Nucleic acid-lipid particles

Abstract

The present invention relates to compositions comprising nucleic acid-lipid particles, wherein the nucleic acid-lipid particles are characterized by encapsulation of the nucleic acid in a lipid bilayer. The present invention further relates to said compositions for use in preventing and / or treating disease, particularly for use in preventing and / or treating cancer. The present invention further relates to methods for producing compositions comprising said nucleic acid-lipid particles.

Description

[Technical Field]

[0001] Technical Field The present invention relates to nucleic acid-lipid particles, particularly for use in therapy, such as treating or preventing cancer, infectious disease, or autoimmune disease. [Background technology]

[0002] Background of the Invention Nucleic acid adjuvants have attracted attention because they can induce strong cellular immune responses to antigens, which is important in cancer therapy, for example. However, the physicochemical properties, pharmacokinetics, and toxicity profiles of nucleic acid adjuvants have limited their application in cancer immunotherapy. This is particularly because nucleic acids are hydrophilic, highly negatively charged molecules and have poor cell penetration ability. When administered intravenously, nucleic acids have a short half-life and are rapidly degraded by nucleases in the blood. These undesirable aspects often force the use of high doses, and may even induce non-specific and toxic effects, especially under systemic administration.

[0003] Current nucleic acid delivery is mostly based on the use of cationic lipids, cationic polymers, or multivalent cations. These cationic molecules interact with the negatively charged phosphate groups of nucleic acids, allowing for encapsulation in the formation of nano- or microparticles. Cationic liposomes and cationic polymers have been widely used for in vitro transfection of cells. They are based on molecules that are positively charged at physiological pH (7.4). They have been successful in protecting nucleic acids from nuclease degradation and enhancing cellular uptake.

[0004] However, some of these delivery systems are highly toxic in vivo and not physiologically stable in plasma, limiting their application for systemic administration of nucleic acids. Recently, novel lipid nanoparticles (LNPs) have been developed for systemic administration of nucleic acids (U.S. Patent No. 8,058,069 B2). They are based on ionizable cationic lipids and are positively charged at acidic pH (<6.5) but not at physiological pH (7.4). The inclusion of ionizable cationic lipids allows for efficient encapsulation and intracellular release of nucleic acids. They have been successfully used in humans for siRNA delivery and in mRNA COVID-19 vaccines. Although LNPs are serum stable, the ionizable cationic lipids used in LNP technology can negatively impact RNA stability (Packer et al., Nat Commun Vol 12, 2021, ref. 6777) and potentially induce dose-limiting inflammatory responses, as demonstrated in preclinical and clinical studies (Ndeupen et al., iScience. 2021 Dec 17;24(12):10347; Tahtinen et al., Nat Immunol Vol 23, 2022, pp. 532-542). For example, cationic lipids (as well as cationic polymers and multivalent cations) activate intracellular inflammasomes, such as NLRP3, which promote IL-1β expression and induce cell death. Such inflammatory responses may limit the broader application of the technology, particularly outside of the vaccination field, given the need for systemic application and the frequent use of nucleic acids in gene therapy and therapeutic protein production. Additionally, the inflammatory profile of NLPs is undesirable for nucleic acid adjuvants, which are inherently proinflammatory and already limit therapeutic duration due to the induction of potentially toxic nonspecific effects. Furthermore, inflammasome activation and IL-1β-mediated inflammation are undesirable for cancer therapy because they have been found to suppress antitumor immunity and lead to the progression of human melanoma, gastric cancer, and breast cancer (Yu et al., Sig Transduct Target Ther Volume 6, 2021, ref. 128).

[0005] Multivalent cations such as calcium and magnesium have been used to complex nucleic acids with neutral and anionic liposomes (Kapoor et al., International Journal of Pharmaceutics, Volume 432, Issues 1-2, 2012, p80-90). However, these multivalent cations can cause liposome aggregation, making their production difficult, affecting particle stability and their biological properties. In addition, they can also reduce the stability and activity of nucleic acids (Dallas, A et al., Artificial nucleases, 2004, p61-88). This method is mainly used in in vitro tests and has not been used in clinical applications. Furthermore, Michanek et al. (Biochimica et Biophysica Acta 1798, 2010, p829-838) investigated how tRNA, when added to preformed nanoparticles, associates with liposomes in the absence of divalent cations, but found that tRNA is only adsorbed to the outer surface of liposomes to a low degree and in a weak manner. Therefore, the lipid composition used is not suitable for pharmaceutical carrier applications.

[0006] It is an object of the present invention to overcome one or more of the above-mentioned or other problems. Summary of the Invention [Problem to be solved by the invention]

[0007] Summary of the Invention The present inventors have developed a method for producing stable anionic liposomes that encapsulate nucleic acids specifically within their lipid bilayer(s). The encapsulation of nucleic acids by each nucleic acid-lipid particle is - Higher stability of nucleic acids; - a stronger association of nucleic acids with particles, which is important for achieving therapeutic functionality, for example, when compared to nucleic acids adsorbed to particles (Michanek et al. (Biochimica et Biophysica Acta 1798, 2010, p829-838); and - a higher degree of resistance of the nucleic acid to the action of nucleases when the particles are released into the patient's bloodstream; and - Avoidance of increased vascular permeability and potential immunotoxic reactions due to the lack of substantial localization of RNA on the nanoparticle surface (Fischer, et al. Blood, The Journal of the American Society of Hematology 110.7, 2007, p2457-2465), This makes it possible.

