Submicron Particles

Abstract

The present invention relates to submicron particles comprising a payload molecule and a lipid structure surrounded by an outer layer comprising an amphiphilic copolymer. The invention extends to a method for producing the submicron particles. The invention also includes pharmaceutical compositions and vaccines comprising the submicron particles and medical uses thereof.

Description

[Technical field]

[0001] The present invention relates to submicron particles, and in particular to the submicron particles themselves which contain payload molecules such as nucleic acids or small molecule drugs. The invention extends to methods of making the submicron particles, pharmaceutical compositions and vaccines which contain the submicron particles, and medical uses thereof. [Background technology]

[0002] Vaccines are one of the most cost-effective ways to prevent infectious diseases. The World Health Organization reported that vaccination could prevent 2 to 3 million deaths per year. Traditional vaccines contain attenuated viruses, or purified signature proteins of the virus. But more recently, RNA vaccines are promising candidates due to their rapid development and low-cost manufacturing. RNA vaccines are among the global frontrunners in the race for clinical trials against COVID-19, including already approved messenger RNA (mRNA) vaccines from Pfizer / BioNTech and Moderna, as well as Imperial College's self-amplifying RNA (saRNA) vaccine, which is in clinical trials. RNA vaccines have also been shown to be effective against influenza. 1 , rabies virus 2 , HIV-1 3 , Zika virus 4 and Ebola virus 5 Various other vaccine indications, including infectious diseases such as polio, bronchitis, and bronchitis, as well as cancer vaccines. 6~9 They have been used preclinically in. mRNA-based vaccines typically encode the antigen of interest and contain 5' and 3' untranslated regions (UTRs), whereas saRNA-based vaccines not only encode the antigen but also the viral replication machinery that allows for intracellular RNA amplification and abundant protein expression. 10 saRNA allows for the production of large amounts of antigen from extremely small doses (10-100 times less than mRNA) due to intracellular replication of antigen-encoding RNA. 11The dose-sparing qualities of saRNA vaccines may facilitate scale-up and production of large numbers of vaccine doses.

[0003] Although mRNA and saRNA show great promise as new classes of vaccines and therapeutics, their clinical translation and commercialization are still limited due to two major challenges: one is the difficulty with the intracellular delivery problem, because RNA has (1) poor endocytosis due to its large molecular weight and negative charge that induces repulsion to the cell membrane, (2) limited intracellular protein expression due to catalytic hydrolysis / enzymatic degradation caused by endosomal trapping, and (3) poor antigen loading and maturation of antigen-presenting cells (APCs).

[0004] Another major problem is that RNA vaccines need to be stored and transported in an extremely challenging cold chain, as RNA is extremely fragile and can easily degrade in exposed environments. Any breakdown in the “cold chain” can significantly reduce the efficacy of RNA vaccines. For example, the world’s first approved COVID-19 vaccine, Pfizer / BioNTech’s mRNA vaccine, has a major obstacle that requires storage at -70°C. At refrigerated temperatures of 2-8°C, mRNA vaccines can only be stable for five days (Pfizer.com, 2020 / 11 / 20). Similarly, Moderna’s mRNA, already approved against COVID-19, needs to be stored at -20°C. This makes it extremely difficult for RNA vaccines to reach the speed and scale of deployment required for vaccination.

[0005] Lyophilization can enhance RNA stability by avoiding aqueous conditions. However, the ice crystals formed during lyophilization present physical stress to the nucleic acid (sequence) and other components of the formulation, which can lead to damage to the nucleic acid (e.g., strand breakage, loss of supercoiling, etc.) and irreversible aggregation or precipitation of the RNA-added formulation. Both of these lead to an irreversible decrease in the efficacy of the RNA formulation, limiting the rapid development of RNA vaccines and therapeutics.

[0006] Therefore, the development of nucleic acid vaccines and therapeutics (be it saRNA, mRNA or DNA) that are stable at 2-8 °C and in non-refrigerated systems (especially the latter) is crucial for supply, distribution and deployment, but is very challenging. Given all these challenges, it is more urgent than ever to develop novel platforms with low cost and safety profile for efficient nucleic acid delivery and improved stability not only at refrigerated but also at ambient temperatures.

[0007] During the past decades, advances in bioengineering and nanotechnology have given rise to several delivery techniques. Liposomes, a widely tested RNA delivery system, need to be positively charged to electrostatically trap RNA, and a specific membrane charge density threshold has been identified as a requirement to ensure efficient endosomal escape. 12~14 However, the payload within liposomes can easily leak out, and the lipid membrane can be disrupted when interacting with negatively charged cell membranes. In addition, the surface charge of liposomes affects their aggregation behavior and the adsorption of serum proteins when injected in vivo. This causes rapid clearance of RNA and reduces transfection efficiency in vivo. The use of amphiphilic diblock copolymers to generate polymer vesicles, known as polymersomes, is another strategy to create structures for encapsulation. Polymersomes have also been tested by researchers for oligonucleotide delivery, as they have higher mechanical strength and toughness than liposomes. 15、16However, the synthesis of cationic polymers requires very difficult procedures due to the complex biological requirements. In addition, efficient delivery of mRNA or saRNA is even more difficult due to the molecular weight, which is much larger than that of oligonucleotides, and the instability of RNA. Although some reported formulations have shown efficient mRNA or saRNA delivery effects, 17、18 However, thermal stability remains a challenge as they were either produced and used immediately or kept at -80°C for later use.

[0008] The present invention arises out of the inventors' research which seeks to overcome problems associated with the prior art. Summary of the Invention

[0009] According to a first aspect of the present invention there is provided a submicron particle comprising a payload molecule and a lipid structure surrounded by an outer layer comprising an amphiphilic copolymer.

[0010] Advantageously, the inventors were surprised to observe that submicron particles can be used for efficient nucleic acid delivery (including saRNA, mRNA or DNA) and non-cold chain storage. This delivery system simultaneously addresses many, if not all, of the intended design requirements, including good biocompatibility, ease of manufacture, small size, controlled surface charge, high RNA loading efficiency, endosomolytic ability, low cost, and excellent stability. Furthermore, submicron particles can be produced using FDA approved amphiphilic polymers (e.g., PEG-PCL) and cationic or ionizable lipids. Both materials are low cost and readily available.

[0011] The amphiphilic copolymer forms a capsule that functions as a colloidal stable shell. The lipid structures can self-assemble into aggregates, which are then enveloped in the capsule core. Nucleic acids such as RNA (e.g., mRNA or saRNA) can be efficiently encapsulated in this nanocontainer by electrostatic interactions with cationic or ionizable lipids. The submicron particles provide dual protection for RNA, i.e., (1) efficient condensation of RNA, and (2) an outer vesicle membrane that is more mechanically stable and less permeable than lipid bilayers. Compared to lipid nanoparticles reported to date, the mechanical strength of the hydrophilic shell composed of polymers is much higher than that composed of lipid bilayers, which can significantly improve the colloidal stability of the submicron particles and allow for better protection of RNA during storage and application.

[0012] Moreover, advantageously, submicron particles can be prepared by a one-pot method based on a few minutes of mixing, stirring and solvent evaporation. The physicochemical and biological properties of submicron particles (e.g., particle size, surface charge, transfection efficiency and stability) can be easily adjusted by simply changing the mixing ratio.

[0013] The term "submicron" may be understood to mean that the particles of the invention have a largest maximum dimension of less than 1 μm. More preferably, the largest maximum dimension of the particles is less than 900 nm, less than 800 nm, less than 700 nm or less than 600 nm, most preferably less than 500 nm, less than 400 nm, less than 300 nm or less than 200 nm. Submicron particles may have a largest dimension of 10-900 nm, 20-800 nm, 30-700 nm or 40-600 nm, more preferably 50-500 nm or 60-400 nm, most preferably 80-300 nm or 100-200 nm. The largest dimension of the submicron particles may correspond to the Z-average size determined using a Zetasizer μV instrument.

[0014] The payload molecule may be encapsulated by the lipid structure.

[0015] The payload molecule may be a biomolecule and / or an active pharmaceutical ingredient (API). The API may be a hydrophobic API or a hydrophilic API. The API may be a macromolecule or a small molecule. It may be understood that a small molecule may be considered to be a molecule having a molecular weight of less than 900 Daltons. In some embodiments, a small molecule may have a molecular weight of less than 800 Daltons, less than 700 Daltons, less than 600 Daltons, less than 500 Daltons, or less than 400 Daltons. Similarly, a macromolecule may be considered to be a molecule having a molecular weight of at least 900 Daltons.

[0016] In a preferred embodiment, the payload molecule is a biomolecule. For example, the biomolecule may be or may include an amino acid, a peptide, an affimer, a protein, a glycoprotein, a lipopolysaccharide, an antibody or a fragment thereof, or a nucleic acid.

[0017] The nucleic acid may be DNA, RNA, or a DNA / RNA hybrid sequence. Preferably, the nucleic acid is DNA or RNA.

[0018] Most preferably, nucleic acid is RNA.RNA can be single-stranded or double-stranded.RNA can be selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), antisense RNA (asRNA), RNA aptamer, interfering RNA, microRNA (miRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA) and small RNA.

[0019] Preferably, the RNA is a self-amplifying RNA (saRNA) or messenger RNA (mRNA). Those skilled in the art will appreciate that self-amplifying RNA may contain the basic elements of mRNA (cap, 5'UTR, 3'UTR, and poly(A) tail of variable length) but may be significantly longer (e.g., 9-12 kb).

[0020] The nucleic acid sequence, preferably RNA, can be at least 10 bases long, at least 20 bases long, at least 50 bases long, at least 100 bases long, at least 200 bases long, at least 300 bases long, at least 400 bases long, at least 500 bases long, at least 600 bases long, at least 700 bases long, at least 800 bases long, or at least 900 bases long. In a preferred embodiment, the RNA is saRNA or mRNA.

[0021] The nucleic acid sequence, preferably RNA, most preferably saRNA or mRNA, can be at least 1000 bases in length, at least 2000 bases in length, at least 3000 bases in length, at least 4000 bases in length, at least 5000 bases in length, at least 6000 bases in length, at least 7000 bases in length, at least 8000 bases in length, at least 9000 bases in length at least 10000 bases in length, at least 11000 bases in length or at least 12000 bases in length.

[0022] In one embodiment, the nucleic acid sequence is at least 6000 bases in length. In one embodiment, the RNA is at least 6000 bases in length. In a preferred embodiment, the saRNA is at least 6000 bases in length.

[0023] In an alternative embodiment, the nucleic acid sequence is at least 900 bases in length. In one embodiment, the RNA is at least 900 bases in length. In a preferred embodiment, the mRNA is at least 900 bases in length.

[0024] The nucleic acid sequence, preferably RNA, most preferably saRNA, may be 5000 to 20000 bases long, 5000 to 15000 bases long, 5000 to 14000 bases long, 5000 to 13000 bases long, 5000 to 12000 bases long, 5000 to 11000 bases long, 5000 to 10000 bases long, 6000 to 20000 bases long, 6000 to 15000 bases long, 6000 to 14000 bases long, 6000 to 13000 bases long, 6000 to 12000 bases long, 6000 to 11000 bases long, 6000 to 10000 bases long, 7000 to 20000 bases long, 7000 to 15000 bases long, 7000 to 14000 bases long, The length of the nucleic acid sequence may be 7000-13000 bases, 7000-12000 bases, 7000-11000 bases, 7000-10000 bases, 8000-20000 bases, 8000-15000 bases, 8000-14000 bases, 8000-13000 bases, 8000-12000 bases, 8000-11000 bases, 8000-10000 bases, 9000-20000 bases, 9000-15000 bases, 9000-14000 bases, 9000-13000 bases, 9000-12000 bases, 9000-11000 bases, or 9000-10000 bases.

[0025] Alternatively, the nucleic acid sequence, preferably RNA, most preferably mRNA, can be 50 to 10,000 bases in length, 100 to 9,000 bases in length, 200 to 8,000 bases in length, 300 to 7,000 bases in length, 400 to 6,000 bases in length, 500 to 6,000 bases in length, 600 to 5,000 bases in length, 700 to 4,000 bases in length, 800 to 3,000 bases in length, or 900 to 2,000 bases in length.

[0026] In one embodiment, the nucleic acid sequence is 6,000 to 15,000 bases long. The nucleic acid sequence may be 8,000 to 12,000 bases long. The RNA may be 6,000 to 15,000 bases long. The RNA may be 8,000 to 12,000 bases long. Preferably, the saRNA is 6,000 to 15,000 bases long. Preferably, the saRNA is 8,000 to 12,000 bases long.

[0027] In alternative embodiments, the nucleic acid sequence is 400-14000, 500-10000, 600-7500, 700-5000, 800-4000, or 900-2000 bases long. The RNA may be 400-14000, 500-10000, 600-7500, 700-5000, 800-4000, or 900-2000 bases long. Preferably, the mRNA is 400-14000, 500-10000, 600-7500, 700-5000, 800-4000, or 900-2000 bases long.

[0028] One of skill in the art will understand that when the nucleic acid is double-stranded, for example double-stranded RNA, "base length" refers to the length of base pairs.

[0029] The nucleic acid may code for at least a portion of a virus. The virus may be a SARS-CoV-2 virus or an influenza virus. The nucleic acid may code for a SARS-CoV-2 spike protein, more preferably a prefusion stabilized SARS-CoV-2 spike protein. Alternatively, the nucleic acid may code for an H1 hemagglutinin of an influenza virus. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is saRNA or mRNA.

[0030] The submicron particle preferably comprises a plurality of lipid structures. For example, the submicron particle may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 lipid structures. The plurality of lipid structures may be surrounded by an outer layer comprising an amphiphilic copolymer.

[0031] The or each lipid structure may be a lipid nanoparticle or a liposome. Preferably, the or each lipid structure is a lipid nanoparticle.

[0032] The or each lipid structure may comprise a cationic lipid or an ionizable lipid. In some embodiments, the or each lipid structure may comprise a plurality of lipids. At least one of the plurality of lipids may comprise a cationic lipid or an ionizable lipid. The cationic lipid or an ionizable lipid may be a multivalent cationic lipid. The cationic lipid or an ionizable lipid may be a pH-sensitive lipid. The cationic lipid or an ionizable lipid may comprise a positively charged nitrogen atom or an ionizable nitrogen atom. The cationic lipid or an ionizable lipid may exhibit a positive charge in an acidic solution. A solution may be considered to be acidic if it has a pH of less than 7 at 20°C, more preferably less than 6.5 at 20°C. A solution may be considered to be acidic if it has a pH of 3.5-7 at 20°C, or 4-7 at 20°C, more preferably 4.5-6.5 at 20°C. The cationic or ionizable lipid can be dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), ethylphosphatidylcholine (ethyl PC), didodecyldimethylammonium bromide (DDAB), 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), N4-cholesteryl-spermine (GL67), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), DLin-MC3-DMA, 1,2-dioleoyl-3-dimethylammonium propane (DODAP), or heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102).

[0033] In some embodiments, the lipid structure may comprise cationic lipids or ionizable lipids such as DOTAP. The lipid structure may comprise at least 1% by weight, at least 10% by weight, at least 20% by weight, at least 30% by weight, at least 40% by weight, at least 50% by weight, at least 60% by weight, at least 70% by weight, at least 80% by weight, at least 90% by weight, or at least 99% by weight of cationic lipids or ionizable lipids. In some embodiments, the lipid structure may consist of cationic lipids or ionizable lipids. Alternatively, the lipid structure may comprise 1-99% by weight cationic or ionizable lipids, 10-90% by weight cationic or ionizable lipids, 20-85% by weight cationic or ionizable lipids, 30-80% by weight cationic or ionizable lipids, 40-75% by weight cationic or ionizable lipids, 50-70% by weight cationic or ionizable lipids, or 55-65% by weight cationic or ionizable lipids.

[0034] In some embodiments, the lipid structure may include a sterol, such as cholesterol. The lipid structure may include at least 1% by weight, at least 5% by weight, at least 10% by weight, at least 15% by weight, at least 20% by weight, at least 25% by weight, at least 30% by weight, at least 35% by weight, or at least 40% by weight of sterol. The lipid structure may include less than 99% by weight of sterol, less than 90% by weight of sterol, less than 80% by weight of sterol, less than 70% by weight of sterol, less than 60% by weight of sterol, less than 50% by weight of sterol, or less than 45% by weight of sterol. The lipid structure may include 1-99% by weight of sterol, 10-90% by weight of sterol, 15-80% by weight of sterol, 20-70% by weight of sterol, 25-60% by weight of sterol, 30-50% by weight of cholesterol, or 35-45% by weight of sterol.

[0035] In some embodiments, the lipid structure may include a combination of a cationic lipid or ionizable lipid, such as DOTAP, and a sterol, such as cholesterol. The weight ratio of cationic lipid or ionizable lipid to sterol may be 1:99 to 99:1, 10:90 to 90:10, 20:80 to 85:15, 30:70 to 80:20, 40:60 to 75:22, 50:50 to 70:30, or 55:45 to 65:35. The weight ratio of cationic lipid or ionizable lipid to sterol may be about 60:40.

[0036] Submicron particles may have an N / P molar ratio of at least 1:50, at least 1:20, at least 1:10, at least 1:5, at least 1:2, at least 1:1, at least 2:1, at least 3:1, at least 4:1 or at least 5:1, more preferably at least 6:1, at least 8:1 or at least 10:1, most preferably at least 11:1, at least 13:1, at least 15:1, at least 16:1, at least 17:1 or at least 18:1. Submicron particles may have an N / P molar ratio of less than 1000:1, less than 500:1, less than 250:1, less than 100:1, less than 50:1, less than 40:1, less than 30:1, less than 28:1, less than 26:1, less than 24:1, less than 22:1, less than 21:1 or less than 20:1. Submicron particles may have an N / P molar ratio of 1:50-1,000:1, 1:10-500:1, 1:5-250:1, 1:2-100:1, 1:1-50:1, 2:1-40:1, 5:1-30:1, 8:1-28:1, 10:1-26:1, 12:1-24:1, 14:1-22:1, 16:1-20:1, or 17:1-19:1. In a preferred embodiment, the submicron particles have an N / P ratio of 11:1-18:1. The N / P molar ratio may be understood to be the ratio of cationic amines in the lipid structure to anionic phosphates in the payload molecule.

[0037] The amphiphilic copolymer is preferably an amphiphilic block copolymer.

[0038] It may be understood that an amphiphilic copolymer may comprise at least one hydrophilic portion and at least one hydrophobic portion. In some embodiments, an amphiphilic copolymer comprises or consists of one hydrophilic portion and one hydrophobic portion. The or each hydrophilic portion may comprise or be a polyether, an amino acid-based polymer or polypeptide, poly(2-methyloxazoline) (PMOXA), and / or a derivative thereof. The or each hydrophobic portion may comprise or be a polyester, an acid-labile polycarbonate, poly(ethylethylene) (PEE), poly(butadiene) (PBD), poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), poly(styrene) (PSt), and / or a derivative thereof.

[0039] Preferably, the amphiphilic copolymer is biodegradable.Thus, the or each hydrophobic portion may comprise or be a polyester, an acid labile polycarbonate and / or a derivative thereof.

