloride:sorbitol (1:1 mokmol) plus y-CD powder at final concentration 2.5 % (w:w), (5) 10 pL of choline chloride:sorbitol (1:1 mokmol) plus y-CD powder at final concentration 2.5 % (w:w), plus 2 pL water, (6) 10 pL of choline chloride:sorbitol (1:1 mokmol) plus y-CD powder at final concentration 2.5 % (w:w), plus 6 pL water, (7) 10 pL of choline chloride:sorbitol (1:1 mokmol), (8) choline chloride:sorbitol (1:1 mokmol) plus y-CD powder at final concentration 2.5% (w:w), (9) 10 pL of choline chloride:sorbitol (1:1 mokmol) plus y-CD powder at final concentration 2.5 % (w:w) and (10) 10 pL water. 0.5 pL of mRNA-LNP (0.3 mg / ml) Flue mRNA, Pfizer Lipid formulation, (Cat. No. CT0072-LNP, CATUG, China) was added to each tube and mixed in a figure-8 movement. Samples 7, 8 and 9 were heated for 30 minutes at 80 °C to test LNP thermostability. The remaining tubes 1-6 were incubated for 30 minutes at 22 °C. Then, a luciferase potency assay was then carried out as set out in Example 6.

[0605] It was found that the addition of 2.5% (w:w) of y-CD in choline chloride:sorbitol (1:1 mokmol) improved the stability of mRNA-LNP in the presence of 20 % water (131-times more) or following heating for 30 minutes at 80°C (6.5-times more) compared with choline chloride:sorbitol (1:1 mokmol) without y-CD as measured by a Luciferase assay as set out in Example 6. Taken together these results demonstrate that the potency of LNPs in choline chloride:sorbitol (1:1 mokmol) is significantly better than water and that 2.5 % (w:w) y-CD in choline chloride:sorbitol (1:1 mokmol) is significantly better than choline chloride:sorbitol (1:1 mokmol).

[0606] Example 39: Prophetic Experimental Protocol for Preparing Internal LNPs with DES-1 (Choline chloride:Urea (1:2 mohmol) and Cyclodextrin.

[0607] To Choline chloride:Urea (1:2 mokmol) (DES-1) may be added either a-CD, 0-CD or y-CD in the range of 0.01-10 % (w:w), preferably 0.1-5 % and most preferably 0.5-3 % (w:w). The resulting mixture may be encapsulated into LNPs as set out in Example 12 and 13 at 20 °C using a final Citrate buffer concentration of 10 mM, pH 4, and a 2-step 4 °C dialysis using Citrate buffer 10 mM, pH 4 as the first buffer (4 hours) and Tris-HCI Saline pH 7.0 as the secondary dialysis buffer (16 hours) which may also serve as the LNP storage buffer.

[0608] Alternatively, it is contemplated that a 10 mM Citrate encapsulation buffer at pH 6 and a dialysis buffer of 10 mM Tris-HCI at pH 7.4 may be used. In order to increase the DES-CD content in the LNP formulation to a total of 75 % volume, the Ethanol Lipid and Aqueous DES- CD / mRNA phases may be combined at 42 °C instead of 20 °C to reduce DES viscosity. It is expected that such 75 % DES-CD filled LNPs will have suitable potency and potentially increased stability during storage. A pH 4 encapsulation buffer and 2-step dialysis may be preferred for formulating DES-l-CD into LNPs.

[0609] It is expected that LNPs could stably encapsulate all three types of CD (a, 0 and y) when mixed in advance with Choline chloride:Urea (1:2 mokmol) with good RNA encapsulation efficiency, RNA integrity and LNP diameter sizes.

[0610] It will be appreciated that the above embodiments have been described by way of example only.

[0611] Other variants or use cases of the disclosed techniques may become apparent to the person skilled in the art once given the disclosure herein. The scope of the disclosure is not limited by the described embodiments but only by the accompanying claims.

Claims

Claims1. A method of manufacturing a pharmaceutical composition, wherein the pharmaceutical composition comprises an active ingredient encapsulated in nanoparticles, which method comprises incorporating a deep eutectic solvent into the pharmaceutical composition.