[0008] Liposomes obtainable by the methods of the present invention are particularly attractive for targeted delivery of nucleic acid adjuvants to the immune system. In particular, liposomes encapsulating nucleic acids enhance adjuvant uptake by immune cells (e.g., dendritic cells, macrophages, lymphocytes) followed by improved immune stimulation (e.g., stimulation by Toll-like receptor activation). The inventors have shown that encapsulation in anionic liposomes protects nucleic acids, making them more accessible to target immune cells, amplifying their biological effects. Anionic liposomes containing nucleic acids within the bilayer(s) are biocompatible and physically stable. Incorporating nucleic acid(s) into the bilayer(s) better preserves the content and biological activity of nucleic acids during storage (a shelf life of at least 5 months, rather than several weeks) compared to those not incorporating nucleic acid(s) within the bilayer(s) as in the prior art. The method of the present invention requires minimal or no cationic lipids, thus overcoming the problems associated with cationic liposomes (e.g., toxicity and poor physical stability in vivo). In contrast to cationic nanoparticles, anionic liposomes do not activate inflammasomes (Li, T. et al., Nanomedicine Volume 14, 2018, p279-288) and are naturally immune-tolerant (Benne et al., Journal of Controlled Release, Volume 291, 2018, p135-146).

[0009] The present invention relates to a method for producing stable liposomes with highly efficient encapsulation of nucleic acids in lipid bilayer(s). Microfluidics technology was used to demonstrate the encapsulation of poly(I:C), a model RNA-based nucleic acid adjuvant, in the liposomal bilayer. A provocative observation is that these RNA-liposome formulations appear to require little or no cationic molecules to encapsulate the RNA. This is advantageous because the inclusion of cationic molecules increases the immunogenicity and toxicity of the formulation (Patil et al., AAPS J 6, 13-22 (2004)). Encapsulation of nucleic acids in lipid bilayer(s) is preferably achieved by using a suitable organic solvent with minimal or no cationic molecules. The organic solvent is preferably an ethanol solution with at least 80% ethanol. To achieve efficient encapsulation of nucleic acids into lipid bilayers and stable particles, an organic solvent is mixed with an aqueous solution containing one or more nucleic acid forms, e.g., by providing a laminar flow, e.g., using a relatively low flow rate (e.g., 25-5000 μL / min). Preferably, following the initial mixing of the organic and aqueous solutions, one or more additional aqueous solutions (RNase-free aqueous media) or organic solutions are mixed, e.g., using a relatively low flow rate (e.g., 10-20,000 μL / min or 25-5000 μL / min, preferably 25-500 μL / min). This can increase formulation reproducibility and allow for better control of particle size.

[0010] Without being bound by theory, hydrophobic interactions, particularly in lipid bilayer(s), may be the mechanism for encapsulation of RNA by liposomes. Through mixing with one or more organic solutions, nucleic acid molecules change conformation to expose hydrophobic moieties that can interact with lipid tails, especially under laminar mixing.

[0011] Our findings are surprising because encapsulation of nucleic acids into anionic or neutral liposomes is generally considered to be very inefficient and requires pre-condensation of nucleic acids with cationic molecules (Bailey et al. Biochim Biophys Acta. 2000 Sep 29; 1468(1-2):239-52; Patil et al., AAPS J 6, 13-22, 2004; Kapoor et al., International Journal of Pharmaceutics, Volume 432, Issues 1-2, 2012, p80-90). However, the use of Ca for encapsulating nucleic acids into anionic liposomes has not been shown to be effective. 2+ The use of polyvalent cationic ions such as α- and β-glucan ions leads to the formation of aggregates, significantly increasing the average size of liposomes to values ​​significantly higher than about 220 nm (Kapoor et al., and Patil et al.). This is disadvantageous when nucleic acid-containing liposomes are administered as injections, since a subsequent sterile filtration step must be introduced into the manufacturing process. Sterile filtration is performed by passing the formulation through a sterilizing-grade filter with a size of 0.22 μm. Any particles larger than 220 nm may significantly reduce the filtration rate or ultimately cause filter clogging, negatively affecting the manufacturability of the formulation. The present invention enables the efficient encapsulation of nucleic acids into nucleic acid-lipid particles, such as anionic liposomes, with an average size of less than 220 nm, making them suitable for the sterile filtration step. In addition, in contrast to prior art thin-film methods, the microfluidics method used in the present invention allows for scale-up of production. The inventors also surprisingly found that a lower dose of nucleic acid is required to achieve a therapeutic effect compared to prior art methods.