[0040] The acid labile polycarbonate can be poly(trimethylene carbonate) (PTMC), poly(2,4,6-trimethoxybenzylidene pentaerythritol carbonate) (PTMBPEC) or a derivative thereof. The amino acid based polymer or polypeptide can be poly(L-glutamic acid) (PGA), poly-L-lysine (PLL) or a derivative thereof.

[0041] Any suitable polyether may be used. In some embodiments, the or each polyether may be polyethylene glycol (PEG), oligo(ethylene glycol) (oligoEG) or a derivative thereof. For example, derivatives of PEG may include poly(ethylene glycol) methyl ether acrylate (mPEGA), and poly(ethylene glycol) methyl ether methacrylate (mPEGMA). Alternatively, the polyether may be a polyether disclosed in applicant's earlier patent application GB2009720.0. For example, the hydrophilic portion may comprise or consist of:

[0042] [ka]

[0043] It can be understood that n is an integer.

[0044] Likewise, any suitable polyester may be used. In some embodiments, the or each polyester may be selected from the list consisting of polycaprolactone (PCL), polylactic acid (PLA), polyglycolide (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(ε-decalactone) (PDL) or derivatives thereof. Alternatively, the polyester may be a polyester disclosed in the applicant's earlier patent application GB2009720.0. For example, the hydrophobic portion may comprise or consist of:

[0045] [ka]

[0046] It can be understood that m and n are integers.

[0047] The hydrophilic portion may comprise less than 60%, less than 50%, less than 45%, less than 40%, less than 35% or less than 32% by weight of the amphiphilic copolymer. The hydrophilic portion may comprise 5-60% by weight of the amphiphilic copolymer, more preferably 10-50% or 20-45% by weight of the amphiphilic copolymer, and most preferably 25-40%, 28-35% or 30-32% by weight of the amphiphilic copolymer.

[0048] The hydrophobic portion may comprise at least 40%, at least 50%, at least 55%, at least 60%, at least 65% or at least 68% by weight of the amphiphilic copolymer. The hydrophobic portion may comprise 40-95% by weight of the amphiphilic copolymer, more preferably 50-90% or 55-80% by weight of the amphiphilic copolymer, and most preferably 60-75%, 65-72% or 68-70% by weight of the amphiphilic copolymer.

[0049] The amphiphilic copolymer may have a molecular weight of at least 1,000 Da, at least 2,000 Da, at least 3,000 Da, at least 4,000 Da, or at least 5,000 Da. Preferably, the amphiphilic copolymer has a molecular weight of at least 6,000 Da or at least 7,000 Da. In some embodiments, the amphiphilic copolymer has a molecular weight of at least 8,000 Da, at least 10,000 Da, at least 12,000 Da, at least 14,000 Da, at least 15,000 Da, or at least 16,000 Da.

[0050] The amphiphilic copolymer may have a molecular weight of less than 100,000 Da, less than 80,000 Da, less than 70,000 Da, less than 60,000 Da, or less than 50,000 Da. Preferably, the amphiphilic copolymer has a molecular weight of less than 40,000 Da or less than 30,000 Da. Most preferably, the amphiphilic copolymer has a molecular weight of less than 25,000 Da, less than 20,000 Da, less than 18,000 Da, less than 17,000 Da, or less than 16,000 Da.

[0051] The amphiphilic copolymer may have a molecular weight of 1,000 to 100,000 Da, 2,000 to 80,000, 3,000 to 70,000, 4,000 to 60,000, 5,000 to 50,000, more preferably 6,000 to 40,000, 7,000 to 50,000, 8,000 to 25,000, 10,000 to 20,000, 12,000 to 18,000, 14,000 to 16,000 Da, 15,000 to 17,500 Da, or 16,000 to 17,000 Da.

[0052] The hydrophobic moiety may have a molecular weight of at least 1,000 Da, at least 2,000 Da, at least 3,000 Da, at least 4,000 Da, or at least 5,000 Da. Preferably, the hydrophobic moiety has a molecular weight of at least 6,000 Da or at least 7,000 Da. In some embodiments, the hydrophobic moiety has a molecular weight of at least 8,000 Da, at least 10,000 Da, or at least 11,000 Da.

[0053] The hydrophobic portion may have a molecular weight of 1,000 to 70,000, 2,000 to 60,000, 3000 to 50,000, more preferably 4,000 to 40,000, 5,000 to 50,000, 6,000 to 25,000, 7,000 to 20,000, 8,000 to 18,000, 9,000 to 15,000 Da, 10,000 to 13,000 Da, or 11,000 to 12,000 Da.

[0054] The molecular weight of the amphiphilic copolymers defined above is determined by the number average molecular weight (M n ) can be understood as

[0055] The molecular weight of the amphiphilic copolymer, the molecular weight of the hydrophobic portion, and / or the molecular weight of the hydrophilic portion can be determined using NMR or gel permeation chromatography (GPC). Methods for using NMR and GPC can be as described in the Examples. In some embodiments, the molecular weight can be determined by NMR, preferably 1 Determined using 1 H NMR.

[0056] The weight ratio of amphiphilic copolymer to payload molecule may be at least 5:1, at least 10:1, at least 20:1, at least 30:1 or at least 40:1, more preferably at least 50:1 or at least 55:1, and most preferably at least 60:1. The weight ratio of amphiphilic copolymer to payload molecule may be less than 1000:1, less than 500:1, less than 250:1, less than 200:1 or less than 150:1, more preferably less than 125:1, less than 100:1, and most preferably less than 85:1. The weight ratio of amphiphilic copolymer to payload molecule may be from 5:1 to 1000:1, 10:1 to 500:1, 20:1 to 250:1, 30:1 to 200:1, or 40:1 to 150:1, more preferably from 50:1 to 125:1, or from 55:1 to 100:1, and most preferably from 60:1 to 85:1.

[0057] The weight ratio of amphipathic copolymer to cationic or ionizable lipid may be at least 1:10, at least 1:8, at least 1:6, at least 1:4, at least 1:2 or at least 1:1.5, more preferably at least 1:1, at least 1.5:1, at least 1.75:1, at least 2:1, at least 2.2:1 or at least 2.4:1, and most preferably at least 2.5:1. The weight ratio of amphipathic copolymer to cationic or ionizable lipid may be less than 50:1, less than 20:1, less than 15:1, less than 10:1, less than 8:1 or less than 6:1, more preferably less than 5.5:1, less than 5:1, less than 4.5:1, less than 4:1, less than 3.5:1 or less than 3:1, and most preferably less than 2.7:1. The weight ratio of amphiphilic copolymer to cationic lipid or ionizable lipid may be 1:10-50:1, 1:8-20:1, 1:6-15:1, 1:4-10:1, 1:2-8:1, or 1:1.5-6:1, more preferably 1:1-5.5:1, 1.5:1-5:1, 1.75:1-4.5:1, 2:1-4:1, 2.2:1-3.5:1, or 2.4:1-3:1, and most preferably 2.5:1-2.7:1.

[0058] The outer layer comprising the amphiphilic copolymer preferably comprises a thickness of at least 0.5 nm, at least 1 nm or at least 1.5 nm, more preferably at least 2 nm or at least 2.5 nm, and most preferably at least 3 nm. The outer layer comprising the amphiphilic copolymer preferably comprises a thickness of less than 25 nm, less than 20 nm or less than 15 nm, more preferably less than 10 nm or less than 7.5 nm, and most preferably less than 5 nm. The outer layer comprising the amphiphilic copolymer preferably comprises a thickness of 0.5 to 25 nm, 1 to 20 nm, or 1.5 to 15 nm, more preferably 2 to 10 nm, or 2.5 to 7.5 nm, and most preferably 3 to 5 nm.

[0059] The submicron particle may further comprise at least one stabilizing molecule. The at least one stabilizing molecule may be surrounded by an outer layer comprising the amphiphilic copolymer. Alternatively or additionally, the at least one stabilizing molecule may be disposed outside the outer layer comprising the amphiphilic copolymer.

[0060] The weight ratio of stabilizing molecules to payload molecules can be at least 1:1, at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, or at least 95:1. In some embodiments, the weight ratio of stabilizing molecules to payload molecules can be at least 100:1, at least 250:1, at least 500:1, at least 1,000:1, at least 2,000:1, at least 3,000:1, at least 4,000:1, or at least 5,000:1. The weight ratio of stabilizing molecules to payload molecules can be less than 100,000: 1, less than 50,000: 1, less than 10,000: 1, less than 9,000: 1, less than 8,000: 1, less than 7,000: 1, or less than 6,500: 1. In some embodiments, the weight ratio of stabilizing molecules to payload molecules can be less than 5,000: 1, less than 1,000: 1, less than 500: 1, less than 400: 1, less than 350: 1, less than 300: 1, less than 250: 1, less than 200: 1, less than 175: 1, less than 150: 1, less than 125: 1, or less than 110: 1. The weight ratio of stabilizing molecules to payload molecules can be from 1:1 to 100,000:1, 2:1 to 50,000:1, 4:1 to 10,000:1, 6:1 to 9,000:1, 8:1 to 8,000:1, 10:1 to 7,000:1, or 20:1 to 6,500:1. In some embodiments, the weight ratio of stabilizing molecules to payload molecules can be 6:1 to 5,000:1, 8:1 to 1,000:1, 10:1 to 500:1, 20:1 to 400:1, 30:1 to 350:1, 40:1 to 300:1, 50:1 to 250:1, 60:1 to 200:1, 70:1 to 175:1, 80:1 to 150:1, 90:1 to 125:1, or 95:1 to 110:1. In alternative embodiments, the weight ratio of stabilizing molecules to payload molecules can be from 90:1 to 100,000:1, 100:1 to 50,000:1, 500:1 to 25,000:1, 1,000:1 to 10,000:1, 2,000:1 to 9,000:1, 3,000:1 to 8,000:1, 4,000:1 to 7,000:1, or 5,000:1 to 6,500:1.These weight ratios may relate to the total weight of stabilizing molecules to payload molecules, i.e., they may include the weight of any stabilizing molecules surrounded by the outer layer as well as any stabilizing molecules disposed outside of the outer layer.

[0061] In the embodiment where at least one stabilizing molecule is surrounded by an outer layer comprising an amphiphilic copolymer, it may be encapsulated in the lipid structure.Alternatively or additionally, at least one stabilizing molecule surrounded by an outer layer comprising an amphiphilic copolymer may be disposed outside the lipid structure.In the embodiment where the submicron particle comprises a plurality of lipid structures, at least one stabilizing molecule may be disposed between the plurality of lipid structures.

[0062] The weight ratio of stabilizing molecules surrounded by the outer layer to payload molecules can be at least 1:1, at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, or at least 95:1. The weight ratio of stabilizing molecules surrounded by the outer layer to payload molecules can be less than 100,000:1, less than 50,000:1, less than 10,000:1, less than 5,000:1, less than 1,000:1, less than 500:1, less than 400:1, less than 350:1, less than 300:1, less than 250:1, less than 200:1, less than 175:1, less than 150:1, less than 125:1, or less than 110:1. The weight ratio of stabilizing molecules to payload molecules surrounded by the outer layer can be 1:1 to 100,000:1, 2:1 to 50,000:1, 4:1 to 10,000:1, 6:1 to 5,000:1, 8:1 to 1,000:1, 10:1 to 500:1, 20:1 to 400:1, 30:1 to 350:1, 40:1 to 300:1, 50:1 to 250:1, 60:1 to 200:1, 70:1 to 175:1, 80:1 to 150:1, 90:1 to 125:1, or 95:1 to 110:1.

[0063] Alternatively or additionally, the at least one stabilizing molecule may be disposed outside the outer layer comprising the amphiphilic copolymer. The at least one stabilizing molecule disposed outside the outer layer comprising the amphiphilic copolymer may be disposed at a concentration of at least 1 mg / ml, at least 5 mg / ml, at least 10 mg / ml or at least 50 mg / ml, more preferably at least 100 mg / ml, at least 150 mg / ml, at least 200 mg / ml or at least 220 mg / ml, most preferably at least 240 mg / ml. The at least one stabilizing molecule disposed outside the outer layer comprising the amphiphilic copolymer may be disposed at a concentration of less than 100,000 mg / ml, less than 50,000 mg / ml, less than 10,000 mg / ml, less than 5,000 mg / ml or less than 1,000 mg / ml, more preferably less than 750 mg / ml, less than 500 mg / ml, less than 300 mg / ml or less than 280 mg / ml, most preferably less than 260 mg / ml. The at least one stabilizing molecule disposed on the outside of the outer layer comprising an amphiphilic copolymer may be disposed at a concentration of 1-100,000 mg / ml, 5-50,000 mg / ml, 10-10,000 mg / ml, 25-5,000 mg / ml, or 50-1,000 mg / ml, more preferably 100-750 mg / ml, 150-500 nm / ml, 200-300 nm / ml, or 220-280 nm / ml, and most preferably 240-260 mg / ml.

[0064] In a preferred embodiment, the submicron particle comprises at least one stabilizing molecule surrounded by an outer layer comprising an amphiphilic copolymer, the at least one stabilizing molecule being disposed outside of the outer layer comprising the amphiphilic copolymer.

[0065] The or each stabilising molecule may be a carbohydrate and / or a polyol.

[0066] The carbohydrate may be referred to as a sugar. The carbohydrate may be a monosaccharide, which may be selected from the group consisting of glucose, galactose, fructose, mannose and xylose, or a pharmaceutically acceptable complex, salt, solvate, tautomeric form, stereoisomer or polymorph thereof. Alternatively, the carbohydrate may be a disaccharide, which may be selected from the group consisting of trehalose, sucrose, lactose, maltose, isomaltose, lactitol, lactulose, mannobiose and isomalt, or a pharmaceutically acceptable complex, salt, solvate, tautomeric form, stereoisomer or polymorph thereof. In a further alternative, the carbohydrate may be a trisaccharide, which may be selected from the group consisting of nigerotriose, maltotriose, melezitose, maltotriulose, raffinose and kestose, or a pharmaceutically acceptable complex, salt, solvate, tautomeric form, stereoisomer or polymorph thereof. In a further alternative, the carbohydrate may be a polysaccharide which may be selected from the group consisting of dextran, amylose, amylopectin, glycogen, galactogen, inulin, callose, cellulose, chitosan and chitin or a pharma-ceutically acceptable complex, salt, solvate, tautomeric form, stereoisomer or polymorph thereof.

[0067] The carbohydrate may be a polyol which may be selected from the group consisting of sorbitol, mannitol, glycerol, alpha-D-glucopyranosyl-1-6-sorbitol, alpha-D-glucopyranosyl-1-6-mannitol, malto-oligosaccharides, hydrogenated malto-oligosaccharides, starch and cellulose or a pharma- ceutically acceptable complex, salt, solvate, tautomeric form, stereoisomer or polymorph thereof.

[0068] Alternatively or additionally, the polyol can be an oligomer containing multiple hydroxyl groups, a polymer containing multiple hydroxyl groups, or a pharma- ceutically acceptable complex, salt, solvate, tautomer, stereoisomer or polymorph thereof.

[0069] The at least one stabilizing molecule may include at least two different stabilizing molecules. Each stabilizing molecule may be a carbohydrate. In one embodiment, the first stabilizing molecule may be a disaccharide (e.g., trehalose) and the second stabilizing molecule may be a polysaccharide (e.g., dextran).

[0070] In some embodiments, the carbohydrate is a disaccharide, most preferably trehalose, or a pharma- ceutically acceptable complex, salt, solvate, tautomeric form, stereoisomer or polymorph thereof. The trehalose may be synthetic or natural trehalose.

[0071] The submicron particle may include at least one targeting ligand or targeting moiety. The at least one targeting ligand or targeting moiety may be disposed on the outer surface of the submicron particle. Thus, the at least one targeting ligand or targeting moiety may be disposed on the outer surface of the outer layer that includes the amphiphilic copolymer.

[0072] The at least one targeting ligand or targeting moiety may be or may include at least one of a peptide, a protein, an aptamer, a carbohydrate, an oligosaccharide, folic acid or folate, and an antibody or an antigen-binding fragment thereof, a vitamin or a derivative thereof. The peptide may be a G protein-coupled receptor (GCR), Arg-Gly-Asp (RGD), or a derivative thereof. The protein may be a lectin, transferrin, or a derivative thereof. The aptamer may be an RNA aptamer against an HIV glycoprotein, or a derivative thereof. The carbohydrate may be as defined above. In particular, the carbohydrate may be mannose, glucose, galactose, or a derivative thereof. The antibody may be monoclonal or polyclonal. The antibody may be an anti-Her2 antibody, an anti-EGFR antibody, or a derivative thereof. The vitamin may be vitamin D.

[0073] The submicron particles may be stored in a solution. The solution may be an aqueous solution. The submicron particles may be present at a concentration such that the concentration of the payload molecule is at least 0.001 μg / ml, at least 0.01 μg / ml, at least 0.05 μg / ml, at least 0.1 μg / ml, at least 0.5 μg / ml, at least 1 μg / ml, at least 5 μg / ml, at least 10 μg / ml, at least 15 μg / ml or at least 20 μg / ml. The submicron particles may be present at a concentration such that the concentration of the payload molecule is less than 500 mg / ml, less than 100 mg / ml, less than 10 mg / ml, less than 5 mg / ml, less than 1 mg / ml, less than 500 μg / ml, less than 200 μg / ml, less than 100 μg / ml, less than 50 μg / ml or less than 30 μg / ml. The submicron particles may be present in a concentration such that the concentration of the payload molecule is 0.001 μg / ml to 500 mg / ml, 0.01 μg / ml to 100 mg / ml, 0.05 μg / ml to 50 mg / ml, 0.1 μg / ml to 10 mg / ml, 0.5 μg / ml to 5 mg / ml, 1 μg / ml to 1 mg / ml, 5 to 500 μg / ml, 10 to 200 μg / ml, 15 to 100 μg / ml, 20 to 50 μg / ml, or 20 to 30 μg / ml.

[0074] Alternatively, the submicron particles can be freeze-dried.

[0075] Preferably, the submicron particles of the first aspect are thermally stabilized.

[0076] It will be understood that the expression "thermostabilization" or "thermally stabilized" may mean that the submicron particle substantially retains its biological activity (e.g., induces an immune response and / or protein expression in a subject administered thereto) when stored at a certain temperature for a certain period of time. Although the inventors do not wish to be bound by any hypothesis, they believe that the thermostabilization effect may be achieved by stabilizing the lipid structure in the formulation, for example, by preventing or reducing its aggregation, by stabilizing the payload molecule (preferably RNA) itself, and / or by stabilizing the submicron particle to have improved colloidal stability. Whether or to what extent the functional activity of the payload molecule (preferably RNA) is retained may be determined, for example, by detecting the presence of immunospecific antibodies (e.g., IgG) produced against the antigen of interest encoded by the RNA construct and / or by detecting the expression of the protein of interest.