2. The method according to claim 1, wherein the deep eutectic solvent includes no more than one component having a net charge.

3. The method according to claim 1 or claim 2, wherein the deep eutectic solvent is free of carboxylic acids.

4. The method according to any preceding claim, comprising combining the deep eutectic solvent with a cyclic polysaccharide, optionally wherein the deep eutectic solvent is combined with the cyclic polysaccharide before incorporating the deep eutectic solvent into the pharmaceutical composition.

5. Use according to claim 4, wherein the cyclic polysaccharide is present in an amount in the range 1 to 5 %, optionally 2 to 3 %, by weight based on the total weight of the deep eutectic solvent and cyclic polysaccharide.

6. The method according to claim 4 or claim 5, wherein the cyclic polysaccharide is a cyclodextrin selected from a-cyclodextrin, p-cyclodextrin, and y-cyclodextrin.

7. The method according to claim 6, wherein: i) the cyclic polysaccharide is y-cyclodextrin and is present in an amount in the range 0.5 to 12.5 %, optionally 0.6 to 10 %, 1 to 3 %, or 1.25 to 2.5 % by weight based on the total weight of the deep eutectic solvent and cyclic polysaccharide; orii) the cyclic polysaccharide is a-cyclodextrin and is present in an amount in the range 5 to 10 % by weight based on the total weight of the deep eutectic solvent and cyclic polysaccharide; or iii) the cyclic polysaccharide is p-cyclodextrin and is present in an amount in the range 0.5 to 5 %, optionally 1.25 to 5 %, further optionally 2.3 to 2.7 % by weight based on the total weight of the deep eutectic solvent and cyclic polysaccharide.

8. The method according to any preceding claim, comprising: preparing a dispersion of the nanoparticles, the nanoparticles having the active ingredient encapsulated therein; and subsequently mixing the dispersion with the deep eutectic solvent to obtain a mixture comprising the nanoparticles dispersed in a medium; optionally wherein the method further comprises drying the mixture.

9. The method according to claim 8, further comprising, before mixing the dispersion with the deep eutectic solvent, mixing the dispersion with an additive.

10. The method according to claim 9, wherein the additive is selected from: i) glycerol; ii) a serum; iii) a protein or peptide, optionally selected from an albumin such as bovine serum albumin; a globulin, such as an antibody, e.g. IgG; gelatin; a silk protein; or collagen; iv) a polyalkylene glycol, such as polyethylene glycol; v) a buffer, such as ammonium acetate; vi) a cyclic polysaccharide; and vii) combinations thereof.

11. The method according to claim 10, wherein the additive comprises glycerol.

12. The method according to claim 11, wherein the glycerol is mixed with the dispersion at a dispersion to glycerol ratio in the range 1:1 to 1:20 by volume.

13. The method according to any of claims 10 to 12, wherein the cyclic polysaccharide comprises a cyclodextrin, optionally selected from a-cyclodextrin, p-cyclodextrin, and y- cyclodextrin; further optionally wherein the cyclic polysaccharide comprises y-cyclodextrin.

14. The method according to claim 13, wherein the cyclic polysaccharide is present in the dispersion in an amount in the range 0.5 to 15 % by weight based on the total weight of the dispersion; optionally wherein: i) the cyclic polysaccharide is y-cyclodextrin and is present in an amount in the range 0.6 to 10 %, 1 to 3 %, or 1.25 to 2.5 % by weight; or ii) the cyclic polysaccharide is a-cyclodextrin and is present in an amount in the range 5 to 10 % by weight; or iii) the cyclic polysaccharide is p-cyclodextrin and is present in an amount in the range 0.5 to 5 %, 1.25 to 5 %, or 2.3 to 2.7 % by weight.

15. The method according to any of claims 8 to 14, wherein mixing the dispersion with the deep eutectic solvent comprises mixing the dispersion with an excess of the deep eutectic solvent.

16. The method according to claim 15, wherein the deep eutectic solvent is mixed with the dispersion at a deep eutectic solvent to dispersion ratio of >4:1, optionally >8:1, >10:1, >15:1, or >20:1.