[0012] Structural characterization of liposomes obtained by the methods described herein indicates that the RNA-containing nanoparticles possessed a typical liposomal vesicle structure, and notably, the RNA cargo appears to be primarily embedded within the hydrophobic regions of the liposomal bilayer. Liposomes of the present invention typically have an aqueous core and one or more lipid bilayers containing RNA. The organization of lipids within the bilayer (lamellar phase) is energetically most favorable and provides a highly stable structure. In comparison, prior art nucleic acid-LNPs have a solid core containing RNA, cationic lipids, and cholesterol with a low water content (27% water volume vs. total core volume) and a reverse hexagonal phase (H) compared to conventional liposomal particles. II ) and lipids organized in a matrix (Arteta et al. 2018, PNAS Vol. 115|No. 15). Cationic ions (e.g., Ca 2+ The complexation of nucleic acids with anionic liposomes using α-hydroxybenzoates (H ) induces the lipids to change from a lamellar phase to a physically less stable inverted hexagonal phase (H ). II ) (Barraan-Berdon et al., Langmuir, 2014, vol. 30, no. 39, p. 11704-11713). The RNA-containing liposomes of the present invention were physically stable during storage at 5°C and -20°C for 5 months. The localization of nucleic acids in the water-free lipid bilayer may explain the maintenance of nucleic acid content and biological activity during storage. Experiments have shown that anionic RNA-containing liposomes remain intact, without aggregating or releasing their contents, and protect the RNA from nuclease degradation when incubated with human plasma at 37°C. Immunostimulatory activity was tested ex vivo using mouse splenocytes and in vivo in healthy and tumor-bearing mice. Poly(I:C) encapsulation enhances the activation of dendritic cells, T cells, and NK cells, and the production of antiviral cytokines, without significant toxicity.

[0013] Liposomes obtainable by this method are stable in plasma, stable during storage, and can be efficiently taken up by immune cells. Liposomes with encapsulated nucleic acids, for example, in the bilayer(s), have been shown to have desirable physical properties for clinical applications such as cancer therapy, including desirable size distribution, polydispersity, and charge. Furthermore, the inventors have discovered that fine-tuning the lipid composition of liposomes can direct immune activity to achieve either preferentially immunostimulatory or immunosilent effects. Incorporation of poly(I:C) into anionic liposomes has been shown to enhance the vaccine efficacy of liposomes containing ovalbumin peptide models. Intravenous administration of liposome-encapsulated poly(I:C) according to the present invention, either as monotherapy or in combination with immune checkpoint inhibitor antibodies (ICI), in multiple mouse tumor models has been shown to delay tumor growth and improve overall mouse survival, unlike ICI or free poly(I:C) in combination as monotherapy. In summary, the characteristics of the particles / liposomes of the present invention make them attractive for targeted delivery to the immune system (e.g., of nucleic acid adjuvants) and / or specifically to the spleen of a subject. It has been found that the addition of anionic lipids to particles improves uptake by splenic dendritic cells. In one experiment, we evaluated uptake and activation by ex vivo splenic conventional type 1 dendritic cells (cDC1), a cell population that expresses TLR3 and upregulates maturation markers in the presence of poly(I:C). In this experiment, we sought to understand the effect of EPG concentration on particle uptake and maturation by cDC1. We selected five formulations containing increasing concentrations of EPG (0, 4, 17, 33, and 67% molar ratios), decreasing concentrations of EPC, and a constant concentration of cholesterol (33% molar ratio). Increasing the molar ratio of EPG was shown to enhance nanoparticle uptake by cDC1 cells.

[0014] In one aspect, the present invention relates to a method for preparing a composition comprising nucleic acid-lipid particles, e.g., comprising at least 2, 10, 100, 1000, 10,000, 100,000 or more particles, the method comprising: a) providing one or more organic solutions comprising at least one anionic lipid and / or at least one neutral lipid; b) providing one or more aqueous solutions containing one or more (types of) nucleic acids; c) combining said one or more organic solutions with said one or more aqueous solutions, preferably by laminar mixing, thereby producing a composition comprising nucleic acid-lipid particles; Includes.

[0015] In one aspect, the present invention relates to a composition comprising an anionic nucleic acid-lipid particle, the anionic nucleic acid-lipid particle comprising: - with one or more (types of) nucleic acids; - one or more (anionic) lipid bilayers comprising (and / or formed by) at least one anionic lipid and / or at least one neutral lipid, Includes.

[0016] Preferably, at least a portion of the one or more nucleic acids is encapsulated by the anionic nucleic acid-lipid particle, and / or less than 10% by weight of the one or more nucleic acids is exposed on the exterior of the anionic nucleic acid-lipid particle, and / or less than 10, 5, or 1% by weight of the one or more nucleic acids is covalently bound to lipids contained by the anionic nucleic acid-lipid particle.

[0017] Preferably, the nucleic acid-lipid particles do not contain cationic lipids, cationic polymers, or multivalent cationic ions (e.g., Ca2+), or contain cationic molecules such as cationic lipids, cationic polymers, or multivalent cationic ions (e.g., Ca2+) at a maximum of 20 mol% relative to the total lipid molar amount of the nucleic acid-lipid particles. Cationic molecules / ions may lead to undesired aggregation of the particles and / or undesired degradation of one or more nucleic acids.

[0018] In one aspect, the present invention relates to a composition according to the present invention for use in preventing and / or treating a disease. [Means for solving the problem]

[0019] Detailed Description of the Invention Compositions of the Invention The present invention provides compositions comprising nucleic acid-lipid particles, e.g., at least 2, 10, 100, 1000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 or for compositions containing more particles, the nucleic acid-lipid particles are - with one or more nucleic acids; - one or more lipid bilayers comprising (and / or formed by) at least one anionic lipid and / or at least one neutral lipid, wherein the nucleic acid-lipid particle is anionic.