[0077] Preferably, the submicron particles are thermally stabilized after storage at temperatures above -100°C, above -80°C, above -60°C, above -40°C or above -20°C, more preferably above -15°C, and most preferably above -10°C. Preferably, the submicron particles are thermally stabilized after storage at temperatures above -5°C, more preferably above 0°C, and most preferably above 1°C. Most preferably, the submicron particles are thermally stabilized after storage at temperatures above 2°C, more preferably above 3°C, and most preferably above 4°C. Even more preferably, the submicron particles are thermally stabilized after storage at temperatures above 5°C, more preferably above 6°C, and most preferably above 7°C.

[0078] The submicron particles may be thermally stabilized after storage at temperatures below 100° C., below 80° C., below 60° C., below 50° C., below 40° C., below 35° C. or below 30° C. The submicron particles may be thermally stabilized after storage at temperatures below 25° C., below 20° C. or below 15° C. The submicron particles may be thermally stabilized after storage at temperatures below 10° C., below 8° C. or below 7° C.

[0079] The submicron particles may be thermally stabilized upon storage at temperatures between -100° C. and 100° C., between -80° C. and 90° C., between -60° C. and 80° C., between -40° C. and 70° C., between -20° C. and 60° C., between -20° C. and 50° C., between -20° C. and 40° C., between -20° C. and 35° C., between -20° C. and 30° C., between -15° C. and 25° C., or between -10° C. and 20° C. The submicron particles may be thermally stabilized upon storage at temperatures between -5° C. and 15° C., between 0° C. and 10° C., between 1° C. and 9° C., or between 2° C. and 8° C.

[0080] According to a second aspect, there is provided a method of producing a submicron particle, the method comprising contacting a payload molecule, a cationic or ionizable lipid, and an amphiphilic copolymer to produce the submicron particle.

[0081] Advantageously, the method provides a one-pot method for providing the submicron particles of the first aspect.

[0082] Preferably, the payload molecule, the cationic or ionizable lipid, and the amphipathic copolymer are contacted simultaneously. The payload molecule, the cationic or ionizable lipid, and the amphipathic copolymer may be understood to be contacted simultaneously if they are all present in the same reaction mixture.

[0083] The payload molecule, the cationic or ionizable lipid, and the amphipathic copolymer may be as defined in relation to the first embodiment. Furthermore, the payload molecule, the cationic or ionizable lipid, and the amphipathic copolymer may be provided in a ratio as defined in relation to the first embodiment.

[0084] The amphiphilic copolymers may be synthesised using any method known in the art, for example, the amphiphilic copolymers may be synthesised using the methods defined in the applicant's earlier patent application, GB2009720.0, although it will be appreciated that alternative methods may be used.

[0085] The method may include providing a first solution comprising a cationic lipid or ionizable lipid and an amphiphilic copolymer. The first solution may include an organic solvent. The organic solvent may be an ether, an alcohol, or a nitrile. The ether may be a cyclic ether. The organic solvent may be tetrahydrofuran (THF), ethanol, methanol, and acetonitrile.

[0086] The method can include providing a second solution comprising the payload molecule. The second solution can comprise water, preferably ribonuclease (RNase) free water.

[0087] Contacting the payload molecule, the cationic or ionizable lipid, and the amphipathic copolymer may include combining the first and second solutions to generate a reaction mixture, thereby contacting the payload molecule, the cationic or ionizable lipid, and the amphipathic copolymer.

[0088] The method may include stirring the reaction mixture.

[0089] The method may include contacting the payload molecule, the cationic or ionizable lipid, and the amphipathic copolymer for at least 15 seconds, at least 30 seconds, at least 45 seconds, or at least 1 minute. The method may include contacting the payload molecule, the cationic or ionizable lipid, and the amphipathic copolymer for 15 seconds to 30 minutes, 30 seconds to 10 minutes, 45 seconds to 5 minutes, or 1 to 2 minutes.

[0090] The method may include contacting the payload molecule, the cationic or ionizable lipid, and the amphiphilic copolymer at a temperature between 0°C and 75°C, between 5°C and 50°C, between 10°C and 30°C, between 15°C and 25°C, or between 19°C and 21°C.

[0091] The method may subsequently include removing the organic solvent, which may be removed by rotary evaporation.

[0092] Contacting the payload molecule, the cationic lipid or ionizable lipid, and the amphipathic copolymer can include contacting the payload molecule, the cationic lipid or ionizable lipid, the amphipathic copolymer, and a stabilizing molecule. The payload molecule, the cationic lipid or ionizable lipid, the amphipathic copolymer, and the stabilizing molecule may be contacted simultaneously.

[0093] Advantageously, in the resulting submicron particles, the stabilizing molecules are surrounded by an outer layer comprising the amphiphilic copolymer.

[0094] The stabilising molecule may be as defined in relation to the first aspect.

[0095] In embodiments in which the method includes providing a second solution, the second solution may further comprise a stabilizing molecule.

[0096] The weight ratio of stabilizing molecules to payload molecules in the second solution and / or in the reaction mixture can be at least 1:1, at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1 or at least 95:1. The weight ratio of stabilizing molecules to payload molecules in the second solution and / or in the reaction mixture can be less than 100,000:1, less than 50,000:1, less than 10,000:1, less than 5,000:1, less than 1,000:1, less than 500:1, less than 400:1, less than 350:1, less than 300:1, less than 250:1, less than 200:1, less than 175:1, less than 150:1, less than 125:1, or less than 110:1. The weight ratio of stabilizing molecules to payload molecules in the second solution and / or in the reaction mixture can be 1:1 to 100,000:1, 2:1 to 50,000:1, 4:1 to 10,000:1, 6:1 to 5,000:1, 8:1 to 1,000:1, 10:1 to 500:1, 20:1 to 400:1, 30:1 to 350:1, 40:1 to 300:1, 50:1 to 250:1, 60:1 to 200:1, 70:1 to 175:1, 80:1 to 150:1, 90:1 to 125:1, or 95:1 to 110:1.

[0097] Alternatively or additionally, the method may include contacting the obtained submicron particles with stabilizing molecules. The stabilizing molecules may be as defined in relation to the first aspect. Thus, at least one stabilizing molecule may be disposed outside the outer layer comprising the amphiphilic copolymer.

[0098] The method may include, after removing the organic solvent, contacting the resulting submicron particles with a stabilizing molecule.

[0099] The method may comprise contacting the resulting submicron particles with a stabilising molecule at a concentration to obtain a concentration of stabilising molecule as defined in relation to the first aspect.

[0100] The method may comprise storing the submicron particles in a solution. The solution may be as defined in relation to the first aspect.

[0101] Alternatively, the method may include drying the submicron particles. Preferably, drying the submicron particles includes freeze-drying the submicron particles.

[0102] According to a third aspect, there is provided a submicron particle obtained or obtainable by the method of the second aspect.

[0103] In a fourth aspect, there is provided a pharmaceutical composition comprising the submicron particles of the first or third aspect and a pharma- ceutically acceptable vehicle.

[0104] In a fifth aspect, there is provided a method of preparing a pharmaceutical composition according to the fourth aspect, the method comprising contacting submicron particles of the first or third aspect with a pharma- ceutically acceptable vehicle.

[0105] In a sixth aspect, there is provided a submicron particle of the first or third aspect, or a pharmaceutical composition of the fourth aspect, for use as a medicament.

[0106] In a seventh aspect, there is provided a method of treatment comprising administering or having administered to a subject in need thereof a therapeutic amount of submicron particles of the first or third aspect, or a pharmaceutical composition of the fourth aspect.

[0107] In an eighth aspect there is provided a vaccine composition comprising the submicron particles of the first or third aspect or the pharmaceutical composition of the fourth aspect.

[0108] The vaccine may include a suitable adjuvant.

[0109] The vaccine may be a vaccine for COVID-19. The vaccine may be a vaccine for influenza virus.

[0110] In a ninth aspect, there is provided a submicron particle of the first or third aspect, a pharmaceutical composition of the fourth aspect, or a vaccine of the eighth aspect, for use in stimulating an immune response in a subject.

[0111] The immune response may be stimulated against a protozoan, a bacterial, a viral, a fungal or a cancer. The virus may be COVID-19. The virus may be an influenza virus.

[0112] In a tenth aspect of the invention there is provided a method of vaccinating a subject, the method comprising administering or having administered to a subject in need thereof a therapeutic amount of submicron particles of the first or third aspect, a pharmaceutical composition of the fourth aspect or a vaccine of the eighth aspect.

[0113] The submicron particles, pharmaceutical compositions or vaccines of the present invention can be combined into compositions having several different forms, in particular depending on the manner in which the composition is used.Thus, for example, the composition can be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle solution, transdermal patch, liposomal suspension, or any other suitable form that can be administered to a person or animal in need of treatment.It will be understood that the vehicle of the pharmaceutical product according to the present invention must be well tolerated by the subject to which it is given.

[0114] The submicron particles, pharmaceutical compositions or vaccines of the present invention may be incorporated into a sustained or delayed release device. Such a device may be inserted, for example, above or below the skin, and the pharmaceutical agent may be released over a period of weeks or months. The device may be placed at least adjacent to the treatment site.

[0115] However, in a preferred embodiment, the medicament according to the invention may be administered to a subject by injection into the bloodstream, muscle, skin, or directly into the site requiring treatment. Injection may be intravenous (bolus or infusion), subcutaneous (bolus or infusion), intradermal (bolus or infusion), intramuscular (bolus or infusion), intrathecal (bolus or infusion), epidural (bolus or infusion) or intraperitoneal (bolus or infusion).

[0116] It will be understood that the amount of submicron particle, pharmaceutical composition or vaccine required will be determined by its biological activity and bioavailability, which will depend on the mode of administration, the physicochemical properties of the submicron particle, pharmaceutical composition or vaccine, and whether it is being used as a monotherapy or in a combination therapy.

[0117] The frequency of administration is also affected by the half-life of the active agent in the subject being treated.The optimal dosage to be administered can be determined by those skilled in the art and varies depending on the submicron particles, pharmaceutical composition or vaccine being used, the strength of the pharmaceutical composition, the mode of administration, and the type of treatment.Additional factors that depend on the specific subject being treated, including subject age, weight, sex, diet, and administration time, will require the dosage to be adjusted.

[0118] The dosage required can depend on many factors, including but not limited to, the active agent being administered, the disease being treated and / or vaccinated against, the subject being treated, and the like.

[0119] Generally, depending on the active agent used, doses of 0.001 μg / kg body weight to 10 mg / kg body weight, or 0.01 μg / kg body weight to 1 mg / kg body weight of the submicron particles, pharmaceutical compositions or vaccines of the invention may be used. Dose may be understood to relate to the amount of payload molecules delivered.

[0120] The dose may be given as a single administration (e.g., a single injection). Alternatively, the submicron particles, pharmaceutical composition or vaccine may require multiple administrations. By way of example, the submicron particles, pharmaceutical composition or vaccine may be administered as two or more doses of 0.07 μg to 700 mg (i.e., assuming a body weight of 70 kg). Alternatively, a sustained release device may be used to provide the patient with an optimal dose of the submicron particles, pharmaceutical composition or vaccine according to the present invention without the need to administer repeated doses. The route of administration may incorporate an intravenous injection route, an intradermal injection route, a subcutaneous injection route, an intramuscular injection route, an intrathecal injection route, an epidural injection route or an intraperitoneal injection route.

[0121] Known procedures, e.g., procedures conventionally used by the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), may be used to form the submicron particles, pharmaceutical compositions or vaccines according to the invention, specific formulations, and precise treatment regimens (e.g., drug doses and frequency of administration).

[0122] A "subject" may be a vertebrate, a mammal or a livestock animal. Thus, the compositions and medicaments according to the invention may be used to treat any mammal, such as livestock (e.g., horses), pets, or for other veterinary purposes. Most preferably, however, the subject is a human.

[0123] A "therapeutically effective amount" of a submicron particle, pharmaceutical composition or vaccine is any amount, as described above, necessary to produce a therapeutic effect when administered to a subject.

[0124] For example, a therapeutically effective amount of the submicron particles, pharmaceutical compositions and vaccines of the present invention may contain from about 0.001 mg to about 800 mg of the payload molecule, preferably from about 0.01 mg to about 500 mg of the payload molecule.

[0125] A "pharmaceutically acceptable vehicle" as referred to herein is any known compound or combination of known compounds known to those skilled in the art to be useful in formulating a pharmaceutical composition.

[0126] In one embodiment, the pharma- ceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder, capsule, or tablet. A solid pharma- ceutically acceptable vehicle may contain one or more substances that may act as flavorings, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet disintegrants. The vehicle may also be an encapsulating material. In a powder, the vehicle is a finely divided solid mixed with a finely divided active agent according to the present invention. In a tablet, the active agent (e.g., the submicron particles of the present invention) may be mixed with a vehicle having the necessary compression properties in a suitable ratio and compressed into the desired shape and size. The pharmaceutical vehicle may be a gel, and the composition may be in the form of a cream, etc.

[0127] Alternatively, the pharmaceutical vehicle may be liquid and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used to prepare solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The submicron particles according to the present invention may be dissolved or suspended in a pharma- ceutically acceptable liquid vehicle, such as water, an organic solvent, a mixture of both, or a pharma-ceutically acceptable oil or fat. The liquid vehicle may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity adjusters, stabilizers or osmolality adjusters. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as described above, such as cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric and polyhydric alcohols, such as glycols) and their derivatives, and oils (such as fractionated coconut oil and peanut oil). For parenteral administration, the vehicle may be an oily ester, for example, ethyl oleate and isopropyl myristate. In sterile liquid form compositions for parenteral administration, sterile liquid vehicles are useful. The liquid vehicle for pressurized compositions may be halogenated hydrocarbon or other pharma- ceutically acceptable propellants.

[0128] Liquid pharmaceutical compositions that are sterile solutions or suspensions can be utilized, for example, by intramuscular, intrathecal, epidural, intraperitoneal, intravenous and subcutaneous injection. The submicron particles of the present invention can be prepared in any suitable sterile injectable medium.

[0129] The submicron particles may be administered by inhalation, for example, the submicron particles may be provided in the form of an aerosol.

[0130] The submicron particles and / or pharmaceutical compositions of the invention may be orally administered in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., sufficient saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monooleate, polysorbate 80 (oleic acid ester of sorbitol and its anhydride copolymerized with ethylene oxide), and the like. The submicron particles of the invention, and / or pharmaceutical compositions according to the invention may also be orally administered in the form of a liquid or solid composition. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, as well as liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0131] All of the features described in this specification (including the accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above embodiments in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0132] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: [Brief description of the drawings]