17. The method according to any of claims 8 to 16 further comprising, before the mixing, heating the deep eutectic solvent to reduce viscosity of the deep eutectic solvent during the mixing.

18. The method according to any of claims 8 to 17, further comprising: incubating the mixture for a time interval; and subsequently eliminating the deep eutectic solvent from the medium or separating the nanoparticles from the medium.

19. The method according to claim 18, wherein the time interval is in the range 10 seconds to 24 hours, optionally 30 seconds to 19 hours.

20. The method according to claim 19, wherein the time interval is in the range 20 seconds to 90 minutes, optionally 30 seconds to 1 hour, further optionally 1 to 30 minutes.

21. The method according to claim 20, wherein the time interval is in the range 1 to 15 minutes, optionally wherein the time interval is about 1 minute.

22. The method according to any of claims 18 to 21, wherein the incubation is at a temperature in the range 18 to 40 °C, optionally about 20 °C or about 37 °C.

23. The method according to any of claims 18 to 22, wherein eliminating the deep eutectic solvent comprises diluting the medium with an excess of a diluent to convert the deep eutectic solvent of the medium into a solution.

24. The method according to claim 17, wherein the diluent is selected from: i) water; ii) a cell culture medium, such as Dulbecco's modified Eagle's medium; iii) an aqueous buffer, such as phosphate buffered saline, ammonium acetate, a citrate buffer, or TRIS; iv) a serum; v) a solution of a protein or a peptide, optionally selected from an albumin such as bovine serum albumin; a globulin, such as an antibody, e.g. IgG; gelatin; a silk protein; or collagen; vi) a pharmaceutically-acceptable excipient, such as glycerol or polyethylene glycol; vii) an aqueous solution of a cyclic polysaccharide, optionally a cyclodextrin, further optionally a-cyclodextrin, p-cyclodextrin, or y-cyclodextrin; and viii) combinations thereof, such as a combination of the cell culture medium and the serum.

25. The method according to any of claims 18 to 24, comprising separating the nanoparticles from the medium.

26. The method according to claim 25, wherein the nanoparticles are separated from the medium by ultrafiltration, ultracentrifugation, tangential flow filtration, or dialysis.

27. The method according to any preceding claim, wherein the deep eutectic solvent comprises a hydrogen bond donor and a hydrogen bond acceptor, wherein the hydrogen bond acceptor has a structure of:wherein:X- is a counterion, such as a halide;R1is H or OH;R2is selected from H, CH3, Cl, Br; andR3, R4, and R5are each independently selected from Cl to C3 alkyl groups, optionally wherein R3, R4, and R5are each methyl groups.

28. The method according to claim 27, wherein the hydrogen bond acceptor is a choline salt, optionally choline chloride.

29. The method according to claim 27 or claim 28, wherein the hydrogen bond donor is a sugar or sugar alcohol.

30. The method according any of claims 1 to 26, wherein the deep eutectic solvent comprises an amino acid and a sugar or sugar alcohol, optionally wherein the amino acid is proline.

31. The method according to any of claims 1 to 26, wherein the deep eutectic solvent comprises: a hydrogen bond acceptor selected from ectoine, hydroxyectoine, homoectoine, and hydroxyhomoectoine, and salts thereof; and a hydrogen bond donor which is a sugar or sugar alcohol; optionally wherein: i) the hydrogen bond acceptor is ectoine; and / or ii) the nanoparticles comprise a virus or bacterium.

32. The method according to any of claims 29 to 31, wherein the sugar alcohol has 5 or 6 carbon atoms.

33. The method according to claim 32, wherein the sugar alcohol is selected from arabitol, optionally D(+)arabitol or L(-)arabitol; sorbitol; xylitol; ribitol; and combinations thereof.

34. The method according to any of claims 27 to 33, wherein the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is in the range 1:3 to 3:1, optionally 1:2 to 2:1, further optionally 1:0.8 to 1:1.2; or is about 1:1.