[0020] Preferably, at least a portion of the one or more nucleic acids are encapsulated by the nucleic acid-lipid particle, and more preferably, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20% by weight of the one or more nucleic acids are encapsulated (or localized) by the nucleic acid-lipid particle, such as within the outer boundary of the nucleic acid-lipid particle.

[0021] Additionally or alternatively, at least a portion, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20% by weight, or all of the one or more nucleic acids, preferably in the form of a nucleic acid-lipid complex, are contained within the hydrophobic region of one or more lipid bilayers (e.g., where the hydrophobic portion of the one or more nucleic acids interacts with the lipid tails of one or more lipid bilayers). Preferably, at least a portion, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20% by weight, or all of the one or more nucleic acids are not in contact with the aqueous medium (e.g., contained in the core or surrounding the nucleic acid-lipid particle).

[0022] Additionally or alternatively, less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20% by weight (or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20% by weight) of the one or more nucleic acids is exposed on the exterior (or adsorbed on the exterior) of the nucleic acid-lipid particle.

[0023] Additionally or alternatively, less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20% by weight (or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20% by weight) of the one or more nucleic acids is contained within the (aqueous) core of the nucleic acid-lipid particle. The term "aqueous core" can refer to the aqueous medium surrounded by one or more lipid bilayers.

[0024] The term "encapsulated" can be understood to mean that at least a portion of one or more nucleic acids (i.e., at least some (preferably all) molecules of one or more nucleic acids) are enclosed (or confined) within the outer boundary of the (anionic) nucleic acid-lipid particle. Preferably, some or all of the one or more nucleic acids (i.e., at least some (preferably all) molecules of one or more nucleic acids) are not in (direct) contact with the (aqueous) medium surrounding the (anionic) nucleic acid-lipid particle.

[0025] In particular, at least a portion of one or more nucleic acids may be present in or contained within one or more lipid bilayers. For example, compositions are provided that include nucleic acid-lipid particles, the nucleic acid-lipid particles comprising: - with one or more nucleic acids; - one or more lipid bilayers comprising at least one anionic lipid and / or at least one neutral lipid; Including, the nucleic acid-lipid particles are anionic; At least a portion of the one or more nucleic acids is present within one or more lipid bilayers.

[0026] Preferably, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90% by weight of the one or more nucleic acids in the nucleic acid-lipid particles are present in the one or more lipid bilayers, based on the total weight of the one or more nucleic acids in the nucleic acid-lipid particles. 6 ~1×10 12 The particles comprise between 1000 and 10000 particles / ml.

[0027] The composition may be a lipid composition. Alternatively, the composition may be a solid composition, such as a powder composition (e.g., a composition comprising lyophilized nucleic acid-lipid particles). Additionally or alternatively, the composition may be a frozen formulation.

[0028] As used herein, the term "nucleic acid" refers to a polymer containing at least two nucleotides, preferably at least two deoxyribonucleotides and / or ribonucleotides, in either single-stranded or double-stranded form. Thus, the term encompasses DNA, RNA, and DNA / RNA hybrids. DNA may be in the form of, for example, an antisense molecule, a plasmid DNA, a pre-condensed DNA, a PCR product, a vector (P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, a chromosomal DNA, or derivatives and combinations of these groups. RNA may be in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), lncRNA, gRNA, mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. The term "nucleic acid" encompasses synthetic, naturally occurring, or non-naturally occurring nucleic acids containing nucleotide analogs or modified backbone residue(s) or linkage(s), which may have similar or different binding properties to a reference nucleic acid lacking said modifications or variations. Examples of such analogs include, but are not limited to, cyclic dinucleotides, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group.Nucleotides are linked to each other through phosphate groups. "Bases" include purines and pyrimidines, and further include the naturally occurring compounds adenine, thymidine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that introduce new reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides. The "nucleic acids" of the present invention may be naturally occurring or non-naturally occurring (i.e., "nucleic acid analogs"). Thus, the term "nucleic acid" as used herein may be substituted with "nucleic acid and / or nucleic acid analog." A "nucleic acid analog" may be a polymer of nucleotides (e.g., at least two nucleotides) with at least one modification in one or more of, for example, the phosphate backbone, the pentose sugar, and one of the four nucleobases. A "nucleic acid analog" may be, for example, synthetic, i.e., produced by chemical synthesis. A "nucleic acid analog" can be a molecule that is structurally similar to a naturally occurring nucleic acid but has modifications in the molecular backbone, e.g., in the case of peptide nucleic acids (PNAs), morpholinos, and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs), threose nucleic acids (TNAs), and hexitol nucleic acids (HNAs), and lipid nucleic acids. As used herein, the term "RNA analog" can refer to a nucleic acid analog in which the pentose sugar is ribose, for example. As used herein, an "RNA analog" is preferably structurally similar to dsRNA. When referring to "one or more" nucleic acids, this can refer to one or more different types or forms of nucleic acid, e.g., selected from those listed above, or to one or more nucleic acids with different constituent residues or different sequences. For example, one or more can refer to at least one, two, three, four, five, or ten nucleic acids. Thus, the term "nucleic acid" can refer to one (type of) nucleic acid that can exist as multiple (identical) nucleic acid molecules. One or more nucleic acids can also be non-coding nucleic acids.