[0133] [Figure 1] (A) Schematic diagram of the preparation procedure of polymer-encapsulated lipid nanoparticles (PE-LNPs) and (B) PE-LNP-mediated intracellular delivery of RNA, including messenger RNA (mRNA) or self-amplifying RNA (saRNA, its self-amplifying step is shown in brackets). PE-LNPs were prepared using PEG5k-PCL10k and DOTAP unless specified. [Diagram 2] (A) 1H-NMR spectrum of PEG5k-PCL10k polymer; (B) dynamic light scattering (DLS) size distribution of PE-LNP 18-80 composed of PEG5k-PCL10k, DOTAP and saRNA; and (C) DLS size, zeta potential and polydispersity (PDI) of various PE-LNPs. [Diagram 3] Figure 1 shows the encapsulation efficiency of PE-LNPs with different N / P molar ratios (PEG5k-PCL10k / saRNA weight ratio = 65) as determined by RiboGreen Assay. [Figure 4] SEM and Cryo-TEM micrographs of PE-LNP 5'-65', PE-LNP 11'-65', PE-LNP 18'-65' and PE-LNP 18'-80' are shown. The apostrophe symbol indicates the absence of payload in PE-LNP. [Diagram 5] PE-LNP 11'-65'. (A) DLS size and (B) PDI changes upon thermal gradient for PE-LNP 11'-80', PE-LNP 18'-65' and PE-LNP 18'-80'. The apostrophe symbol indicates the absence of payload in PE-LNP. [Figure 6](A) is a schematic diagram of a uniform polymer-lipid hybrid membrane showing a similar distance between NBD (donor dye) and Rhod (acceptor dye) and therefore a similar fluorescence resonance energy transfer (FRET) efficiency compared to a pure lipid membrane. (B-D) show the fluorescence spectra and FRET efficiency of PE-LNP (PE-LNP 5'-65', PE-LNP 11'-65' and PE-LNP 18'-65') and DOTAP liposomes. The apostrophe symbol suggests the absence of payload in PE-LNP. The small numbers after DOTAP in each sample group, i.e., 143, 358 and 572, respectively, indicate the equimolar concentration of DOTAP present in the corresponding PE-LNP. The large numbers after DOTAP in each sample group, i.e., 186, 401 and 615, respectively, indicate the total molar concentration of DOTAP and PEG5k-PCL10k present in the corresponding PE-LNP. The concentrations of NBD-PE (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) and Rhod-PE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl), ammonium salt) in PE-LNP or DOTAP liposomes were fixed at 4.0 mM, and their fluorescence intensities were measured at an excitation wavelength of 460 nm. The significant difference in FRET efficiency suggests that a uniform polymer-lipid hybrid membrane was not present. [Figure 7](A) is a schematic diagram of the absence of lipid mixing when DOTAP is present in the core of PE-LNP, resulting in minor FRET. (B,C) show the fluorescence spectra and FRET efficiency of PE-LNP 11'-65' and DOTAP liposomes at intervals of 0, 1 and 2 h. The apostrophe symbol suggests the absence of payload in PE-LNP. The number after DOTAP, i.e., 358, indicates the equimolar concentration of DOTAP present in PE-LNP 11'-65'. The concentrations of NBD-PE and Rhod-PE in PE-LNP 11'-65' or DOTAP liposomes were fixed at 4.0 mM, and their fluorescence intensities were measured at an excitation wavelength of 460 nm. The negligible FRET after mixing PE-LNP 11'-65'(NBD-PE) and PE-LNP 11'-65'(Rhod-PE) for 2 h suggests the presence of DOTAP lipids within the core of PE-LNP. [Figure 8] (A, B) Graphs of HEK293 cell transfection efficiency of saRNA-added PE-LNPs with different N / P molar ratios in the absence of fetal bovine serum (FBS), and HEK293 cell transfection efficiency of saRNA-added PE-LNPs with different PEG-PCL / saRNA (w / w) ratios in the absence (C, D) or presence (E, F) of FBS, expressed as relative luminescence units (RLU) determined by firefly luciferase (fLuc) assay. The saRNA dose was fixed at 1 μg mL-1. [Figure 9] Comparing HEK293 cell transfection efficiency of saRNA-loaded PE-LNP 18-80 (saRNA in nanoparticle core) and saRNA-bound PE-LNP 18-80 M2 (saRNA on nanoparticle surface) in (C) RNase-free water or (D) RNase-free PBS in (A) the absence or (B) presence of FBS, and after 5 days of storage at 4 °C. The saRNA dose was fixed at 1 μg mL-1. Single (*), double (**), triple (***) and quadruple asterisk symbols (****) indicate p<0.05, p<0.01, p<0.001 and p<0.0001, respectively, and NS represents no significant difference between the two groups. [Figure 10] Figure 1 shows the HeLa cell transfection efficiency of saRNA-loaded PE-LNPs with polymer / saRNA weight ratios (N / P molar ratio = 11 or 18) of (A) 65 and (B) 80. saRNA doses of 0.1, 0.5, 1, 3, 5 and 7 μg mL-1 were tested in a firefly luciferase (fLuc) assay. [Figure 11] (A) Fluorescence microscopy images showing Jurkat cells transfected with various PE-LNPs supplemented with messenger RNA (mRNA) encoding green fluorescent protein (GFP) in RPMI-1640 medium; (B, C) percentage of GFP-expressing Jurkat cells compared to negative control (cells treated with medium only) measured using a Cell Viability Analyzer after 4 h of PE-LNP treatment in OPTIMEM medium, and mean fluorescence intensity (MFI) and (D) flow cytometry analysis of cell viability. The mRNA dose was fixed at 2 μg mL-1. Single (*) and quadruple asterisk symbols (****) indicate p<0.05 and p<0.0001, respectively, and NS represents no significant difference between the two groups. [Figure 12] (A) Confocal microscopy images showing the intracellular distribution of PE-LNP 18-80 (coloaded with 1 μg mL-1 saRNA and 50 μg mL-1 FITC) in HEK293 cells over 4 h at 4 or 37 °C. The yellow line indicates the cross section used to generate the intensity profile shown in (B) using ImageJ. (C) Confocal microscopy images showing the intracellular distribution of saRNA and FITC coloaded PE-LNP 18-80 in HEK293 cells in the presence of the endocytosis inhibitor MβCD. (D) Flow cytometry analysis of endocytosis of saRNA and FITC coloaded PE-LNP 18-80 in the presence of various endocytosis inhibitors. Double asterisks (**) indicate p<0.01. [Figure 13](A) Hemolysis after 1 h incubation at 37 °C with various PE-LNPs spiked with 1 μg mL-1 saRNA; and (B) graph of viability of HEK293 cells treated with saRNA-spiked PE-LNPs over 24 h compared to negative control (cells treated with media only) measured using the alamarBlue assay. Double asterisks (**) indicate p<0.01. [Figure 14] (A) Visualization of fLuc bioluminescence and (B) quantification of fLuc expression in Balb / C female mice 7 days after intramuscular (IM) injection of 5 μg of fLuc saRNA formulated inside or on the surface of PE-LNPs into both hind quadriceps; (C) Schematic of immunogenicity experiment in Balb / C female mice immunized IM in one hind quadriceps with 1 μg of saRNA encoding the H1 hemagglutinin of Cal / 09 virus (HA saRNA) formulated inside various PE-LNPs and boosted 4 weeks later with the same formulation; (D) HA antigen-specific IgG antibody titers after immunization of mice with prime and boost of HA saRNA-supplemented jetPEI and various PE-LNPs by IM injection; and (E) change in body weight after intranasal (IN) challenge with Cal / 09 influenza virus in IM-injected Balb / C female mice. A single asterisk (*) indicates p<0.05. [Figure 15] Shown are SARS-CoV-2 (COVID-19) specific IgG antibody titers following immunization of mice with primed SARS-CoV-2 saRNA-spiked PE-LNP 11-65 and PE-LNP 18-65 by IM injection. [Figure 16] Graphs of saRNA-loaded PE-LNP 11-65 composed of PEG5k-PCL8.5k and PEG5k-PCL10k, respectively: (A) DLS size of fresh samples, (B) PDI and (C) Zeta potential, and (D) HEK293 cell transfection efficiency of samples after 21 days storage in aqueous solution at 4 °C. The saRNA dose was fixed at 1 μg mL-1. [Figure 17]Figure 1 shows stable storage of 40 μg mL-1 saRNA formulated in PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80, respectively, in aqueous solution at 4 °C. The graph shows HEK293 cell transfection efficiency after 3 months of storage in aqueous solution at 4 °C. The saRNA dose was fixed at 1 μg mL-1. [Figure 18] Figure 2 shows the HEK293 cell transfection efficiency of 40 μg mL-1 saRNA formulated in PE-LNP 11-65 after storage in aqueous solution at room temperature for 21 days. The saRNA dose was fixed at 1 μg mL-1. NS indicates no significant difference between the two groups. [Figure 19] Stable storage of 40 μg mL-1 saRNA formulated in PE-LNP 11-65, 18-65 and 18-80, respectively, in aqueous solution at room temperature. Changes in (A) transfection efficiency, (B) DLS size, (C) PDI and (D) zeta potential were recorded after 28 days of storage at room temperature in aqueous solution. For transfection efficiency, the saRNA dose was fixed at 1 μg mL-1. [Figure 20] A schematic diagram of the structure and preparation process of saRNA and trehalose co-loaded PE-LNPs is shown. [Figure 21] Figure 1 shows stable storage at 4 °C of 40 μg mL-1 of saRNA formulated in PE-LNP 11-65 in aqueous solution containing trehalose: (i) saRNA and trehalose co-loaded PE-LNP 11-65 (trehalose / saRNA weight ratio = 100 for coencapsulation, then mixed with additional trehalose to replenish total trehalose to 250 mg mL-1); (ii) saRNA-loaded PE-LNP 11-65 mixed with 250 mg mL-1 of external trehalose. The graph shows HEK293 cell transfection efficiency after 383 days of storage in aqueous solution at 4 °C. The saRNA dose was fixed at 1 μg mL-1. [Figure 22]Stable storage of saRNA at 40 μg mL-1 with trehalose co-addition in PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80 at room temperature in aqueous solution (trehalose / saRNA weight ratio = 100 for co-encapsulation, followed by mixing with additional trehalose to replenish total trehalose to 250 mg mL-1). After storage at room temperature in aqueous solution for 28 days, the changes in (A) transfection efficiency, (B) DLS size, (C) PDI and (D) zeta potential were recorded. For transfection efficiency, the saRNA dose was fixed at 1 μg mL-1. [Figure 23] Figure 1 shows the effect of lyophilization conditions by varying the concentration of saRNA formulated in PE-LNP in the presence of 200 mg mL-1 of external trehalose. The graph shows the HEK293 cell transfection efficiency of lyophilized PE-LNP 11-65 after immediate rehydration with RNase-free water. Freshly prepared PE-LNP 11-65 (without trehalose) at an equivalent saRNA dose of 1 μg mL-1 was used as a negative control. Single (*) and triple asterisk symbols (***) indicate p<0.05 and p<0.001, respectively. [Figure 24] Figure 1 shows the effect of lyophilization conditions by varying the concentration of external trehalose of PE-LNP 11-65 spiked with 40 μg mL-1 saRNA. The graph shows the HEK293 cell transfection efficiency of lyophilized PE-LNP 11-65 after immediate rehydration with RNase-free water. Freshly prepared PE-LNP 11-65 (without trehalose) with an equivalent saRNA dose of 1 μg mL-1 was used as a negative control. Single (*) and double asterisk symbols (**) indicate p<0.05 and p<0.01, respectively. [Diagram 25]Figure 1 shows stable storage at 4°C after lyophilization of 40 μg mL-1 of saRNA formulated in PE-LNP 11-65 in the presence of 250 mg mL-1 of external trehalose. The graph shows HEK293 cell transfection efficiency of lyophilized PE-LNP 11-65 after 28 days of storage at 4°C and rehydration with RNase-free water. Freshly prepared PE-LNP 11-65 (without trehalose) at an equivalent saRNA dose of 1 μg mL-1 was used as a negative control. NS indicates no significant difference between the two groups. [Figure 26] The effect of lyophilization conditions by varying the trehalose / saRNA weight ratio is shown. To prepare saRNA and trehalose co-loaded PE-LNP 11-65, trehalose was pre-dissolved in 40 μg mL-1 of saRNA solution in RNase-free deionized water at various trehalose / saRNA weight ratios. Additional trehalose was then added to the exterior of the resulting nanoparticles to replenish the total trehalose (both inside and outside the nanoparticles) to 250 mg mL-1. The graph shows the HEK293 cell transfection efficiency of lyophilized saRNA and trehalose co-loaded PE-LNP 11-65 after immediate rehydration with RNase-free water. Freshly prepared PE-LNP 11-65 (without trehalose) with an equivalent saRNA dose of 1 μg mL-1 was used as a negative control. Triple (***) and quadruple asterisk symbols (****) indicate p<0.001 and p<0.0001, respectively. [Figure 27]Figure 1 shows stable storage at 4 °C after lyophilization of 40 μg mL-1 of saRNA formulated in PE-LNP 11-65 in the presence of trehalose. The graph shows HEK293 cell transfection efficiency of two different PE-LNP 11-65 samples after lyophilization, storage at 4 °C for 380 days, and subsequent rehydration: (i) PE-LNP 11-65 with saRNA and trehalose (trehalose / saRNA weight ratio = 100 for coencapsulation, then mixed with additional trehalose to replenish total trehalose to 250 mg mL-1); (ii) PE-LNP 11-65 with saRNA mixed with 250 mg mL-1 of external trehalose. The saRNA dose was fixed at 1 μg mL-1. [Figure 28] 40 μg mL-1 of saRNA formulated in trehalose-containing PE-LNP 11-65 was shown to be stable for storage at 40°C after lyophilization. The graph shows the HEK293 cell transfection efficiency of three different PE-LNP 11-65 samples: (i) saRNA and trehalose co-loaded PE-LNP 11-65 after lyophilization, storage at 40°C for 0, 1, 3, 5 or 7 days, and rehydration without removing external trehalose (trehalose / saRNA weight ratio = 100 for coencapsulation, followed by mixing with additional trehalose to replenish total trehalose to 250 mg mL-1); (ii) saRNA-loaded PE-LNP 11-65 mixed with 250 mg mL-1 external trehalose after lyophilization, storage at 40°C for 0, 1, 3, 5 or 7 days, and rehydration without removing external trehalose; (iii) freshly prepared saRNA-loaded PE-LNP 11-65 (no trehalose) as a negative control. The saRNA dose was fixed at 1 μg mL-1. Single (*), double (**), triple (***) and quadruple asterisk symbols (****) indicate p<0.05, p<0.01, p<0.001 and p<0.0001, respectively. [Figure 29]Stable storage at 40°C after lyophilization of 40 μg mL-1 of saRNA formulated in PE-LNP 11-65 in the presence of internal and external trehalose. The graph shows HEK293 cell transfection efficiency of saRNA and trehalose co-loaded PE-LNP 11-65 (trehalose / saRNA weight ratio = 100 for coencapsulation, followed by mixing with additional trehalose to replenish total trehalose to 250 mg mL-1) after lyophilization, storage at 40°C for 14 days, and rehydration without removing external trehalose. Freshly prepared saRNA-loaded PE-LNP 11-65 (no trehalose) with an equivalent saRNA dose of 1 μg mL-1 was used as a negative control. NS represents no significant difference between the two groups. [Diagram 30] 40 μg mL-1 of saRNA formulated in trehalose-containing PE-LNP 11-65 was shown to be stable for storage at 40°C after lyophilization. The graph shows the HEK293 cell transfection efficiency of three different PE-LNP 11-65 samples: (i) saRNA and trehalose co-loaded PE-LNP 11-65 after lyophilization, storage at 40°C for 0, 1, 3, 5 or 7 days, rehydration, and then removal of external trehalose by ultrafiltration centrifugation (trehalose / saRNA weight ratio = 100 for coencapsulation, followed by mixing with additional trehalose to replenish total trehalose to 250 mg mL-1); (ii) saRNA-loaded PE-LNP 11-65 mixed with 250 mg mL-1 external trehalose after lyophilization, storage at 40°C for 0, 1, 3, 5 or 7 days, rehydration, and then removal of external trehalose by ultrafiltration centrifugation; (iii) freshly prepared saRNA-loaded PE-LNP 11-65 (no trehalose) as a negative control. The saRNA dose was fixed at 1 μg mL-1. Single (*) and double asterisk symbols (**) indicate p<0.05 and p<0.01, respectively. [Diagram 31](A) Confocal microscopy images of HEK293 cells at 4 hours after treatment with freshly prepared PE-LNP 11-65 with saRNA and calcein, or PE-LNP 11-65 with saRNA, trehalose, and calcein, and 2 hours after treatment, and (B) MFI of calcein in the confocal microscopy images analyzed by ImageJ. Quadruple asterisks indicate p<0.0001. [Diagram 32] (A) Graphs showing the percentage and MFI of GFP-expressing HEK293 cells treated with freshly prepared mRNA and trehalose co-loaded PE-LNP 11-65 (trehalose / mRNA weight ratio = 100 for coencapsulation, then mixed with additional trehalose to replenish total trehalose at 10 mg mL-1) and saRNA-loaded trehalose-free PE-LNP 11-65, respectively, as measured by flow cytometry. Single (*) and double asterisk symbols (**) indicate p<0.05 and p<0.01, respectively. [Diagram 33] (A) Transfection efficiency of saRNA-loaded PE-LNP 11-65 formulated with cholesterol, (B) Zeta potential, (C) PDI and (D) DLS size of PE-LNP 11'-65' formulated with cholesterol.

[0134] Example 1 - Synthesis of PEG-PCL copolymers with various molecular weights Stannous octoate (Sn(Oct)) as a catalyst 2 PEG-PCL copolymers with various molecular weights were synthesized by ring-opening polymerization of ε-caprolactone (ε-CL) initiated by mPEG-OH using mPEG-OH. 5K -OH, 470 mg ε-CL (or 200 mg mPEG 2K -OH, 530 mg ε-CL) and 50 mg Sn(Oct). 2 was dissolved in 5 mL of anhydrous toluene and the reaction was heated to 110 °C for 48 h under a dry nitrogen atmosphere. 22After the reaction, the mixture was degassed and cooled to room temperature. The resulting product was then precipitated using excess cold diethyl ether. The polymer was filtered and dried under vacuum at room temperature to a constant weight.

[0135] Characterization of PEG-PCL copolymers Using a Jeol 400MHz NMR spectrometer 1 H-NMR spectrum (CDCl 3 (at ) were recorded to characterize the chemical composition and degree of polymerization of the PCL blocks. Gel permeation chromatography (GPC) was used with an Agilent 1260 Infinity II to determine the molecular weight and molecular weight distribution of the polymers.

[0136] Results and Discussion Figure 1 is a schematic diagram of the preparation procedure of polymer-encapsulated lipid nanoparticles (PE-LNPs) and PE-LNP-mediated intracellular delivery of RNA, including mRNA and saRNA. PEG-PCL is a biodegradable and biocompatible polymer, and both PEG and PCL are approved by the FDA for human application. As reported in the literature, the molecular weight of the amphiphilic polymer plays an important role in the self-assembled structure. Amphiphilic copolymers could self-assemble into robust vesicular structures when the hydrophilic block accounted for 25-40% of their molecular weight. 23~25 The composition and molecular weight of the amphiphilic copolymer can be varied, which allows the formation of vesicles with various surface properties, film thickness and stability. n Since PEG having the formula (I) is one of the most widely used hydrophilic blocks in drug delivery systems, commercially available PEG (M n = 2,000 and 5,000 Da) were selected as initiators. Stannous octanoate (Sn(Oct) 2 We successfully synthesized PEG-PCL copolymers with two different molecular weights by ring-opening polymerization of ε-CL using PEG-PCL. 1 The H-NMR spectrum is shown in Figure 2A.1 The caprolactone unit (-COCH) at 4.06 ppm in the H-NMR spectrum 2 CH 2 CH 2 CH 2 CH 2 O-) methylene peak and ethylene glycol unit (-CH 2 CH 2 The number average molecular weight (M n ) were 7,873, 14,473, and 16,580 Da, respectively, which were in good agreement with the theoretical molecular weights (Table 1). 2k -PCL 5k Copolymer, PEG 2k -PCL 8.5k Copolymers and PEG 5k -PCL 10k The copolymers were successfully synthesized. Table 1 also shows that the PEG copolymers 2k -PCL 5k Copolymer, PEG 2k -PCL 8.5k Copolymers and PEG 5k -PCL 10k The molecular weights of the copolymers as well as their relatively low polydispersity (D = M w / M n ) is shown.

[0137] Table 1: PEG 2k -PCL 5k , PEG 5k -PCL 8.5k and PEG 5k -PCL 10k Theoretical molecular weight (M n,th ) and 1 H-NMR(M n,NMR ) and GPC(M n,GPC 、M w,GPC , polydispersity D=M w,GPC / Mn,GPC ) molecular weight measured by

[0138] [Table 1]

[0139] Example 2 - Preparation of saRNA-loaded PE-LNPs PE-LNPs were prepared by a simple one-pot method. Typically, 650 μg of PEG was added. 5k -PCL 10k and 250 μg of DOTAP were co-dissolved in 0.5 mL of THF, and 10 μg of saRNA was dissolved in 1 mL of RNase-free water. The organic and aqueous solutions were quickly mixed, and the mixture was kept stirring at room temperature for 1-2 min. Then, after removing the THF by rotary evaporation, saRNA-loaded PE-LNP 11-65 (N / P=11, polymer / saRNA (w / w)=65) was obtained. Various PE-LNPs with various N / P molar ratios or polymer / saRNA weight ratios were similarly prepared. Both chips and glassware should be treated before use to ensure RNase-free conditions.

[0140] Characterization of PE-LNPs The size distribution (Z-average) and zeta potential (based on the Smulochowski model) of the various PE-LNPs were determined at 25°C using a Zetasizer μV instrument (Malvern, UK) and ZETA PALS, respectively. The thermal stability of the PE-LNPs was assessed by measuring the size and polydispersity index (PDI) upon a temperature increase from 25°C to 85°C by high-throughput dynamic light scattering (HT-DLS) using a DynaPro Plate Reader III (Wyatt, UK).

[0141] The morphology of PE-LNPs was visualized using SEM. A drop of PE-LNP suspension was placed on a graphite surface. After drying, the sample was coated with gold using an Ion Sputter. The sample was then observed using a JSM-6400 scanning electron microscope (JEOL Ltd, Tokyo, Japan) at an accelerating voltage of 20 kV.

[0142] PE-LNP samples for Cryo-TEM (Tecnai F20 G2 at 200 kV) were prepared on lacey carbon-coated copper grids (Structure Probe Incorporation, PA) using a semi-automated Vitrobot system (Vitrobot Mark II, FEI). Briefly, 4 μL of 1 mg mL PE-LNPs were prepared on lacey carbon-coated copper grids (Structure Probe Incorporation, PA). -1 A PE-LNP solution of 100 μl was cast onto a carbon grid. The grid was then transferred to a Vitrobot chamber at 100% humidity and 20°C. Rapid immersion of the grid into liquid ethane after blotting for 1 s effectively vitrified the sample. To prevent the formation of crystalline ice, the sample was kept under -170°C using a Gatan 626 cryoholder until successful transfer to the Cryo-TEM instrument. Images were obtained at a defocus of approximately 4,000 nm.