35. The method according to claim 27 or claim 28, wherein the hydrogen bond donor has a structure of:wherein:A is selected from O, S, and NH;R3is selected from: H; -NH2; a Cl to C6 alkyl, alkenyl, or haloalkyl group; and -NH-(CH2)nCH3, where n is 0 or an integer from 1 to 5; andR4is H and or a Cl to C3 alkyl group.

36. The method according to claim 35, wherein the hydrogen bond donor is urea.

37. The method according to claim 35 or claim 36, wherein the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is in the range 1:1.5 to 1:2.5, or is about 1:2.

38. The method according to any preceding claim, comprising: preparing a first phase, the first phase comprising the active ingredient; preparing a second phase, the second phase comprising an encapsulant material; and combining the first phase and the second phase to form the nanoparticles having the active ingredient encapsulated therein; wherein the first phase and / or the second phase further comprises the deep eutectic solvent; optionally wherein the first phase further comprises the deep eutectic solvent.

39. The method according to claim 38, wherein preparing the first phase comprises: preparing an aqueous solution of the active ingredient; and mixing the deep eutectic solvent with the aqueous solution.

40. The method according to claim 39, wherein the deep eutectic solvent is mixed with the aqueous solution at a deep eutectic solvent to aqueous solution ratio of at least 1:1 by volume.

41. The method according to claim 39 or claim 40, wherein the aqueous solution further comprises one or more of: i) saline; ii) a cryoprotectant, optionally a sugar such as sucrose; iii) a buffer, optionally selected from: Tris, PIPES, MES, HEPES, MOPSO, CAPS, CAPSO, BICINE, BIPES, BAPTA, citrate, phosphate, imidazole, glycine, acetate, and combinations thereof; and iv) a cyclic polysaccharide, optionally a cyclodextrin, further optionally a-cyclodextrin, P-cyclodextrin, or y-cyclodextrin.

42. The method according to claim 41, wherein: the buffer is present in the aqueous solution at a concentration of 1 to 100 mM, optionally 1 to 10 mM; and / or the buffer comprises citrate.

43. The method according to any of claims 39 to 42, wherein the aqueous solution has a pH in the range 1 to 9, optionally 4 to 6.5, further optionally 5.5 to 6.5.

44. The method according to any of claims 38 to 43, wherein the second phase is an organic phase.

45. The method according to claim 44, wherein the organic phase comprises a lipid and a water-miscible organic solvent.

46. The method according to claim 45, wherein the water-miscible organic solvent comprises ethanol.

47. The method according to any of claims 38 to 46, wherein the first and second phases are combined at a temperature in the range 18 to 75 °C, optionally 25 to 55 °C, and further optionally 25 to 42 °C.

48. The method according to any of claims 38 to 47, further comprising, after combining the first phase and the second phase, removing excess deep eutectic solvent from the composition.

49. The method according to claim 48, wherein the excess deep eutectic solvent is removed by dialysis against a dialysate; optionally wherein the ratio of the composition to the dialysate is at least 1:200 or at least 1:1000 by volume.

50. The method according to claim 49, wherein the dialysate comprises water and optionally one or more of:1) saline;2) a cryoprotectant, such as sucrose; and3) a buffer, optionally selected from: Tris, PIPES, MES, HEPES, MOPS, MOPSO, CAPS, CAPSO, BICINE, BIPES, BAPTA, citrate, phosphate, imidazole, glycine, and acetate.

51. The method according to claim 50, wherein the dialysate comprises the buffer at a concentration in the range 1 to 100 mM, optionally 1 to 10 mM.

52. The method according to any of claims 49 to 51, wherein the dialysate has a pH in the range 1 to 8, optionally 3 to 7.

53. The method according to any of claims 49 to 52, wherein the dialysis is performed at a temperature in the range 4 to 37 °C, optionally 4 to 18 °C.

54. The method according to any of claims 49 to 53, wherein the dialysis comprises performing two or more rounds of dialysis.

55. The method according to claim 54, wherein the dialysis comprises: performing a first round of dialysis against a first dialysate, the first dialysate having a pH in the range 3.5 to 4.5 and optionally a pH of about 4; and subsequently performing a second round of dialysis against a second dialysate, the second dialysate having a pH in the range 7 to 8, and optionally a pH of about 7.4.