[0029] Preferably, the one or more nucleic acids are contained in a particle or one or more lipid bilayers thereof in the form of a nucleic acid-lipid complex, preferably stabilized by hydrophobic interaction(s) between the lipid and the non-polar portion of the nucleic acid. Additionally or alternatively, the one or more nucleic acids are contained / encapsulated in a particle or one or more lipid bilayers thereof, and the encapsulation is not, or substantially not, due to electrostatic interaction(s).

[0030] Nucleic acids in the context of the present invention may have any length, for example at least 10, 100, 1000 bp (base pairs), or at least 10, 1000 kb (kilobases), and / or less than 1000 kb, less than 10 kb, less than 1 kb, but preferably have a length of between 100 bp and 100 kb, preferably between 0.1 and 20 kb.

[0031] In a preferred embodiment, the nucleic acid is an immunological adjuvant. In the context of the present invention, the term "immunological adjuvant" refers to a substance or compound that modulates, preferably increases, the immune response (natural or acquired) to another immunological drug, such as a vaccine and / or a cancer immunotherapy. This typically means that the strength of the immunological drug is increased and / or less immunological drug is required to achieve similar effectiveness of the immune response compared to when the immunological adjuvant is not used. "Immunological adjuvants" preferably mimic the activity of pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides, molecular cages of antigens, components of bacterial cell walls, and nucleic acids such as single-stranded RNA, double-stranded RNA, single-stranded DNA, unmethylated CpG dinucleotide-containing DNA, or DNA-RNA hybrids. Additionally or alternatively, the term "immunological adjuvant" as used herein may refer to a substance or compound that further enhances the immune response nonspecifically or specifically through binding to pattern recognition receptors (PRRs), including, but not limited to, C-type lectin receptors (CLRs), RIG-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD) proteins, stimulator of interferon genes (STING), and Toll-like receptors (TLRs). Preferably, the present disclosure is not for use as a SARS-Cov2 vaccine. Preferably, the nucleic acid-lipid particles according to the present invention do not contain a SARS-Cov2 antigen.

[0032] In a preferred embodiment, the immunological adjuvant and / or one or more nucleic acids are TLR agonists, preferably agonists of one or more of the endosomal TLRs, more preferably TLR3, 7, 8 and / or 9. A "Toll-like receptor" in the context of the present invention may be TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13, preferably TLR3. The immunological adjuvant and / or TLR agonist preferably comprises one or more CpG (cytosine followed by a guanine residue), CpG motif, methylated CpG, CpG oligodeoxynucleotide (ODN, e.g., 5-25 bp)) (e.g., CpG1018, a TLR9 agonist), poly(I:C) (i.e., polyinosinic acid:polycytidylic acid, a TLR3 agonist), poly(I:C) 12 Poly(G:C) (TLR3 agonist), poly(A:U) (TLR3 agonist), poly(U) (TLR7 / 8 agonist). The CpG ODN may be class A, class B, or class C CpG ODN.

[0033] As used herein, the term "CpG" refers to a nucleic acid (analog) in which a cytosine is followed by a guanine linked by a phosphate bond and the pyrimidine ring of the cytosine is unmethylated. As used herein, the term "methylated CpG" refers to methylation of the cytosine on the pyrimidine ring, preferably at the 5-position of the pyrimidine ring. As used herein, the term "CpG motif" refers to a pattern of bases that includes an unmethylated central CpG, surrounded by at least one flanking base (on both the 3' and 5' sides of the central CpG). As used herein, the term "CpG ODN" refers to an oligodeoxynucleotide that includes one or more unmethylated CpGs and is preferably at least about 10 nucleotides in length. A "CpG ODN" is preferably single-stranded. The entire CpG ODN may be unmethylated, but a portion may also be unmethylated. In one embodiment, at least the C of the 5'CG3' is unmethylated. A "CpG ODN" may be a class A, class B, or class C CpG ODN. Those skilled in the art are familiar with different classes and types of CpG ODN that can be used in the context of the present invention (e.g., Zhang et al. Pharmaceuticals. 2021 Dec 28; 14(1): 73.).

[0034] In a preferred embodiment, a "nucleic acid" in the context of the present invention is a TLR3 agonist. TLR3 interacts with the ribose phosphate backbone of double-stranded RNA and does not have specific sequence requirements. Preferably, the "nucleic acid" is a TLR3 agonist, such as poly(I:C) (i.e., polyinosinic acid:polycytidylic acid) and poly(I:C 12 Poly(I:C) is one or more of the following: poly(I:C) and poly(I:C) (poly(I:C)), and "poly(I:C)" according to the present invention preferably includes single-stranded polyinosinic acid (poly I) and single-stranded polycytidylic acid (poly C), which preferably do not associate by hydrogen bonds or covalent bonds upon administration, as well as double-stranded or complexed poly I / poly C. Additionally or alternatively, poly(I:C) is preferably a mismatched double-stranded RNA, where one strand is a polymer of inosinic acid and the other is a polymer of cytidylic acid. Poly(I:C) 12Poly(A:U) is preferably poly(I:C) with a U mismatch at every 12th base of the C strand. Poly(A:U) (polyadenylic-polyuridylic acid) or poly(G:C) (polycytidylic-guanylic acid) can be used as alternative analogs of poly(I:C) in the context of the present invention.