[0143] The saRNA encapsulation efficiency of PE-LNPs was determined by RiboGreen Assay (Quant-iT™ RiboGreen™ RNA Assay Kit, Thermo Fisher). TE buffer and aqueous RiboGreen working solutions were prepared with and without 0.5% Triton X-100 according to the manufacturer's instructions. A calibration curve of fluorescence intensity versus saRNA concentration was established. The free (non-spiked) saRNA concentration (C unloaded) was determined as follows: samples were diluted to appropriate concentrations using TE buffer without Triton X-100, mixed with RiboGreen working solution without Triton X-100, and incubated in the dark for 15 min. Samples were then pipetted into a black 96-well plate with each well containing 200 μL of the final mixture. Fluorescence intensity measurements were then taken in triplicate by a spectrofluorometer (GloMax® Discover Microplate Reader, Promega, USA) at excitation and emission wavelengths of 480 and 520 nm, respectively. Total saRNA concentration (C) was determined following a similar procedure using TE buffer to dissolve the nanoparticles and RiboGreen working solution containing 0.5% Triton X-100. total ) was measured. The encapsulation efficiency of the system could be calculated according to the following formula:

[0144]

number

[0145] Results and Discussion Various saRNA-loaded PE-LNPs with various N / P molar ratios or polymer / saRNA (w / w) weight ratios were obtained. The PE-LNPs with N / P = 5 and polymer / saRNA (w / w) = 65 are named PE-LNP 5-65. The same rule is followed to assign other PE-LNPs. The size distribution, PDI and zeta potential of PE-LNPs with various compositions are summarized in Figure 2C.

[0146] The saRNA (approximately 9,500 nt) needs to be encapsulated in a carrier that can provide protection and facilitate the intracellular delivery of the saRNA. Figure 2B shows that the saRNA-loaded PE-LNPs were monodisperse. As listed in Figure 2C, PEG 5k -PCL 10k PE-LNPs assembled from PEG showed average hydrodynamic sizes ranging from 113.1 ± 0.3 to 134.2 ± 0.6 nm in diameter and low PDIs ranging from 0.21 ± 0.03 to 0.35 ± 0.02. 2k-PCL 5k PE-LNPs composed of PEG showed larger size (133.9±0.5-149.6±0.4 nm) and higher PDI (0.49±0.09-0.59±0.10). 5k -PCL 10k It was shown that PE-LNPs self-assembled from PEG showed a more stable structure, which could be attributed to the longer hydrophilic PEG chain length and thicker polymer capsule layer.

[0147] The N / P molar ratio is a key factor controlling saRNA loading capacity and endosomal escape efficiency. Increasing the N / P ratio from 5 to 18 increased the 5k -PCL 10k The zeta potential of PE-LNPs (polymer / saRNA weight ratio = 65) composed of PEG increased significantly from +29.0 ± 0.9 mV to +44.5 ± 0.8 mV due to the increase in the amount of cationic lipid. However, when the polymer / saRNA weight ratio of PE-LNPs was increased from 65 to 80, the zeta potential of PEG increased significantly. 5k -PCL 10k The zeta potential of PE-LNPs (N / P=18) composed of PEG decreased from +44.5±0.8mV to +40.2±0.9mV, which may be attributed to the enhanced shielding effect of the polymer encapsulation layer. 2k -PCL 5k PE-LNPs composed of showed the same trend.

[0148] PEG 5k -PCL 10k and DOTAP were used to prepare RNA-loaded PE-LNP mn for further investigation in exemplary studies unless otherwise specified, where the numbers m and n indicate the N / P molar ratio and polymer / RNA weight ratio, respectively. Figure 3 shows the change in encapsulation efficiency of PE-LNPs (polymer / saRNA weight ratio = 65) with various N / P molar ratios. A significant increase of 20% in the encapsulation efficiency was observed when the N / P molar ratio increased from 5 to 11. The encapsulation efficiency of PE-LNP 11-65 was 93.6 ± 3.3%, indicating the extremely high RNA loading capacity of the nanoformulation.

[0149] The counterpart of PE-LNP mn without payload is named as PE-LNP m'-n'. The structure of PE-LNP without RNA loading was investigated by Cryo-TEM. The Cryo-TEM micrograph in Figure 4 shows that PE-LNP exhibited a transparent outer layer. The amphiphilic PEG-PCL polymers could self-assemble into polymersome structures containing a hydrophilic core. The darker outer layer should be due to the hydrophobic PCL polymers with higher contrast. There were many black dots in the core, which are believed to be aggregated DOTAP lipid nanoparticles. DOTAP is amphiphilic, the polar parts prefer water, also called hydrophilic, and the non-polar parts as hydrophobic parts try to move away from water. Since the inner PEG-PCL vesicle structure is hydrophilic, the "hydrophobic effect" of the DOTAP tails could be the reason. 26 (which act to minimize contact between the lipid hydrocarbon tails and the aqueous environment), along with others involving van der Waals interactions and headgroup hydrogen bonds, determine the self-assembly behavior of DOTAP. Therefore, the black dots that exhibited dark contrast due to poor electron transparency are likely composed of the unsaturated hydrophobic hydrocarbon tails present in DOTAP.

[0150] Thus, DOTAP self-assembled into "sponge-like" aggregates that were encapsulated in the aqueous core of polymersomes, dividing the internal space into subcompartments by hydrophobic interactions of the hydrocarbon tails. Compared with liposomes and polymersomes, the internal structure of PE-LNPs offers a significantly higher surface area that is favorable for nucleic acid loading. Furthermore, the PEG-PCL shell can not only prevent RNA degradation but also enhance the stability of the PE-LNP system due to its higher mechanical strength than lipid bilayers.

[0151] We evaluated the thermal stability of PE-LNP 11'-65', 11'-80', 18'-65' and 18'-80'. As shown in Figure 5, the DLS size and PDI of PE-LNP did not respond to temperature changes within the range of 25 °C to 85 °C, demonstrating good structural and colloidal stability upon temperature changes.

[0152] Example 3 - Probing PE-LNP structure by fluorescence resonance energy transfer (FRET) analysis FRET experiments were performed to further investigate the PE-LNP structure using a fluorometer (FluoroMax-4, Horiba Scientific). For the donor / acceptor pair of N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) / N-(lissamine rhodamine B sulfonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (Rhod-PE), the excitation wavelength was set at 460 nm, and emission spectra were collected from 480 to 630 nm.

[0153] PE-LNPs were incorporated with donor NBD-PE (4 mM) and acceptor Rhod-PE (4 mM). Briefly, for various PE-LNPs, e.g., PE-LNP 5'-65', PE-LNP 11'-65', and PE-LNP 18'-65' without added saRNA, NBD-PE and Rhod-PE were co-dissolved in 0.5 mL of THF (containing pre-dissolved PEG-PCL and DOTAP) at the desired ratio, followed by HPLC with 100 ng / mL of HO. 2 The mixture was stirred at room temperature for 1-2 min, and then the THF was removed by rotary evaporation (see PE-LNP Preparation section in Example 2 above). For comparison, a control group without polymer (i.e., DOTAP liposomes) was prepared. The molar concentration of DOTAP in the control liposomes was the same as the molar concentration of DOTAP in the corresponding PE-LNP (or the total molar concentration of both PEG-PCL and DOTAP). For example, DOTAP and PEG in PE-LNP 5'-65' 5k -PCL 10k The molar concentrations of DOTAP-143 and DOTAP-186 were 143 mM and 43 mM, respectively. Therefore, the corresponding control liposomes were denoted as DOTAP-143 and DOTAP-186, respectively. In addition, each donor-labeled (only NBD-PE) PE-LNP was also prepared to measure the FRET efficiency E according to the following formula: 1 We calculated:

[0154]

number

[0155] In the formula, I D and I DA are the donor fluorescence intensities at 530 nm of PE-LNPs labeled with only donor NBD-PE and PE-LNPs co-labeled with both donor NBD-PE and acceptor NBD-PE, respectively.

[0156] In another experiment, PE-LNP 11'-65' was labeled separately with NBD-PE (4 mM) and Rhod-PE (4 mM). Then, PE-LNP 11'-65' (NBD-PE only) was mixed with PE-LNP 11'-65' (Rhod-PE only) in equal volume with shaking at 100 rpm for 5 min, 1 h, and 2 h, respectively. Control DOTAP liposomes were also labeled separately with NBD-PE (4 mM) and Rhod-PE (4 mM), respectively, and the molar concentration of DOTAP in the control liposomes was equivalent to that of the corresponding PE-LNP. Blank PE-LNP and DOTAP liposomes without labeling were prepared for comparison. FRET efficiency E at specific time points 2 was calculated according to the following formula:

[0157]

number

[0158] In the formula, I D is the donor fluorescence intensity at 530 nm of PE-LNPs labeled by NBD-PE after mixing with the blank sample, and I DM is the donor fluorescence intensity of PE-LNPs labeled with NBD-PE after mixing with PE-LNPs labeled with Rhod-PE.

[0159] Results and Discussion To further verify that lipid nanoparticles are formed at the core of the nanostructure, FRET analysis using NBD-PE and Rhod-PE pairs was applied to investigate the spatial arrangement of PEG-PCL and DOTAP. The efficiency of energy transfer between the donor fluorophore NBD (4.0 mM) and the acceptor fluorophore Rhod (4.0 mM) depends on their distance on the membrane layer. The most common structures formed by mixing amphiphilic polymers with lipids are hybrid vesicle structures with mixed membrane compositions. 27~29 As shown in Figure 6B, excitation of the donor NBD at 460 nm resulted in a fluorescence spectrum with enhanced emission intensity of the acceptor Rhod in PE-LNP 5'-65', indicating a FRET efficiency of 93.7 ± 0.2%. Assuming that PEG-PCL and DOTAP self-assembled into a uniform hybrid membrane, the FRET efficiency of the control DOTAP-186 liposomes (assembled from DOTAP at the same molar concentration as the combination of polymer and lipid molecules present in PE-LNP 5'-65') should be at a similar level to that of PE-LNP 5'-65', since the distance between the donor NBD and the acceptor Rhod is similar (Figure 6A). Indeed, the FRET efficiency of PE-LNP 5'-65' was significantly higher than that of DOTAP-186 liposomes (85.9 ± 0.3%). Similarly, assuming that PEG-PCL and DOTAP self-assembled into a uniform hybrid membrane, the FRET efficiency of control DOTAP-143 liposomes (assembled from DOTAP at the same molar concentration as DOTAP present in PE-LNP 5'-65') should be higher than that of PE-LNP 5'-65' due to the shorter distance between the donor NBD and the acceptor Rhod. However, DOTAP-143 liposomes self-assembled from relatively few molecules showed a similar level of FRET efficiency (92.9 ± 0.3%) as PE-LNP 5'-65'. A similar trend was observed for PE-LNP 11'-65' and PE-LNP 18'-65' (Figure 6C-Figure 6D). The striking difference in FRET efficiency suggests that a uniform polymer-lipid hybrid membrane was not present in PE-LNP.

[0160] Further studies were then performed to verify the formation of lipid nanoparticles within the core of PE-LNP. PE-LNP 11'-65' was labeled separately with donor NBD-PE (4.0 mM) or receptor Rhod-PE (4.0 mM). PE-LNP 11'-65' (NBD) was then mixed with an equal volume of PE-LNP 11'-65' (Rhod) while shaking at 100 rpm for up to 2 hours. Control DOTAP-358 liposomes (where the number (i.e., 358) indicates the equimolar concentration of DOTAP present in PE-LNP 11'-65') were also labeled separately with donor NBD-PE (4.0 mM) or receptor Rhod-PE (4.0 mM) for comparison. After mixing DOTAP-358 liposomes (NBD) with DOTAP-358 liposomes (Rhod) for incubation under the same conditions, an immediate increase in FRET efficiency to 17.1% was observed, followed by a gradual increase to 22.0% after 1 h (Figure 7B-D). This was due to lipid mixing between DOTAP liposomes, which led to a decrease in the distance between the donor NBD and the acceptor Rhod. However, after mixing PE-LNP 11'-65' (NBD) with PE-LNP 11'-65' (Rhod) for 1 h, no obvious FRET efficiency was observed, followed by only a slight increase to 3.4% after 2 h of prolonged incubation. The insignificant FRET efficiency suggests the formation of lipid nanoparticles within the core of PE-LNP (Figure 7A).

[0161] Example 4 - In vitro HEK293 cell transfection with saRNA-containing PE-LNPs Transfection efficiency of saRNA-loaded PE-LNPs (saRNA within the nanoparticle core) For firefly luciferase (fLuc) assays, 5 × 10 HEK293 cells were cultured at 4 °C for 1 min. 4The cells were seeded into 96-well plates at a density of 100 μg / well and cultured for 48 h in DMEM medium supplemented with 10% (v / v) FBS and 1% (v / v) penicillin / streptomycin to reach 60–80% confluence before transfection. After removing the spent medium, 100 μL of fresh serum-free DMEM medium or complete DMEM medium was added to each well, which was then incubated with various N / P molar ratios and polymer / saRNA (w / w) weight ratios (1 μg mL -1 10 μL of fLuc saRNA-spiked PE-LNPs was added to five replicates containing 10 μL of fLuc saRNA (equivalent to 10 μL of saRNA). After 4 h of incubation, the transfection medium was replaced with fresh complete DMEM medium. fLuc activity, expressed as relative luminescence units (RLU) in 50 μL of medium, obtained from transfected cells after 24 h treatment with 50 μL of fLuc substrate was assayed using a GloMax® Microplate Reader (Promega).

[0162] Transfection efficiency of saRNA-conjugated PE-LNP (saRNA on nanoparticle surface) For comparison, fLuc saRNA-conjugated PE-LNPs were prepared by method 2 (M2). In summary, 650 μg of PEG 5k -PCL 10k and 400 μg of DOTAP were co-dissolved in 0.5 mL of THF. The resulting organic solution was quickly added to 1 mL of Rnase-free water and stirred at room temperature for 1-2 min. The THF was then removed by rotary evaporation to obtain blank PE-LNPs. After that, 10 μg of fLuc saRNA was added to the blank PE-LNP solution. After vortexing for 30 s, fLuc saRNA-conjugated PE-LNPs were obtained. Using the above method in Example 4, the transfection efficiency of fLuc saRNA-conjugated PE-LNPs in the absence or presence of FBS was determined. PEI / fLuc saRNA complexes and fLuc saRNA-loaded DOTAP lipid nanostructures were prepared as controls.

[0163] Transfection efficiency of saRNA-loaded PE-LNPs and saRNA-bound PE-LNPs after storage at 4°C 100 μL of RNase-free PBS (10×) buffer solution (or RNase-free water) was added to 900 μL of fLuc saRNA-added PE-LNP solution or fLuc saRNA-bound PE-LNP solution, and the resulting samples were stored at 4° C. Then, HEK293 cells were transfected with these samples that were freshly prepared or stored at 4° C. for 5 days, and the transfection efficiency was measured according to the above method in Example 4.

[0164] Results and Discussion The effects of PEG-PCL molecular weight, N / P molar ratio and polymer / saRNA (w / w) weight ratio on the transfection efficiency of various PE-LNPs in HEK293 cells were evaluated. Figures 8A and 8B show the transfection efficiency of PE-LNPs with a fixed polymer / saRNA (w / w) weight ratio of 65 but with different N / P molar ratios. PEG 2k -PCL 5k For PE-LNPs composed of PEG, the transfection efficiency increased by nearly four orders of magnitude when the N / P molar ratio was increased from 5 to 16, and then decreased as the N / P molar ratio was further increased. 5k -PCL 10k PE-LNPs composed of showed a similar trend in transfection efficiency, but with higher transfection efficiency at a low N / P molar ratio of 5 and peak transfection efficiency at an N / P molar ratio of 18.

[0165] Figures 8C and 8D show the transfection efficiency of PE-LNPs with a fixed N / P molar ratio but varying polymer / saRNA weight ratios. 2k -PCL 5k For PE-LNPs composed of PEG, the transfection efficiency increased by two orders of magnitude when the polymer / saRNA weight ratio was increased from 40 to 65. Further increase in the polymer / saRNA weight ratio to 90 resulted in only minor changes in transfection efficiency (Figure 8C).5k -PCL 10k For PE-LNPs composed of PEG, the transfection efficiency was at a similar level within the range of polymer / saRNA weight ratios tested (40–90) (Figure 8D). 5k -PCL 10k This may be due to the enhanced formation of a thick polymer shell layer obtained from PEG-LNP. The resulting thick polymer shell layer could provide improved protection of the saRNA, which was confirmed by the slight change in transfection efficiency between PE-LNP samples at polymer / saRNA weight ratios of 65 to 90 in the absence (Figure 8D) and presence (Figure 8F) of FBS. In contrast, PEG-LNPs were not shown to be effective in preventing the formation of saRNA. 2k -PCL 5k PE-LNPs constructed from PEG showed significantly reduced transfection efficiency in the presence of FBS (Figure 8E). Many other cationic nucleic acid delivery systems with high in vitro transfection efficiency showed low in vivo efficacy due to the adsorption of serum proteins, so the high serum compatibility due to the improved stability of PE-LNPs with a thick polymer shell layer is preferred. Therefore, in further studies, PEG-LNPs were used as the transfection medium. 5k -PCL 10k We selected PE-LNP composed of

[0166] RNA molecules are highly unstable and can be easily hydrolyzed / degraded. To compare the stability of RNA added to the core and bound to the surface of PE-LNPs, two different RNA addition methods were used. As shown in Figure 9A, saRNA-added PE-LNP 18-80 and saRNA-bound PE-LNP 18-80 showed comparable transfection efficiency in the absence of FBS, which was one order of magnitude higher than PEI / saRNA complexes and 4-5 orders of magnitude higher than saRNA-added DOTAP lipid nanostructures. It is interesting to note that while saRNA-added PE-LNP 18-80 was FBS-compatible, the presence of FBS reduced the transfection efficiency of saRNA-bound PE-LNP 18-80 by one order of magnitude (Figure 9B). In comparison, the transfection efficiency of PEI / saRNA complexes was reduced by three orders of magnitude upon incubation with FBS. That is, saRNA-added PE-LNP 18-80 showed a transfection efficiency four orders of magnitude higher than PEI / saRNA complexes in the presence of FBS.

[0167] We further evaluated the stability of RNA attached to the surface of PE-LNPs after being added to the cores and stored in RNase-free water or PBS at 4°C. As shown in Figure 9C and Figure 9D, after 5 days of storage in liquid formulation, saRNA-loaded PE-LNP 18-80 showed no decrease in transfection efficiency, while the transfection signal of saRNA-bound PE-LNP 18-80 was still relatively high (>10 6 ) was half an order of magnitude lower compared to freshly prepared samples.

[0168] These results suggest that the PE-LNP system exhibits favorable serum stability and that high encapsulation of biological payloads, especially RNA molecules that are easily hydrolyzed / degraded, into the nanoparticle core may provide optimal protection. This is because the hydrophilic PEG corona of PE-LNPs can reduce protein absorption, and the hydrophobic PCL layer can protect the RNA from the harsh external environment.