56. The method according to claim 55, wherein the first dialysate comprises a buffer at a concentration of 1 to 15 mM, optionally about 10 mM.

57. The method according to claim 55 or claim 56, wherein the second dialysate comprises a buffer at a concentration of 1 to 10 mM, optionally about 10 mM.

58. The method according to any of claims 55 to 57, wherein the first dialysate is a citrate buffer; and the second dialysate is a Tris buffer.

59. The method according to claim 48, wherein the excess deep eutectic solvent is removed using tangential flow filtration, ultrafiltration, or ultracentrifugation.

60. The method according to any preceding claim, wherein the active ingredient comprises a biomolecule.

61. The method according to claim 60, wherein the active ingredient comprises a nucleic acid, a protein, and / or a polysaccharide.

62. The method according to claim 61, wherein the biomolecule is a nucleic acid, optionally an RNA.

63. The method according to any preceding claim, wherein the composition comprises two or more active ingredients, such as a combination of a nucleic acid and a protein or a combination of a nucleic acid and an antibiotic.

64. The method according to any preceding claim, wherein the nanoparticles are selected from: lipid-based nanoparticles, such as liposomes, emulsified lipid nanoparticles, lipid nanoparticles, a nanostructured lipid carrier, or solid lipid nanoparticles; polymeric nanoparticles, such as dendrimers, micelles, polymeric micelles, polymersomes, cubosomes, nanospheres, or charge altering release transporters; inorganic nanoparticles, such as silica nanoparticles, carbon nanotubes, or metallic nanoparticles; and protein-based nanoparticles, such as virus-like particles or an attenuated virus.

65. The method according to claim 64, wherein the nanoparticles are lipid-based nanoparticles.

66. The method according to claim 65, wherein the nanoparticles are lipid nanoparticles or liposomes; optionally wherein the nanoparticles are lipid nanoparticles.

67. The method according to claim 65 or claim 66, wherein the active ingredient comprises a nucleic acid, wherein the nanoparticles include a cationic lipid, and wherein the nanoparticles have an N / P ratio of at least 7, optionally 10 to 20, further optionally 12 to 18.

68. The method according to any preceding claim, wherein the composition further comprises a liquid carrier, the nanoparticles being dispersed in the liquid carrier.

69. The method according to claim 61, wherein the liquid carrier includes at least one of: i) water; ii) a cell culture medium, such as Dulbecco's modified Eagle's medium; iii) an aqueous buffer, such as phosphate buffered saline, ammonium acetate, a citrate buffer, or TRIS; iv) a serum; v) a solution of a protein or a peptide, optionally selected from an albumin such as bovine serum albumin; a globulin, such as an antibody, e.g. IgG; gelatin; a silk protein; or collagen; vi) a pharmaceutically-acceptable excipient, such as glycerol or polyethylene glycol; vii) a deep eutectic solvent; viii) a cyclic polysaccharide, optionally a cyclodextrin, further optionally a- cyclodextrin, p-cyclodextrin, or y-cyclodextrin; and viii) combinations thereof, such as a combination of the cell culture medium and the serum, or a mixture of a deep eutectic solvent and a cyclic polysaccharide.

70. The method according to any preceding claim, wherein the deep eutectic solvent is selected from: choline chloride : urea; choline chloride : xylitol; choline chloride : sorbitol; choline chloride : arabitol;N,N,N-trimethylglycine : xylitol;N,N,N-trimethylglycine : arabitol;N,N,N-trimethylglycine : urea; proline : sorbitol; ectoine : sorbitol; choline chloride : mannose; and choline chloride : ribitol.

71. A pharmaceutical composition obtainable by the method according to any preceding claim.

72. A pharmaceutical composition comprising: an active ingredient encapsulated in nanoparticles; a carrier, wherein the nanoparticles are dispersed in the carrier; and a deep eutectic solvent; wherein the deep eutectic solvent is: a) present in the nanoparticles; and / or b) present in the carrier.