[0035] In one embodiment, the nucleic acid-lipid particle is preferably a liposome, more preferably an anionic liposome. As used herein, the term "liposome" refers to an (aqueous) compartment enclosed by at least one phospholipid bilayer. A "liposome" is an encapsulated vesicle formed by a lipid bilayer that preferably encloses an aqueous compartment. Typically, water-soluble compounds are entrapped in the aqueous phase / core of the liposome, as opposed to lipophilic compounds, which are typically entrapped in the core / center of the lipid bilayer. As used herein, the term "liposome" encompasses unilamellar vesicles (UVs) or small unilamellar vesicles (SUVs), which contain a single lipid bilayer, or multilamellar vesicles (MLVs), which contain two or more central bilayers, each separated from the next by a water layer. The nucleic acid-lipid particles or liposomes disclosed herein may have a substantially spherical shape.

[0036] Preferably, the lipid bilayer(s) of the particles of the invention are at least 30, 40, 50, 60, 70, 80, 90% arranged in a lamellar phase structure. One skilled in the art can characterize the lipid phase assembly and overall structure of the lipid particle using electron microscopy, nuclear magnetic resonance (NMR), small angle X-ray scattering (SAXS), and / or small angle neutron scattering (SANS).

[0037] The liposomes and / or nucleic acid-lipid particles disclosed herein preferably have a core-shell structure (with a lipid shell) and / or a core encapsulated by one or more lipid bilayers, as determined, for example, by small-angle neutron scattering (SANS), and / or the core comprises at least 30, 40, 50, 60, 70, 80, or 90% by volume of aqueous medium / solvent relative to the total core volume. The aqueous medium / solvent content within the core compartment can be determined by small-angle neutron scattering (SANS) solvent contrast modulation techniques using the calculated neutron scattering length density (SLD) values ​​of the components and their volume fractions (as described in the Experimental Section). Additionally and / or alternatively, the aqueous compartment comprises up to 25, 20, 15, 10, 5, 4, 2, or 1% by weight of nucleic acid relative to the total nucleic acid of the nucleic acid-lipid particle.

[0038] In preferred embodiments, the at least one anionic lipid and / or the at least one neutral lipid included in one or more lipid bilayers disclosed herein is preferably a phospholipid or a sphingolipid.

[0039] The phospholipid content is preferably calculated according to the measurement of phosphate content. The Rouser assay (Rouser et al. Lipids 5, 494-496 (1970)) is a preferred method for determining phosphate content. The Rouser assay is based on the hydrolysis of phospholipids to orthophosphate by incubation with concentrated perchloric acid during heating, followed by spectrophotometric quantification of inorganic phosphate after reaction with (ammonium) molybdate and ascorbic acid in a heated water bath. The preferred protocol for performing the Rouser assay is as follows: Dilutions of liposome samples and known amounts of 0.5 mM KH2PO4 standard solution were transferred to clean glass tubes, and the solvent was completely evaporated using a heat block at 220 °C. Next, 0.3 mL of perchloric acid was added to each tube, and the tubes were placed in the heat block for 60 minutes, or until the yellow color disappeared. After cooling, 1 mL of water, 0.5 mL of molybdate solution, and then 0.5 mL of ascorbic acid solution were added and vortexed. The tubes were placed in a heated water bath for 5 minutes and then allowed to cool. The absorbance of the samples and standards was then measured at 797 nm using a spectrophotometer microplate reader (BMG SPECTROstar Nano, De Meern, The Netherlands).

[0040] As used herein, the term "lipid bilayer" can refer to a two-layer arrangement of lipid molecules. The bilayer arrangement may be at least partially enabled by amphipathic lipids and therefore may contain polar and non-polar regions. The polar regions typically consist of phosphate groups, acidic groups, and / or tertiary or quaternary ammonium salts and may have a net negative (anionic), neutral, or positive (cationic) surface charge at physiological pH, depending on the composition of the lipid head groups. The non-polar regions typically consist of one or more fatty acid chains and / or cholesterol, each having at least eight carbons. The lipids that make up the vesicle bilayer may be organized so that the non-polar hydrocarbon "tail" is oriented toward the center of the bilayer, while the polar "head" is oriented toward the inner and outer aqueous phases. If more than one lipid bilayer is present in the particles of the present invention, each lipid bilayer is preferably composed of two lipid monolayers, each of which has a hydrophobic "tail" region and a hydrophilic polar "head" region. When referring to lipid(s) herein, it is preferable to mean lipid(s) capable of forming one or more lipid bilayers. The term "one or more lipid bilayers" can refer to (at least) one, two, three lipid bilayer(s).