[0169] Example 5 - In vitro transfection of interferon-competent HeLa cells with saRNA-loaded PE-LNP Transfection efficiency of saRNA-added PE-LNPs The saRNA encoded by fLuc was formulated inside PE-LNP 11-65 by the same method as described above in Example 2. The saRNA doses were 0.1, 0.5, 1, 3, 5 and 7 μg mL -1 HeLa cells were seeded and transfected according to the method described above in Example 4, where titration was performed by changing the concentration of

[0170] Results and Discussion The clinical application of RNA systems is limited by their high innate immunogenicity in addition to inefficient protein expression. It is important to achieve a good balance between protein expression and innate immune responses. Toll-like receptors (TLRs) 3, 7, 8 and 9 are intracellular sensors of nucleic acids, present in the endoplasmic reticulum, endosomes and lysosomes. Upon detection of foreign nucleic acids, intracellular TLRs activate various signaling pathways and induce the production of cytokines, which may result in the restriction of protein expression or even adverse effects in patients. As HeLa cells express TLR3, we assessed the transfection efficiency into HeLa cells to investigate the ability of PE-LNPs to prevent recognition by intracellular TLRs.

[0171] Figures 10A and 10B show the cell transfection titers of PE-LNPs on interferon-competent HeLa cells. The four formulations tested, PE-LNP 11-65, PE-LNP 18-65, PE-LNP 11-80 and PE-LNP 18-80, shared a similar trend in transfection efficiency, with a maximum of 10 7 Reach maximum transfection efficiency at an RLU of 3 μg mL -1The transfection efficiency increased with saRNA dose until an optimal dose of 1000 μg / ml. All four PE-LNP formulations showed promising levels of transfection efficiency indicating effective prevention of TLR recognition by the PE-LNP structure. This is attributed to the polymer shell, where the hydrophilic block provides a hydration layer to prevent recognition by TLRs and direct interaction between the cationic lipid core and cells, resulting in effective transfection into interferon-competent cells.

[0172] As the dose of saRNA decreases (polymer / saRNA weight ratio = 65, 0.1–5 μg mL -1 0.5 μg mL for polymer / saRNA weight ratio = 80 -1 ~3μg mL -1 ), when the N / P molar ratio was increased from 11 to 18, the transfection efficiency increased by about one order of magnitude. This may be due to the higher N / P molar ratio resulting in more efficient release of the payload in the endosomal compartment. Therefore, this may decrease the possibility of activation of TLRs localized in endosomes.

[0173] These results also demonstrate that PE-LNPs can effectively deliver nucleic acid payloads to a variety of cell types, including human fetal kidney cells (Example 4), cancer cells (Example 5), and T-lymphocyte cells (Example 6).

[0174] Example 6 - PE-LNP-mediated intracellular delivery of mRNA into suspension Jurkat cells To further demonstrate the ability of PE-LNPs to deliver different payloads to different cell lines, we used different PE-LNPs to intracellularly deliver messenger RNA (mRNA) encoding green fluorescent protein (GFP) into suspended Jurkat cells. GFP mRNA-added PE-LNPs were prepared by the same method as described above in Example 2 for saRNA-added PE-LNPs. In summary, Jurkat cells were resuspended by gentle pipetting and then counted by Vi-CELL XR Cell Viability Analyzer (Beckman Coulter, USA). Cells were washed twice and then aliquoted at 2 × 10 in pre-warmed (37 °C) unsupplemented serum-free RPMI-1640 medium. 6 cells mL -1 The cells were resuspended at a density of 100 μL per well. 1 mL of cell suspension was seeded into each well of a 12-well plate. GFP mRNA-loaded PE-LNP was then added dropwise at 2 μg of mRNA per well, followed immediately by gentle pipetting to thoroughly mix the culture. After 4 h of incubation at 37° C., the cells were washed twice and resuspended in 1 mL of complete RPMI-1640 medium for further incubation at 37° C. overnight. A portion of the sample wells was used for viability testing via cell counting with a Vi-CELL XR Cell Viability Analyzer. The remaining sample wells were washed twice with PBS, and the cells were resuspended in 100 μL of PBS containing LIVE / DEAD™ Fixable Aqua Dead Cell Stain (Thermo Fisher, USA) and incubated for 30 min. After washing with PBS, each sample was observed under a fluorescent microscope (EVOS Floid Imaging System, Thermo Fisher) and quantitatively analyzed by flow cytometry (Canto, BD, USA) to examine GFP expression in viable Jurkat cells.

[0175] Results and Discussion Figure 11 shows that GFP expression was minor in Jurkat cells treated with mRNA-added PE-LNP 5-65. With the polymer / mRNA weight ratio fixed at 65, the percentage of GFP-expressing Jurkat cells significantly increased to over 80% as the N / P molar ratio increased from 5 to 11, followed by a further enhancement of about 10% as the N / P molar ratio further increased to 18 (Figure 11B). The mean fluorescence intensity (MFI) in Jurkat cells treated with mRNA-added PE-LNP showed a similar dependence on the change in N / P molar ratio from 5 to 11. However, no significant enhancement was observed with a further increase in the N / P molar ratio to 18 (Figure 11C). Figure 11D shows that Jurkat cells were well tolerated by PE-LNP, with PE-LNP 5-65 and PE-LNP 11-65 exhibiting high cell viability of over 80% 4 hours after treatment. Although a slight increase in cytotoxicity was observed when the N / P molar ratio was increased to 18, PE-LNP 18-65 still exhibited relatively high cell viability at 70.4 ± 1.8%. The results demonstrate that our PE-LNPs can also successfully deliver mRNA to suspension cell lines.

[0176] Example 7 - Mechanism of cellular uptake and endosomal escape of saRNA and FITC co-loaded PE-LNPs 500 μg FITC and 800 μg PEG 5k -PCL 10k saRNA and 400 μg of DOTAP were co-dissolved in 0.5 mL of THF, and 10 μg of saRNA was dissolved in 1 mL of RNase-free water. The organic and aqueous solutions were quickly mixed, and the mixture was kept stirring at room temperature for 1-2 min. THF was removed by rotary evaporation, and free FITC was removed by centrifugation at 3000 rpm for 15 min in an ultrafiltration centrifuge tube (MWCO = 3000) to obtain saRNA and FITC co-loaded PE-LNPs.

[0177] The cellular uptake mechanism of the nanoscale system was investigated by laser scanning confocal microscopy. 5HEK293 cells) were seeded on 35 mm glass-bottom culture dishes and cultured for 24 h. After preincubation at 4°C for 1 h, saRNA and FITC-co-loaded PE-LNPs (final saRNA concentration of 2 μg per dish) were added, and cells were further incubated at 4°C for 4 h. As a control, HEK293 cells were treated with saRNA and FITC-co-loaded PE-LNPs at 37°C for 4 h. After treatment at 4°C or 37°C, cells were washed with PBS and fixed with 4% paraformaldehyde solution for 10 min, and nuclei and lysosomes were stained with Hoechst 33342 (5 μg mL -1 ) and LysoTracker-Red (50 nM) for 5 min. The cells were then imaged using a Leica SP8 inverted confocal microscope, and the fluorescent colocalization of FITC and LysoTracker-Red was analyzed by Image J.

[0178] To further investigate the mechanism of cellular uptake and intracellular trafficking of saRNA and FITC-coloaded PE-LNPs, HEK293 cells were cultured in 6-well plates at 5 × 10 5 The cells were seeded at a density of 10 μg / well for 24 h. First, the cells were treated with the following inhibitors: chlorpromazine hydrochloride (10 μg mL -1 ), methyl-β-cyclodextrin (MβCD, 5 mM), filipin (5 μg mL -1 ), amiloride (1 mM), genistein (40 μg mL -1 ) and nystatin (40 μg mL -1 ) for 1 h, respectively. Then, saRNA and FITC-co-loaded PE-LNPs (containing 2 μg of saRNA) were added to each well and co-incubated with the inhibitor for another 1 h. Finally, the cells were washed three times with pre-cooled PBS solution and analyzed by flow cytometry (Fortessa I).

[0179] The uptake and intracellular transport of saRNA and FITC-co-loaded PE-LNPs were also examined by confocal microscopy. 5Cells) were seeded on 35 mm glass-bottom culture dishes and cultured for 24 h. After preincubation with MβCD (5 mM) for 1 h, saRNA and FITC-co-loaded PE-LNPs (containing 2 μg of saRNA) were added to the culture dishes and co-incubated with MβCD for 4 h. Cells were then washed with PBS and fixed with 4% paraformaldehyde solution for 10 min, and nuclei and lysosomes were stained with Hoechst 33342 (5 μg mL -1 ) and LysoTracker-Red (50 nM) for 5 min. Finally, the cells were imaged using a Leica SP8 inverted confocal microscope.

[0180] Results and Discussion It is important to design nanocarriers that can release plasma membrane-entrapped biomolecules into the cytoplasm by endosomal escape before they are transported to lysosomes for degradation. 30 (Figure 1B). In this study, FITC was co-loaded with saRNA in PE-LNP 18-80 to investigate the endocytic pathway and intracellular trafficking. After 4 h treatment at 37 °C, the strong diffuse green fluorescence of FITC was visualized by confocal microscopy (Figure 12A). ImageJ analysis suggested that there was only a minor signal of fluorescent colocalization between saRNA and FITC co-loaded PE-LNP (green) and endosomes / lysosomes (red), indicating successful PE-LNP-mediated endosomal escape of the payload into the cytoplasm (Figure 12B). In comparison, the intracellular green fluorescence intensity of FITC was significantly reduced to a much lower level after 4 h incubation at 4 °C, suggesting that the cellular uptake of PE-LNP was via an energy-dependent endocytic pathway (Figure 12A and Figure 12B). In addition, various endocytic inhibitors were used to further investigate the specific endocytic mechanism of PE-LNP. Figures 12C and 12D show that the intracellular green fluorescence intensity was significantly decreased after treatment with the inhibitor MβCD, suggesting that lipid-raft-mediated endocytosis is the major cellular uptake pathway for PE-LNPs.

[0181] Example 8 - Hemolysis and cell viability tests The hemolysis method was used to investigate the biocompatibility of saRNA-loaded PE-LNPs. Briefly, defibrinated sheep red blood cells (RBCs) were centrifuged at 1500×g for 10 min at 4° C. and washed three times with PBS. The cell pellet was resuspended in 5% (v / v) red blood cell suspension with PBS. To 0.9 mL of RBC suspension in a centrifuge tube, 100 μL aliquots of various PE-LNPs containing 1 μg of saRNA were added. Treatment of the RBC suspension with deionized water was used as a positive control. After incubation at 37° C. for 1 h, the RBC suspension was centrifuged, 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance (A) was measured at 540 nm using a spectrofluorometer (GloMax® Discover Microplate Reader, Promega, USA). The relative hemolysis was calculated according to the following formula:

[0182]

number

[0183] The cytotoxicity of saRNA-loaded PE-LNPs against HEK293 cells was measured using the alamarBlue assay. HEK293 cells were cultured at 5 × 10 4 Cells were seeded in 96-well plates at a density of 1000 cells / well. After 24 h of incubation, 1 μg mL -1 Cells were treated with various PE-LNPs supplemented with 10 mg / mL saRNA for 4 h. Then, 10 μL of alamarBlue HS reagent (5 mg mL -1 ) was added to each well. After further incubation for 4 hours, the absorbance of each well was measured at 570 nm using a spectrofluorometer (GloMax® Discover Microplate Reader, Promega, USA) according to the manufacturer's instructions. The cytotoxic effect was determined from the absorbance readings.

[0184] Results and Discussion Hemolytic activity and nonspecific cytotoxicity are typical problems associated with cationic carriers due to their strong interaction with negatively altered cell membranes. It is interesting to note that the hemolysis rates of the various PE-LNPs were all less than 10% after 1 h of treatment (Figure 13A), suggesting high biocompatibility. Figure 13B shows that HEK293 cells were well tolerated by saRNA-loaded PE-LNPs. 1 μg mL -1 At a fixed saRNA dose of 1000, PE-LNP 5-65 and PE-LNP 11-65 showed very high cell viabilities of 92.3 ± 1.8% and 86.7 ± 1.4%, respectively, after 24 h of treatment. Although an increase in cytotoxicity was observed when increasing the N / P molar ratio to 18, PE-LNP 18-65 and PE-LNP 18-80 still showed relatively high cell viabilities at 73.0 ± 4.9% and 73.6 ± 7.8%, respectively. These results demonstrate that PE-LNPs have low cytotoxicity and good biocompatibility, which can be attributed to the protection provided by the hydrophilic PEG corona and hydrophobic PCL layer surrounding the biological payload within the core of the PE-LNPs.

[0185] Example 9 - In vivo bioluminescence imaging and immunogenicity In vivo bioluminescence imaging Female BALB / c mice (Charles River, UK) aged 6–8 weeks were housed in groups of n=5 in fully acclimatized rooms with free access to food and water. All animals were handled in accordance with the UK Home Office Animals Scientific Procedures Act of 1986, in accordance with the internal ethical committee, and project and personal licenses approved by the UK government. Animals were given an acclimatization time at least 7 days before the start of the experiment. Mice were injected intramuscularly (IM) into both hind quadriceps muscles with 5 μg of fLuc saRNA formulated inside DOTAP lipid nanostructures (negative control), PE-LNP 5-65, PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80, respectively, or on the surface of PE-LNP 18-80 M2 (prepared by method 2). After 7 days, mice were intraperitoneally injected with 100 μL of XenoLight RediJect D-Luciferin Substrate (Perkin Elmer, UK) and allowed to sit for 10 min. Mice were then anesthetized using isoflurane and imaged for 10 min using an In Vivo Imaging System FX Pro (Kodak Co., Rochester, NY, USA) equipped with Molecular Imaging Software Version 5.0 (Carestream Health, USA). Signals from each injection site were quantified using equal detection areas using Molecular Imaging Software and expressed as RLU.

[0186] In vivo immunogenicity of HA saRNA-loaded PE-LNPs Six to eight week old female BALB / c mice (Charles River, UK) were placed in groups of n=5. Mice were immunized IM in one hind quadriceps with 1 μg saRNA encoding the H1 hemagglutinin of Cal / 09 virus (HA saRNA) formulated inside PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80, respectively, to a total injection volume of 50 μL in 1× PBS and boosted 4 weeks later with the same formulation. jetPEI / HA saRNA complex was used as a control. Blood was collected via tail bleed 4 and 6 weeks after the start of the study. Blood was collected and centrifuged at 10,000×RPM for 5 minutes. Serum was collected and stored at -80°C.

[0187] In vivo immunogenicity of SARS-CoV-2 saRNA-loaded PE-LNPs Six to eight week old female BALB / c mice (Charles River, UK) were placed in groups of n=5. Mice were immunized IM in one hind quadriceps with 1 μg of saRNA encoding the prefusion stabilized SARS-CoV-2 spike protein formulated inside PE-LNP 11-65 and PE-LNP 18-65, respectively, to a total injection volume of 50 μL in 1× PBS. Blood was collected via tail bleed at 4 weeks into the study. Blood was collected and centrifuged at 10,000×RPM for 5 minutes. Serum was collected and stored at -80°C.

[0188] HA specific ELISA The HA antibody titer was quantitatively assessed using an immunoglobulin ELISA protocol. Briefly, 0.5 μg mL HA antibody titer was determined by incubating with 1% (w / v) bovine serum albumin (BSA) / 0.05% (v / v) Tween-20 in PBS. -1HA-coated ELISA plates were blocked. After washing, diluted serum samples were added to the plates, incubated for 2 h, washed, and anti-mouse IgG-HRP (Southern Biotech, UK) was used at a dilution of 1:4000. ELISA plate wells were coated with anti-mouse kappa (1:1000) and lambda (1:1000) light chain (Serotec, UK), blocked with PBS / 1% (w / v) BSA / 0.05% (v / v) Tween-20, washed, and purified IgG (Southern Biotech, UK) was added at 1000 ng mL -1 Standards were prepared by adding 0.01% ethanol starting at 0.01% and titrating through a 5-fold dilution series. Samples and standards were developed using TMB (3,3';5,5'-tetramethylbenzidine) and the reaction was stopped after 5 min with Stop Solution (Insight Biotechnologies, UK). Absorbance was read using a spectrophotometer (VersaMax, Molecular Devices) using SoftMax Pro GxP v5 software.

[0189] SARS-CoV-2 specific ELISA SARS-CoV-2 antibody titers were quantitatively assessed using an immunoglobulin ELISA protocol, following a procedure similar to that of the HA-specific ELISA.

[0190] Influenza burden Three weeks after the boost injection, mice were administered 4.2 × 10 5 Mice were challenged with plaque forming units (pfu) of influenza A (Cal / 09). Mice were anesthetized using isoflurane, challenged intranasally (IN), and weighed daily to determine weight loss. In accordance with the humane endpoint of the challenge protocol, mice were euthanized if they sustained a 20% weight loss for more than 3 days or a 25% weight loss in a single day.

[0191] Results and Discussion To test the saRNA expression efficiency in vivo, luciferase saRNA was formulated into various PE-LNPs and administered to mice by intramuscular injection at just one dose (5 μg / leg). After 7 days, the mice were imaged and the relative fluorescence intensity was quantified. The signal of mice treated with saRNA / DOTAP lipid nanostructures was barely detectable, which may be due to the limited saRNA encapsulation capacity and low stability of saRNA / DOTAP lipid nanostructures. However, all PE-LNP groups showed protein expression. Compared with PE-LNP 5-65, the three formulations PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80 showed significantly higher luciferase expression (p<0.05) (Figure 14A and Figure 14B). Although luciferase expression in PE-LNP 18-80 M2 (prepared by method 2, with saRNA on the nanoparticle surface) was significantly lower (p<0.05) than that of PE-LNP 18-80 (prepared by method 1, with saRNA in the nanoparticle core), they showed similar in vitro transfection efficiency (Figure 9A). This further confirms that the incorporation of saRNA in the core of PE-LNP is preferred over that on the nanoparticle surface. Although the in vitro transfection efficiency of PE-LNP 18-65 was an order of magnitude higher than that of PE-LNP 11-65 (Figure 8B), the in vivo luciferase expression of these two groups showed similar signals. This may be due to the superior cell viability of PE-LNP 11-65 with a lower N / P molar ratio.

[0192] In addition, we evaluated the immunogenicity and protective capacity of saRNA encoding HA formulated inside PE-LNPs after IM injection. One commercially available linear PEI, jetPEI, previously shown to effectively deliver RNA in vivo, was used. 31was used as a positive control. Mice were primed and boosted with 1 μg saRNA formulated with jetPEI, PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80. The boost was administered 4 weeks after the first prime. Two weeks after the boost, mice were challenged IN with Cal / 09 influenza virus and daily body weights were measured to monitor disease pathology (Figure 14C). To distinguish weight loss between groups, 4.2 × 10 5 A relatively high dose of 10000 pfu was used. All mice in the naïve group lost >25% of their body weight after 6 days and had to be euthanized according to the humane endpoint of the challenge protocol, but encouragingly, all mice in the jetPEI and PE-LNP groups were fully protected (Figure 14E). Mice in all three PE-LNP groups lost less than 10% body weight during the entire observation period, indicating good protection with fewer side effects, especially in the PE-LNP 11-65 group, which showed the least amount of weight loss (approximately 8%). The HA IgG antibody titers (Figure 14D) directly reflected the challenge results. All three of these PE-LNP groups, PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80, induced high antibody titers with no significant differences (approximately 1 × 10 after 6 weeks). 5 ng mL -1 ), which was comparable to jetPEI. This was consistent with the in vivo protein expression results shown in FIG. 14B. This indicates that both of these PE-LNP groups reached the threshold of protein expression required to stimulate a robust immune response. In this respect, PE-LNPs offer more flexibility in formulation composition while ensuring efficacy, which should be advantageous for delivering biomolecules, including RNA vaccines and therapeutics.