73. The pharmaceutical composition according to claim 72, which includes a mixture of the deep eutectic solvent and a cyclic polysaccharide, optionally wherein the cyclic polysaccharide is a cyclodextrin, further optionally a-cyclodextrin, p-cyclodextrin, or y- cyclodextrin.

74. A medical device loaded with the pharmaceutical composition of any of claims 71 to 73, which device comprises a microneedle or a transdermal patch.

75. The medical device according to claim 74, wherein the device comprises a microneedle array.

76. A method of manufacturing the medical device of claim 74 or claim 75, which method comprises loading the pharmaceutical composition with the pharmaceutical composition by lyophilisation.

77. The pharmaceutical composition according to any of claims 71 to 73, for use in medicine, optionally veterinary medicine.

78. The pharmaceutical composition for use according to claim 77, wherein the composition delivers the active ingredient to the spleen and / or a ganglion selectively overthe liver.

79. The pharmaceutical composition for use according to claim 77 or claim 78, wherein the deep eutectic solvent increases potency of the active ingredient.

80. The pharmaceutical composition for use according to any of claims 77 to 79, which is to be delivered using a microneedle or a transdermal patch.

81. The pharmaceutical composition for use according to any of claims 77 to 80, which is to be administered by injection, optionally intramuscular injection.

82. Use of a deep eutectic solvent to stabilise an active ingredient of a pharmaceutical composition, wherein the active ingredient is encapsulated in nanoparticles.

83. Use according to claim 82, wherein a mixture of the deep eutectic solvent and a cyclic polysaccharide is used to stabilise the active ingredient.

84. Use according to claim 82, wherein the cyclic polysaccharide is a cyclodextrin selected from a-cyclodextrin, p-cyclodextrin, and y-cyclodextrin.

85. Use according to claim 83 or claim 84, wherein the cyclic polysaccharide is present in an amount in the range 1 to 5 %, optionally 2 to 3 %, by weight based on the total weight of the deep eutectic solvent and cyclic polysaccharide.

86. Use according to and of claims 83 to 85 wherein: i) the cyclic polysaccharide is y-cyclodextrin and is present in an amount in the range 0.5 to 12.5 %, optionally 0.6 to 10 %, 1 to 3 %, or 1.25 to 2.5 % by weightbased on the total weight of the deep eutectic solvent and cyclic polysaccharide; or ii) the cyclic polysaccharide is a-cyclodextrin and is present in an amount in the range 5 to 10 % by weight based on the total weight of the deep eutectic solvent and cyclic polysaccharide; or iii) the cyclic polysaccharide is |3- cyclodextrin and is present in an amount in the range 0.5 to 5 %, optionally 1.25 to 5 % or 2.3 to 2.7 %, by weight based on the total weight of the deep eutectic solvent and cyclic polysaccharide.

87. Use according to any of claims 82 to 86, wherein the deep eutectic solvent comprises: i) a choline derivative, having a structure of:wherein:R1is H, OH, and -OC(O)CH3;R2is selected from H, CH3, Cl, Br;R3, R4, and R5are each independently selected from Cl to C3 alkyl groups; andX- is a counterion, such as a halide; and ii) a sugar or sugar alcohol.

88. Use according to claim 87, wherein the deep eutectic solvent comprises i) a choline salt, optionally a choline halide such as choline chloride; and ii) a sugar alcohol, optionally a sugar alcohol having 5- or 6- carbon atoms, further optionally wherein the sugar alcohol is sorbitol or xylitol.

89. Use according to claim 88, wherein the deep eutectic solvent is choline chloride : sorbitol.

90. Use according to any of claims 82 to 89, wherein the nanoparticles are lipid-based nanoparticles, optionally lipid nanoparticles or liposomes.

91. Use according to any of claims 82 to 90, wherein the active ingredient comprises a biomolecule, optionally a nucleic acid, further optionally an RNA.

92. Use according to any of claims 82 to 91, wherein the deep eutectic solvent is encapsulated in the nanoparticles.

93. Use according to any of claims 82 to 92, wherein the nanoparticles are dispersed in the deep eutectic solvent.

94. Use according to any of claims 82 to 93, comprising transiently contacting the deep eutectic solvent with the pharmaceutical composition.

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