[0041] As used herein, the term "anionic (lipid or particle)" refers to a compound (e.g., a lipid or particle, such as a liposome) that has a net negative charge in an aqueous medium at a physiologically acceptable pH (preferably pH 7.0-7.8, preferably about 7.4), where net negative charge refers to a zeta potential of preferably less than -2 mV, preferably less than -10 mV, and more preferably less than -50 mV, as measured in a medium, e.g., as described below. Additionally or alternatively, the term "anionic (particle)" can refer to a particle (e.g., a liposome) that has at least 0.1 mol% (e.g., at least 1, 5, 10, 20, 30, 40, 50, or 60 mol%) of at least one anionic lipid relative to the total lipid molar amount of the particle.

[0042] The inventors have found that increasing the proportion of anionic lipids leads to more anionic surfaces. The inclusion of anionic lipids appears to play a role in promoting colloidal stability. Formulations without anionic lipids tend to aggregate after a few days of storage.

[0043] The term "anionic lipid" in the context of the present invention refers to a lipid that has a negative net charge in aqueous solution at a pH of 3 to 9, preferably a pH of 5 to 8, more preferably a pH of 6.0 to 7.5, and most preferably a pH of 7.4. In the context of the present invention, the at least one anionic lipid may be, but is not limited to, diacylglycerol phosphatidic acid (1,2-distearoyl-sn-glycero-3-phosphate (DSPA); 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA); 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA); 1,2-dilauroyl-sn-glycero-3-phosphate (DLPA); 1,2-dioleo ... diacylglycerol phosphoglycerols (1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG); 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG); 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG); 1,2-dilauroyl-sn-glycero-3-phospho-(1' The anionic lipid may be one or more selected from the group consisting of DPPG (dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DLPG); 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG)), phosphatidylglycerol [e.g., egg phosphatidylglycerol (EPG)], cardiolipin, diacylphosphatidylinositol, diacylphosphatidylserine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, gangliosides (e.g., GM1, GM2, GM3), sulfoglycosphingolipids, fatty acids, and anionic modifying groups attached to natural lipids. Preferably, the anionic lipid is EPG. Preferably, the nucleic acid-lipid particles according to the present invention are free of DPPG or contain at most 5, 4, 3, 2, 1, or 0.5 mol% of DPPG relative to the total lipid molar amount of the particle.

[0044] The term "neutral lipid" in the context of the present invention refers to a lipid that carries (substantially) no net charge in aqueous solution at pH 5-9, preferably pH 6-8, more preferably pH 6.5-7.5, and most preferably pH 7.4. In the context of the present invention, neutral lipids include, but are not limited to, phosphatidylcholine [e.g., egg phosphatidylcholine (EPC)], diacylglycerol phosphocholine (L-α-phosphatidylcholine, hydrogenated (soy) (HSPC); diacylglycerol phosphocholine (L-α-phosphatidylcholine, (soy) (soy PC)), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dipalmitoyl-sn-glycero-3-phosphocholine ( DPPC); 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diacylglycerol phosphoethanolamines (1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE); 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPP) The neutral lipid may be one or more selected from the group consisting of E; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), ceramide, sphingosine, sphingomyelin, cephalin, glycolipid-like glycoglycerolipids (monogalactosyldiacylglycerol (MGD), digalactosyldiacylglycerol (DGD), sulfoquinovosyldiacylglycerol (SQDG)) and glycosphingolipids (α-galactosylceramide, β-mannoseceramide), cholesterol and other sterols, and mono-, di-, or triacylglycerols. Preferably, the neutral lipid is EPC. Additionally or alternatively, at least one neutral lipid may include glycolipids that can assist in immune system targeting.

[0045] The term "cationic lipid" in the context of the present invention means a lipid that has a net positive charge in aqueous solution at pH 3-9, preferably pH 5-8, more preferably pH 6.0-7.5, and most preferably pH 7.4.

[0046] In the context of the present invention, cationic lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTMA"); N-(2,3-dioleyloxy)propyl)-N,N-dimethylammonium chloride ("DODMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTAP"); N-(2,3-dioleoyloxy)propyl)-N,N-dimethylammonium chloride ("DODAP"); 3-(N-(N',N'-dimethylaminoethane)caca N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA); 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA); 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA); 2-{4-[(3b)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propanamine (CLinDMA).

[0047] In one embodiment, the (anionic or neutral) lipid disclosed herein is a polysarcosine or polyoxazoline or pegylated lipid (or PEGylated lipid or PEG lipid). As used herein, "pegylated lipid" refers to a lipid molecule comprising a lipid moiety having one or more polyethylene glycol (i.e., PEG) polymer chains covalently and / or noncovalently attached thereto, wherein the PEG moiety has an average molecular weight of about 2,000 to 20,000 daltons.PEGylated lipids include 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG-2000-DSPE); 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG-2000-DOPE); 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[ 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG-2000-DPPE); 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG-2000-DMPE); 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG-2000-DLPE); 1,2-Distearoyl 1,2-Dioctadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (mPEG-5000-DSPE); 1,2-Dioctadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (mPEG-5000-DOPE); 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) )-5000] (mPEG-5000-DPPE); 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (mPEG-5000-DMPE); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (mPEG-5000-DLPE). Preferably, the nucleic acid-lipid particles according to the present invention are free of PEGylated lipids or contain a maximum of 5, 4, 3, 2, 1, or 0.5 mol% of PEGylated lipids relative to the total lipid molar amount of the particle.