[0193] We also evaluated the immunogenicity of nCoV-encoding saRNA formulated inside PE-LNPs after IM injection. Mice were primed with 1 μg of saRNA formulated in PE-LNP 11-65 and PE-LNP 18-65, respectively. As shown in Figure 15, both PE-LNP 11-65 and PE-LNP 18-65 induced high antibody titers after priming, with no significant difference (approximately 1 × 10 after 4 weeks). 4 ng mL -1 This demonstrated that the PE-LNP system can effectively deliver RNA vaccines to combat various diseases, including the global COVID-19 pandemic and the influenza viruses mentioned above.

[0194] Example 10 - Optimization of PE-LNP composition for stable storage of spiked saRNA in aqueous solution at 4°C Optimization of the polymer composition of PE-LNP 11-65 for storage in aqueous solution at 4 °C Following the same procedure as described above in Example 1, PEG 5k -PCL 8.5k and PEG 5k -PCL 10k (Table 1) were synthesized and characterized. Using the same method as described above in Example 2, saRNA was formulated inside PE-LNP 11-65 containing amphiphilic polymers of various molecular weights. The nanoformulations were stored in aqueous solution at 4°C. Then, 1 μg mL of saRNA was analyzed at 0, 7 and 21 days according to the method described above in Example 4. -1 Their in vitro HEK293 cell transfection efficiencies at saRNA doses of 1000 μg / ml were measured.

[0195] Transfection efficiency of various saRNA-loaded PE-LNPs after storage in aqueous solution at 4 °C saRNA was formulated inside PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80 using the same method as described above in Example 2. The nanoformulations were stored in aqueous solution at 4°C. Then, 1 μg mL at 0, 1 and 3 months were analyzed according to the method described above in Example 4. -1Their in vitro HEK293 cell transfection efficiencies at saRNA doses of 1000 μg / ml were measured.

[0196] Results and Discussion As shown in Figure 16, PEG 5k -PCL 8.5k and PEG 5k -PCL 10k Fresh PE-LNP 11-65 samples, each composed of PEG (containing different hydrophobic PCL chain lengths), showed similar DLS size, PDI, zeta potential and transfection efficiency. However, Figure 16D shows that PEG with longer PCL chains showed similar DLS size, PDI, zeta potential and transfection efficiency. 5k -PCL 10k This shows that PE-LNP 11-65, composed of PEG, showed more robust functional stability during storage at 4°C for 21 days. 5k -PCL 8.5k The functional stability of PE-LNP 11-65 constructed from was significantly compromised during storage, as shown by a significant decrease in transfection efficiency after 7 and 21 days of storage at 4 °C. The difference in transfection efficiency during storage suggests that the chain length of the hydrophobic block of the amphiphilic polymer plays an important role in maintaining RNA stability during storage.

[0197] PEG 5k -PCL 10kSince polymers were shown to be a superior option, further investigations were performed on the storage of liquid formulations of saRNA-loaded PE-LNPs in the absence of stabilizing molecules at 4°C. Figure 17 shows the transfection efficiency of PE-LNP 11-65, PE-LNP 18-65, and PE-LNP 18-80 during 3 months of storage in aqueous solution at 4°C. The functional stability of saRNA-loaded PE-LNP 18-65 and PE-LNP 18-80 showed no obvious changes within the first month of storage, whereas a decrease in the transfection efficiency of PE-LNP 11-65 was observed. This result suggests that a high N / P ratio of 18 may provide an even higher level of protection for nucleic acid payloads during storage in aqueous solution, possibly due to the formation of smaller lipid nanostructures within the PE-LNP core.

[0198] Example 11 - Stable storage of saRNA-loaded PE-LNPs in aqueous solution at room temperature Transfection efficiency of saRNA-loaded PE-LNP 11-65 after storage in aqueous solution at room temperature saRNA was formulated inside PE-LNP 11-65 using the same method as described above in Example 2. The nanoformulation was stored in aqueous solution at room temperature. Then, the concentration of saRNA at 1 μg mL after 21 days of storage at room temperature was determined according to the method described above in Example 4. -1 Its in vitro HEK293 cell transfection efficiency at saRNA doses of 11-65 was measured and compared with that of freshly prepared saRNA-supplemented PE-LNP 11-65.

[0199] Transfection efficiency of various saRNA-loaded PE-LNPs after storage in aqueous solution at room temperature Using the same method as described above in Example 2, saRNA was formulated inside PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80, respectively. The nanoformulations were stored in aqueous solution at room temperature. Then, 1 μg mL of saRNA was administered on days 0, 14, 21 and 28 according to the method described above in Example 4. -1 Their in vitro HEK293 cell transfection efficiencies at saRNA doses of 1000 μg / ml were measured.

[0200] Results and Discussion As shown in Figures 18 and 19, 1 μg mL -1 No decrease in transfection efficiency of saRNA formulated inside PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80 was observed after 21 days of storage in aqueous solution at room temperature compared to freshly prepared PE-LNP formulations with comparable saRNA doses. After 28 days, high transfection efficiency was maintained, although a slight decrease was observed compared to the fresh samples. As shown in Figure 19B-D, PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80 also well retained hydrodynamic size, PDI and zeta potential during storage at room temperature. This indicates that amphiphilic PEG 5k -PCL 10k It is further confirmed that the polymer shell layer formed by the self-assembly of the copolymers can improve the stability of the nanoparticles and optimally protect the biological payload, especially the easily hydrolyzed / degraded RNA molecules, from the harsh external environment in liquid formulations.

[0201] Example 12 - Stable storage of saRNA-loaded PE-LNPs in the presence of trehalose in aqueous solution at 4°C Transfection efficiency of PE-LNP 11-65 with trehalose-containing saRNA after storage in aqueous solution at 4°C Using a method similar to that described above in Example 2, saRNA and trehalose-co-loaded PE-LNP 11-65 was prepared by adding saRNA (40 μg mL ) at a trehalose / saRNA (w / w) weight ratio of 100. -1 ) and an aqueous solution of trehalose were used. Total trehalose (both internal and external) was then diluted to 250 mg mL -1 Additional trehalose was mixed with the resulting saRNA and trehalose co-loaded PE-LNP 11-65 to replenish.

[0202] For comparison, an aqueous solution of saRNA (40 μg mL ) was added to the interior of PE-LNP 11-65 using the same method as described above in Example 2. -1The resulting nanoformulation was then added at 250 mg mL -1 The mixture was mixed with external trehalose at a concentration of 0.01g / ml.

[0203] These nanoformulations were stored in aqueous solution at 4 °C for 383 days and then diluted at 1 μg mL -1 Their in vitro HEK293 cell transfection efficiencies at saRNA doses of 11-65 were measured and compared with freshly prepared saRNA-supplemented trehalose-free PE-LNP 11-65.

[0204] Results and Discussion RNA molecules are extremely fragile and can easily degrade in exposed environments, making it necessary to store and transport RNA vaccines and therapeutics in extremely challenging cold chains. The world's first approved COVID-19 vaccine, the Pfizer / BioNTech mRNA vaccine, faces a major obstacle requiring storage at -70°C. Other RNA vaccines have similar thermostability issues, for example, Moderna's mRNA vaccine must be kept at -20°C for storage. This makes it extremely difficult for RNA vaccines to reach the speed and scale of deployment required to ensure herd immunity.

[0205] PE-LNPs formulated with RNA vaccines showed efficient in vivo protein expression and excellent immunogenicity in Example 9 above (Figures 14 and 15). Example 10 above demonstrated that saRNA-added PE-LNP formulations without stabilizing molecules could maintain high transfection efficiency after storage in aqueous solution at 4°C for 3 months (Figure 17). As demonstrated in Example 11 above, after storage in aqueous solution at room temperature for 21 days, saRNA-added PE-LNP formulations showed no decrease in transfection efficiency compared to their freshly prepared counterparts with equivalent saRNA doses (Figures 18 and 19). This was due to the unique PE-LNP nanostructure that provides optimal protection of the RNA payload within the nanoparticle core.

[0206] To further extend the shelf life of RNA nanoformulations in aqueous solution, external and / or internal stabilizing molecules such as trehalose were included (Figure 20) following the method of Example 10. As shown in Figure 21, saRNA and trehalose co-loaded PE-LNP 11-65 and saRNA-loaded PE-LNP 11-65 with mixed external trehalose showed comparable transfection efficiency, which was not significantly different compared to freshly prepared saRNA-loaded PE-LNP 11-65 without trehalose after storage in aqueous solution at 4°C for over a year (383 days).

[0207] Example 13 - Stable storage of saRNA and trehalose co-loaded PE-LNPs in aqueous solution at room temperature Transfection efficiency of PE-LNPs co-loaded with saRNA and trehalose after storage in aqueous solution at room temperature Using the same method as described above in Example 12, saRNA and trehalose-co-loaded PE-LNP 11-65, PE-LNP 16-65, and PE-LNP 18-80 were prepared by adding saRNA (40 μg mL ) at a trehalose / saRNA (w / w) weight ratio of 100. -1 ) and trehalose in water, followed by 250 mg mL -1 The solution was mixed with additional trehalose to replenish the total trehalose at 100°C.

[0208] These nanoformulations were stored in aqueous solution at room temperature and then diluted at 1 μg mL on days 0, 14, 21 and 28. -1 Their in vitro HEK293 cell transfection efficiencies at saRNA doses of 100-200 μg / ml were measured and compared with freshly prepared saRNA-supplemented trehalose-free PE-LNP 11-65, PE-LNP 16-65 and PE-LNP 18-80, respectively.

[0209] Results and Discussion We then proceed to investigate the effect of trehalose as a stabilizing molecule in aqueous solution for storage at room temperature (Figure 20). With fresh saRNA-added PE-LNP without trehalose as the negative control, saRNA and trehalose-added PE-LNP showed comparable transfection efficiency after 4 weeks of storage at 20°C (Figure 22A). Compared with the negative control, saRNA and trehalose-added PE-LNP also maintained good DLS size (Figure 22B), PDI (Figure 22C) and zeta potential (Figure 22D) during 4 weeks of storage at 20°C, suggesting the possibility of long-term storage.

[0210] Example 14 - Optimization of freeze-drying conditions in the presence of trehalose and stable storage of freeze-dried saRNA-loaded PE-LNPs at 4 °C Optimization of freeze-drying conditions for saRNA-loaded PE-LNPs in the presence of external trehalose Using the method described above in Example 2, various concentrations of saRNA (e.g., 20, 40, 60, and 100 μg mL−1) were injected into the interior of PE-LNP 11-65. -1 The resulting nanoparticles were then mixed with 200 mg mL -1 The formulations were frozen in a -80°C freezer, lyophilized for 48 h, and then immediately rehydrated with RNase-free water. To optimize the saRNA concentration during lyophilization, 1 μg mL -1 The DLS particle size distribution and in vitro HEK293 cell transfection efficiency of rehydrated PE-LNPs at saRNA doses of 100 μg / mL were then evaluated. -1 ) was lyophilized and immediately rehydrated with RNase-free water for further analysis by DLS and in vitro transfection. Thus, the optimal concentration of added saRNA and mixed trehalose for lyophilization of PE-LNP 11-65 formulations was identified.

[0211] Lyophilized saRNA-spiked PE-LNPs in the presence of trehalose. Storage of saRNA at 4 °C 250mg mL -1 saRNA-loaded PE-LNP 11-65 containing 100 mg of external trehalose, and saRNA and trehalose co-loaded PE-LNP 11-65 (trehalose / saRNA weight ratio = 100 for coencapsulation, followed by 250 mg of total trehalose mL -1 The solution was freeze-dried and then stored at 4 °C. -1 For measurement of in vitro HEK293 cell transfection efficiency at saRNA doses of 100 μg / ml, lyophilized PE-LNP formulations were rehydrated with RNase-free water.

[0212] Optimization of freeze-drying conditions for saRNA and trehalose-co-loaded PE-LNPs Using the method described above in Example 2, saRNA and trehalose-co-loaded PE-LNP 11-65 were prepared by adding saRNA (40 μg mL ) in RNase-free water at various trehalose / saRNA (w / w) weight ratios (e.g., 25, 50, 100, 200, 400, and 6250). -1 The total trehalose (both internal and external) was then added at 250 mg mL -1 Additional trehalose was mixed with the resulting saRNA and trehalose co-loaded PE-LNP 11-65 to supplement up to 100 μg mL at a trehalose / saRNA (w / w) ratio of 6250 and a saRNA concentration of 40 μg mL . -1 In the specific case of saRNA and trehalose co-loaded PE-LNP 11-65, no additional trehalose had to be added for supplementation. After lyophilization and immediate rehydration with RNase-free water, the colloidal stability of saRNA and trehalose co-loaded PE-LNP 11-65 was tested by DLS and showed a high concentration of 1 μg mL -1 Their in vitro transfection efficiencies at saRNA doses of 1000 μg / ml were analyzed.

[0213] Results and Discussion Another strategy that we have utilized for stable storage of RNA vaccines and therapeutics without the need for extremely challenging cold chains is the lyophilization of RNA-loaded PE-LNPs in the presence of external and / or internal stabilizing molecules such as trehalose (Figure 20). The lyophilization process can potentially significantly reduce the colloidal stability of RNA delivery nanoformulations, resulting in an irreversible loss of efficacy. Other researchers have reported that the colloidal stability of nanoparticles can be improved by simply mixing trehalose on the outside of the nanoparticles during lyophilization, as vitrification of trehalose can immobilize the nanoparticles within a rigid amorphous glassy sugar matrix, thereby dramatically reducing nanoparticle aggregation or rupture. 21 Moreover, because PE-LNPs have a unique structure containing an internal hydrophilic domain, trehalose can also be co-added with RNA into the interior of PE-LNPs to directly interact with RNA. The hydroxyl groups of trehalose can form hydrogen bonds with RNA molecules, thereby minimizing the hydration of RNA during lyophilization. Thus, the thermal stability of RNA can be significantly improved.

[0214] First, 200 mg mL -1 A fixed concentration of external trehalose and various concentrations (20–100 μg mL) were added to the nanoparticle interior. -1 PE-LNP 11-65 containing saRNA was freeze-dried and immediately rehydrated with RNase-free water. The transfection efficiency of the rehydrated PE-LNP 11-65 was then evaluated. Interestingly, Figure 23 shows that the transfection efficiency of 1 μg mL -1 We demonstrate the synergistic effect of external trehalose on HEK293 cell transfection, showing a 1-1.5 order of magnitude improvement in transfection efficiency of the whole lyophilized PE-LNP 11-65 formulation compared to freshly prepared trehalose-free PE-LNP 11-65 at an equivalent saRNA dose of 200 mg mL according to Figure 23. -1As the optimal saRNA concentration for lyophilization of saRNA-loaded PE-LNP 11-65 in the presence of external trehalose, 40 μg mL -1 was selected.

[0215] Additionally, a fixed internal saRNA concentration (40 μg mL -1 PE-LNP 11-65 containing 100 μg / mL trehalose and various concentrations of external trehalose were freeze-dried and immediately rehydrated with RNase-free water. -1 As the optimal external trehalose concentration for lyophilization of PE-LNP 11-65 supplemented with internal saRNA, 250 mg mL -1 As shown in Figure 24, the lyophilized formulation under these conditions produced a 1 μg mL -1 showed the highest transfection efficiency, one order of magnitude higher than freshly prepared trehalose-free PE-LNP 11-65 at a comparable saRNA dose.

[0216] Figure 25 shows the concentration of 40 μg mL -1 of internal saRNA and 250 mg mL -1 Figure 1 shows stable RNA storage at 4 °C after lyophilization of PE-LNP 11-65 formulation containing 1 μg mL external trehalose. After storage at 4 °C for 28 days and then rehydration with RNase-free water, 1 μg mL -1 No reduction in transfection efficiency was observed for the lyophilized formulation compared to freshly prepared trehalose-free PE-LNP 11-65 at an equivalent saRNA dose, suggesting that the combined effect of the intrinsic protection provided by the core-shell PE-LNP nanostructure and external trehalose well preserved RNA stability during storage at 4 °C.

[0217] To further improve RNA stability during storage, we incorporated saRNA (40 μg mL -1 PE-LNP 11-65 was prepared by co-adding saRNA (fixed concentration of saRNA) and trehalose (various trehalose / saRNA weight ratios).

[0218] Total trehalose (both internal and external) was then added at 250 mg mL -1 Additional trehalose was mixed with the resulting saRNA and trehalose co-loaded PE-LNP 11-65 to replenish, and the formulation was subsequently lyophilized. A trehalose / saRNA (w / w) weight ratio greater than 400 was found to result in nanoparticles larger than 300 nm in size after lyophilization and rehydration with RNase-free water. A trehalose / saRNA weight ratio of 100 was the best, resulting in the formation of rehydrated PE-LNP 11-65 with a small uniform DLS size of 167.7 ± 3.6 nm and good monodispersity with a PDI of 0.313 ± 0.017, comparable to freshly prepared trehalose-free PE-LNP 11-65 (size = 131.3 ± 0.5 nm and PDI = 0.265 shown in Figure 2C). This suggests that the presence of both internal and external trehalose well preserved the colloidal stability of PE-LNP 11-65 during the lyophilization and rehydration processes. Furthermore, saRNA and trehalose co-loaded PE-LNP 11-65 showed a high saRNA encapsulation efficiency of 85.2 ± 4.8%, which was comparable to the saRNA encapsulation efficiency of trehalose-free saRNA-loaded PE-LNP 11-65 (93.6 ± 3.2%).

[0219] Total trehalose concentration is 250mg mL -1 While the trehalose / saRNA (w / w) weight ratio was fixed at 50-100, it was preferred to increase the trehalose / saRNA (w / w) weight ratio to 50-100 (i.e., increase the proportion of internal trehalose) for lyophilization of PE-LNP 11-65 formulations. As shown in Figure 26, saRNA and trehalose co-added PE-LNP 11-65 formulations lyophilized under this condition showed a dramatic improvement in transfection efficiency upon immediate rehydration with RNase-free water, which was achieved at concentrations of 1 μg mL -1The transfection efficiency was 1.5 orders of magnitude higher than that of freshly prepared trehalose-free PE-LNP 11-65 at an equivalent saRNA dose of 100. Further increase in the trehalose / saRNA (w / w) weight ratio above 100 resulted in a significant decrease in transfection efficiency, but was still an order of magnitude higher than that of freshly prepared trehalose-free PE-LNP 11-65. This suggests that the coexistence of saRNA and trehalose inside the nanoparticles in combination with the presence of external trehalose may exert a synergistic effect, significantly improving RNA stability and efficacy.

[0220] Figure 27 compares the stability of lyophilized PE-LNP 11-65 with and without internal trehalose. After storage at 4°C for more than one year (380 days) and then rehydration with RNase-free water, PE-LNP 11-65 with saRNA and trehalose showed significantly higher transfection efficiency (p<0.05) than that of fresh samples without trehalose. This can be explained by the fact that trehalose provided a synergistic effect on improving transfection in addition to stabilizing the formulation. The formulation without internal trehalose showed more than two-fold loss in transfection efficiency after storage at 4°C for more than one year. This demonstrates that PE-LNP optimized in the presence of both internal and external trehalose can effectively maintain the functional stability of the lyophilized formulation during long-term storage.

[0221] For further heat stress testing of the lyophilized formulation, saRNA and trehalose co-loaded PE-LNP 11-65 (40 μg mL for co-encapsulation) was used. -1 of saRNA, and trehalose / saRNA weight ratio = 100, followed by 250 mg total trehalose mL -1 The optimized freeze-drying conditions for the 100% lyophilized cellulose (mixed with additional trehalose to replenish) were selected.

[0222] Example 15-Heat stress test of freeze-dried saRNA and trehalose-co-loaded PE-LNP for stable storage at 40°C The optimized saRNA and trehalose co-loaded PE-LNP 11-65 formulations were freeze-dried and then kept for storage at 40°C (tropical conditions). saRNA-loaded PE-LNP 11-65 mixed with external trehalose was freeze-dried as a control. After a period of storage, the freeze-dried PE-LNP 11-65 formulations were rehydrated with RNase-free water and their in vitro transfection efficiencies were measured.

[0223] Results and Discussion To evaluate the potential of RNA and trehalose co-loaded PE-LNP for non-cold chain storage, the optimized saRNA and trehalose co-loaded PE-LNP 11-65 were freeze-dried and kept for storage at 40° C. for 1, 3, 5, 7 or 14 days, respectively. Figure 28 shows that after freeze-drying and then immediate rehydration with RNase-free water (day 0), the transfection efficiency of saRNA and trehalose co-loaded PE-LNP 11-65, as well as saRNA-loaded PE-LNP 11-65 mixed with external trehalose, was about 1.5 orders of magnitude higher than that of freshly prepared PE-LNP 11-65 without trehalose, further confirming the synergistic effect of trehalose. As shown in Figures 31 and 32, no decrease in transfection efficiency was observed for saRNA and trehalose co-added PE-LNP 11-65, or saRNA-added PE-LNP 11-65 mixed with external trehalose, compared with freshly prepared trehalose-free saRNA-added PE-LNP 11-65, even after being kept for storage at a high temperature of 40°C for 14 days, indicating superior thermal stability and efficacy of RNA at room temperature and even tropical conditions.

[0224] We then used ultrafiltration centrifugation to remove free (non-added) external trehalose after freeze-drying, storing at 40°C for 7 days, and rehydrating PE-LNP 11-65 with saRNA and trehalose, and PE-LNP 11-65 with saRNA mixed with external trehalose, respectively. Figure 30 shows that after removal of external trehalose, especially on the 7th day of storage at 40°C, the transfection efficiency of rehydrated PE-LNP 11-65 without internal trehalose was significantly reduced compared to their counterparts without ultrafiltration centrifugation. In comparison, it is interesting to note that after removal of external trehalose, the transfection efficiency of rehydrated PE-LNP 11-65 with saRNA and trehalose co-added to the hydrophilic core of the nanoparticles was comparable to that of freshly prepared PE-LNP 11-65 with trehalose-free saRNA after storage at 40°C for 7 days. These results further confirm the important role of internal trehalose in enhancing the thermal stability and efficacy of RNA co-loaded into PE-LNPs.

[0225] Example 16 - Effect of trehalose on PE-LNP-mediated intracellular delivery of various RNA molecules Using the method described above in Example 12, trehalose and 50 μg mL -1 of calcein and 40 μg mL at a trehalose / saRNA (w / w) weight ratio of 100. -1 saRNA was pre-dissolved in RNase-free deionized water to prepare saRNA, trehalose, and calcein-co-added PE-LNP 11-65, which was then mixed with additional trehalose to give a total volume of 250 mg mL. -1 Total trehalose (both internal and external) was supplemented with 100 mg of saRNA and calcein co-loaded PE-LNP 11-65 (no trehalose) was prepared as a control. The uptake and intracellular trafficking of the two calcein-containing PE-LNP 11-65 formulations were investigated by laser scanning confocal microscopy. HEK293 cells (2 × 10 per dish) were cultured in a 2 mL volume. 5Cells) were seeded on 35 mm glass-bottom culture dishes and cultured for 24 h. saRNA, trehalose and calcein-co-loaded PE-LNP 11-65, or saRNA and calcein-co-loaded PE-LNP 11-65 were added to the culture dishes (2 μg saRNA per dish). After 2 h of treatment, cells were washed and replenished with complete medium for further incubation for 4 h. Cells were imaged using a Leica SP8 inverted confocal microscope, and the mean fluorescence intensity of calcein in the confocal microscopy images was analyzed by ImageJ.

[0226] Using the method described above in Example 12, GFP-expressing mRNA and trehalose-co-loaded PE-LNP 11-65 (trehalose / mRNA weight ratio = 100 for co-encapsulation, followed by 10 mg mL -1 PE-LNP 11-65 with mRNA but no trehalose was prepared as a control. HEK293 cells were plated in 6-well plates at 5 × 10 5 Cells were seeded at 1000 x g / well and cultured for 48 h, and in vitro transfection was quantitatively analyzed by flow cytometry (Canto, BD, USA).

[0227] Results and Discussion As shown in Figures 23-30, the significant enhancement in transfection efficiency of saRNA-added PE-LNPs due to the presence of external / internal trehalose prompted us to further investigate the synergistic effect of added trehalose. As an indicator of cellular uptake and intracellular trafficking, calcein, a membrane-impermeable dye, was co-added inside the two PE-LNP 11-65 formulations. As shown in the confocal microscopy images (Figure 31A), HEK293 cells treated with freshly prepared saRNA, trehalose and calcein co-added PE-LNP 11-65 showed significantly stronger green diffuse staining throughout the cells compared to those treated with freshly prepared saRNA and calcein co-added PE-LNP 11-65. Mean fluorescence intensity (MFI) was analyzed by ImageJ. Figure 31B shows that cells treated with saRNA, trehalose and calcein-co-loaded PE-LNP 11-65 had significantly higher MFI than cells treated with saRNA and calcein-co-loaded PE-LNP 11-65, confirming that the presence of trehalose can enhance cellular uptake and subsequent endosomal escape.

[0228] The effect of trehalose on the intracellular delivery of GFP-encoding mRNA into HEK293 cells by PE-LNP 11-65 was also investigated. As shown in Figure 32, after treatment with freshly prepared mRNA and trehalose-co-loaded PE-LNP 11-65, the percentage of GFP-expressing HEK293 cells increased from 51.9±4.9% to 74.2±2.2%, and the MFI increased 1.7-fold, compared to freshly prepared mRNA-loaded PE-LNP 11-65 without trehalose. This further confirms the synergistic effect of trehalose on the intracellular delivery of various nucleic acids by PE-LNP-based formulations.

[0229] Example 17 Variations in lipid composition of PE-LNP saRNA was formulated inside PE-LNP 11-65 in the presence of cholesterol according to the same method as described above in Example 2, where cholesterol was co-dissolved with DOTAP and PEG-PCL in THF. Cholesterol content (wt%) was defined as the weight percentage of cholesterol relative to cholesterol and DOTAP. The size, PDI and zeta potential of the nanoformulations were evaluated using a Litesizer (Anton Paar, UK). Then, 1 μg mL -1 Their in vitro HEK293 cell transfection efficiencies at saRNA doses of 1000 μg / ml were measured.

[0230] Results and Discussion The preparation method of the PE-LNP system can be easily adapted to incorporate a variety of lipids and combinations thereof into the nanoparticle core, including but not limited to cationic / ionizable lipids with charged groups, and sterols such as cholesterol. We chose cholesterol to be incorporated into the PE-LNP system to further demonstrate the versatility of the nanosystem with respect to lipid composition.

[0231] Figure 33 shows the physicochemical characterization of PE-LNP 11'-65' and the transfection efficiency of saRNA-loaded PE-LNP 11-65 formulated with cholesterol. PE-LNP 11'-65' in the presence of cholesterol exhibited suitable size, PDI and zeta potential for biomolecule delivery. We then proceeded to investigate the effect of cholesterol content on transfection efficiency. A significant increase of >1 in transfection efficiency was observed when increasing cholesterol content from 20 to 40 wt%. This demonstrates that the PE-LNP system is compatible with various lipid molecules and their combinations. The transfection efficiency of the nanoformulations can be tailored for various applications by adjusting the lipid composition.

[0232] conclusion We have developed polymer-encapsulated lipid nanoparticles (PE-LNPs) that achieve efficient intracellular delivery of biomolecules, including RNA, both in vitro and in vivo, and enable stable storage of vaccines and therapeutics without the need for cold chains. This nanoformulation platform has the characteristics of an ideal carrier, including a favorable safety profile, small size, controlled charge, high loading efficiency, efficient endosomolytic activity, excellent colloidal and payload (RNA) stability, and a simple, cost-effective, and easily scalable preparation method.

[0233] PE-LNPs are composed of two main structural components that have been proven to be biocompatible and approved by the FDA: an amphiphilic polymer, such as PEG-PCL, and a cationic or ionizable lipid, such as DOTAP. PE-LNPs can be prepared using a simple one-pot method with easy and rapid mixing and organic solvent evaporation. Cryo-TEM and FRET analysis showed that the inner structure of PE-LNPs consists of lipid nanostructures, which is favorable for efficient payload encapsulation. The self-assembled PEG-PCL outer layer surrounding the inner lipid nanostructures can ensure colloidal stability and serum compatibility and sufficiently protect the function of payloads such as extremely labile RNA. PE-LNPs showed excellent stability and high in vitro transfection efficiency, which can be four orders of magnitude higher than commercial PEI in the presence of FBS.

[0234] Mice transfected with PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80 by IM injection, respectively, showed strong luciferase expression. Moreover, all nanoformulations induced high HA IgG antibody titers, so that all mice were effectively protected against Cal / 09 influenza challenge after immunization with HA saRNA formulated inside PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80, respectively. The average weight loss of mice immunized with HA saRNA-loaded PE-LNP was less than 10%, and PE-LNP 11-65 in particular induced the least amount of weight loss (approximately 8%).

[0235] We have demonstrated two strategies for stable storage of biological payloads at ambient temperature, especially RNA molecules that are easily hydrolyzed / degraded. The optimal protection provided by the core-shell nanoparticle structure allowed for stable storage of RNA-loaded PE-LNPs in aqueous solutions at room temperature. Another strategy for stable RNA storage is lyophilization of RNA-loaded PE-LNPs in the presence of external and / or internal stabilizing molecules such as trehalose. saRNA and trehalose co-loaded PE-LNPs, as well as saRNA-loaded PE-LNPs mixed with external trehalose, showed the desired thermal stability of saRNA after lyophilization, storage at ambient temperatures as high as 40°C (tropical conditions), and rehydration, with the former performing better. These demonstrate that PE-LNP nanoformulations can enable stable storage of vaccines and therapeutics at ambient temperatures without the need for cold chains. This provides a viable solution for improving global distribution of vaccines and therapeutic formulations.

[0236] Furthermore, the PE-LNPs developed by the inventors have been demonstrated to be disseminable for efficient intracellular delivery to various cell types and non-cold chain storage of various vaccines and therapeutics (based on biomolecules including, but not limited to, saRNA and mRNA).

[0237] material mPEG-OH(M n =5,000 and 2,000), ε-caprolactone (ε-CL), toluene, diethyl ether, tetrahydrofuran (THF), 4% paraformaldehyde solution, fluorescein isothiocyanate (FITC), Hoechst, LysoTracker (red), Triton X-100, bovine serum albumin (BSA) and Tween-20 were purchased from Sigma-Aldrich. 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP, chloride salt) was purchased from Avanti Polar Lipids. Trypsin-EDTA (0.25%, w / v), fetal bovine serum (FBS) and 1% penicillin / streptomycin were purchased from Gibco (CA, USA). ONE-Glo™ Luciferase Assay System was obtained from InvivoGen. Phosphate-buffered saline (PBS) and DMEM medium were obtained from Hyclone Laboratories (UT, USA). RNase-free water, RNase-free PBS (10x) and TPCK-trypsin were purchased from Thermo Fisher Scientific (UK). Trehalose Assay Kit was purchased from Abbexa. XenoLight RediJect D-Luciferin Substrate was purchased from Perkin Elmer. Firefly luciferase saRNA and saRNA encoding Cal / 09 virus H1 hemagglutinin were both kindly donated by the group of Professor Robin Shattock, St Mary Hospital and Department of Infectious Disease, Imperial College London.

[0238] cell culture HEK293 (human embryonic kidney) cells and HeLa (human cervical cancer cell line) cells were obtained from ATCC (American Type Culture Collection, Wesel, Germany) and cultured in high glucose DMEM medium (Gibco) supplemented with 10% (v / v) FBS (Gibco) and 1% (v / v) penicillin / streptomycin (Gibco). Jurkat human T lymphocyte cells (Clone E6-1, ATCC® TIB-152™) were cultured in 10% (v / v) FBS, 100 U mL -1 Penicillin, 100 μg mL -1 Cells were cultured in either RPMI-1640 medium supplemented with streptavidin and 2 mM L-glutamine (R10 medium) or OPTIMEM medium. Cells were incubated at 37°C in 5% CO 2 The plates were incubated in a humidified incubator at 4 °C.

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Claims

1. comprising a plurality of lipid structures surrounded by an outer layer containing a payload molecule and an amphiphilic copolymer, wherein the amphiphilic copolymer comprises at least one hydrophilic moiety and at least one hydrophobic moiety, the hydrophilic moiety constitutes 5 to 40% by weight of the amphiphilic copolymer, and the hydrophobic moiety constitutes 60 to 95% by weight of the amphiphilic copolymer, submicron particles.

2. The submicron particles according to claim 1, having a maximum dimension of less than 1 μm.

3. The submicron particles according to claim 1 or claim 2, wherein the payload molecule is a biomolecule and / or an active pharmaceutical ingredient (API).

4. The submicron particles according to claim 3, wherein the biomolecule is a nucleic acid, and the nucleic acid is a DNA, RNA, or DNA / RNA hybrid sequence.

5. The submicron particles according to claim 4, wherein the RNA is self-amplifying RNA (saRNA) or messenger RNA (mRNA).

6. The submicron particles according to claim 1 or 2, wherein the plurality of lipid structures are a plurality of lipid nanoparticles or a plurality of liposomes, preferably a plurality of lipid nanoparticles.

7. The lipid structure contains a cationic lipid or an ionizable lipid, and / or having an N / P molar ratio of 1:50 to 1,000:1, 1:10 to 500:1, 1:5 to 250:1, 1:2 to 100:1, 1:1 to 50:1, 2:1 to 40:1, 5:1 to 30:1, 8:1 to 28:1, 10:1 to 26:1, 12:1 to 24:1, 14:1 to 22:1, or 16:1 to 20:1, the submicron particles according to claim 1 or 2.

8. The weight ratio of the amphiphilic copolymer to the payload molecule is 5:1 to 1000:1, 10:1 to 500:1, 20:1 to 250:1, 30:1 to 200:1, 40:1 to 150:1, 50:1 to 125:1, 55:1 to 100:1, or 60:1 to 85:1, and / or the weight ratio of the amphiphilic copolymer to the cationic lipid or ionizable lipid is 1:10 to 50:1, 1:8 to 20:1, 1:6 to 15:1, 1:4 to 10:1, 1:2 to 8:1, 1:1.5 to 6:1, 1:1 to 5.5:1, 1.5:1 to 5:1, 1.75:1 to 4.5:1, 2:1 to 4:1, 2.2:1 to 3.5:1, 2.4:1 to 3:1, or 2.5:1 to 2.7:

1. The submicron particles according to claim 1 or 2.

9. The amphiphilic copolymer includes at least one hydrophilic portion and at least one hydrophobic portion, the hydrophilic portion constitutes 10 to 35% by weight of the amphiphilic copolymer, and the hydrophobic portion constitutes 65 to 90% by weight of the amphiphilic copolymer. The submicron particles according to claim 1 or 2.

10. The amphiphilic copolymer has a molecular weight of 1,000 to 100,000 Da. The submicron particles according to claim 1 or 2.

11. The submicron particles according to claim 1 or 2 further include at least one stabilizing molecule.

12. At least one stabilizing molecule is surrounded by the outer layer including the amphiphilic copolymer; The weight ratio of the at least one stabilizing molecule to the payload molecule is 1:1 to 100,000:1, 2:1 to 50,000:1, 4:1 to 10,000:1, 6:1 to 5,000:1, 8:1 to 1,000:1, 10:1 to 500:1, 20:1 to 400:1, 30:1 to 350:1, or 40:1 to 300:1; At least one stabilizing molecule is disposed outside the outer layer containing the amphiphilic copolymer, preferably, the at least one stabilizing molecule disposed outside the outer layer containing the amphiphilic copolymer is disposed at a concentration of 1 to 100,000 mg / ml, 5 to 50,000 mg / ml, 10 to 10,000 mg / ml, 25 to 5,000 mg / ml, 50 to 1,000 mg / ml, 100 to 750 mg / ml, 150 to 500 nm / ml, 200 to 300 nm / ml, 220 to 280 nm / ml, or 240 to 260 mg / ml; and / or, The stabilizing molecule or each stabilizing molecule is a carbohydrate and / or a polyol, preferably, the carbohydrate is a monosaccharide, disaccharide, trisaccharide, polysaccharide, starch, cellulose or polyol, preferably, trehalose or a pharmaceutically acceptable complex, salt, solvate, tautomer, stereoisomer or polymorph thereof, the submicron particles according to claim 11.

13. A method for producing submicron particles, comprising contacting a payload molecule, a cationic lipid or an ionizable lipid, and an amphiphilic copolymer to produce the submicron particles, The amphiphilic copolymer includes at least one hydrophilic portion and at least one hydrophobic portion, the hydrophilic portion constitutes 5 to 40% by weight of the amphiphilic copolymer, and the hydrophobic portion constitutes 60 to 95% by weight of the amphiphilic copolymer, the method.

14. - Providing a first solution comprising the cationic lipid or ionizable lipid and the amphiphilic copolymer and an organic solvent, - Providing a second solution comprising the payload molecule and water, - Combining the first and second solutions to produce a reaction mixture, thereby contacting the payload molecule, the cationic lipid or ionizable lipid, and the amphiphilic copolymer comprising; and / or Simultaneously contacting the payload molecule, the cationic lipid or ionizable lipid, and the amphiphilic copolymer includes contacting the payload molecule, the cationic lipid or ionizable lipid, the amphiphilic copolymer, and at least one stabilizing molecule, and / or The method according to claim 13, comprising contacting the obtained submicron particles with at least one stabilizing molecule.

15. Submicron particles obtainable or obtained by the method according to claim 13 or 14.

16. A pharmaceutical composition comprising the submicron particles according to claim 1 or 2 and a pharmaceutically acceptable vehicle.

17. The pharmaceutical composition according to claim 16 for use as a medicament.

18. A vaccine composition comprising the submicron particles according to claim 1 or 2.

19. The vaccine composition according to claim 18 for use in stimulating an immune response in a subject.

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