[0048] Preferably, the at least one anionic / neutral lipid of the present invention is not DOPG, DOPE or DSPG to improve particle stability.

[0049] The nucleic acid-lipid particles disclosed herein preferably comprise: - at least 0.5 mol%, preferably at least 5 mol%, more preferably at least 15 mol% of at least one anionic lipid relative to the total lipid molar amount of the particle; and / or - at least 1 mol%, preferably at least 5 mol%, more preferably at least 15 mol% of at least one neutral lipid relative to the total lipid molar amount of the particle Includes.

[0050] In one embodiment, the nucleic acid-lipid particles disclosed herein comprise at least 10, 20, or 30, or 40, or 50, or 60, or 70, or 80, or 90, or 95, or 99 mol%, or 100 mol% of at least one anionic lipid and / or neutral lipid, based on the total lipid molar amount of the particle.

[0051] In one embodiment, one or more lipid bilayers of a nucleic acid-lipid particle disclosed herein comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 mol % of at least one anionic lipid and / or at least one neutral lipid relative to the total lipid molar amount of the one or more lipid bilayers. Additionally or alternatively, one or more lipid bilayers of a nucleic acid-lipid particle disclosed herein comprise no more than 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 mol % of at least one anionic lipid and / or at least one neutral lipid relative to the total lipid molar amount of the one or more lipid bilayers.

[0052] In a preferred embodiment, the nucleic acid-lipid particles disclosed herein comprise one or more anionic lipids in an amount of 0.5 to 80 mol %, preferably 10 to 40 mol %, and more preferably 10 to 30 mol %, based on the total molar amount of lipid present in the particle.

[0053] In a preferred embodiment, the nucleic acid-lipid particles disclosed herein comprise one or more neutral lipids in an amount of 1 to 99.5 mol%, preferably 20 to 90 mol%, and more preferably 60 to 90 mol%, based on the total molar amount of lipid present in the particle.

[0054] In one embodiment, the nucleic acid-lipid particles disclosed herein comprise no more than 25, 20, 15, 10, 5, 4, 3, 2, 1, 0.5, 0 mol % of at least one cationic lipid relative to the total lipid molar amount of the particle.

[0055] Preferably, the nucleic acid-lipid particles according to the present disclosure comprise: - does not contain cationic lipids and / or contains at most 1, 2, 3, 4, 5 mol% of cationic lipids relative to the total lipid molar amount of the particle; - does not contain a cationic polymer and / or contains at most 1, 2, 3, 4, 5 mol % of a cationic polymer relative to the total lipid molar amount of the particle; and / or - containing no polyvalent cations and / or containing up to 1, 2, 3, 4, 5 mol % of polyvalent cations relative to the total lipid molar amount of the particle.

[0056] In one embodiment, one or more nucleic acids in the nucleic acid-lipid particle are present in one or more lipid bilayers at a weight percentage of at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, or 99% (e.g., 100%) of the total molar amount of nucleic acid in the nucleic acid-lipid particle. The presence of nucleic acid in one or more lipid bilayers may be evident from the absence of nucleic acid in the (aqueous) core. Additionally or alternatively, the presence of nucleic acid in one or more lipid bilayers may alter the neutron contrast of the liposome shell, the flow properties of the bilayer, and / or the thermal properties of the nucleic acid. In the prior art, anionic particles are expected to have even less than a single nucleic acid molecule in the lipid bilayer.

[0057] In one embodiment, the nucleic acid-lipid particles disclosed herein comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 mol% of at least one sterol relative to the molar amount of total lipid (including sterol) in the particle. In one embodiment, the nucleic acid-lipid particles disclosed herein comprise no more than 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 mol% of at least one sterol relative to the molar amount of total lipid (including sterol) in the particle.

[0058] In a preferred embodiment, the nucleic acid-lipid particles disclosed herein contain 5 to 50 mol %, preferably 10 to 40 mol %, and more preferably 30 to 35 mol % of sterol relative to the molar amount of total lipid (including sterol) in the particle.

[0059] As used herein, "sterol" refers to a steroid having a hydroxyl group at the C-3 position and a cholestane-derived skeleton. The term "cholestane-derived skeleton" as used herein refers to a cholestane skeleton in which an unsaturated bond has been introduced. The sterol of the present invention is preferably cholesterol or a cholesterol derivative. As used herein, "cholesterol derivative" refers to at least one selected from the group consisting of, for example, cholesterol, sitosterol (e.g., p-sitosterol), ergosterol, stigmasterol, 4,22-stigmastadien-3-one, stigmasterol acetate, lanosterol, and cycloartenol, or any combination thereof. Additionally or alternatively, preferred sterols in the context of the present invention may be one or more of ergosterol, ergocalciferol, steroidal saponin, vitamin D, campesterol, desmosterol, beta-cholesterol, and estradiol. Sterols may be derived from and / or found naturally in plants and / or animals.

[0060] The present inventors have found that enriching nucleic acid-lipid particles with cholesterol allows for a lower polydispersity index (PDI) and higher encapsulation efficiency of the formulation. Without being bound by theory, cholesterol appears to play a role in stabilizing lipid particles containing RNA. Cholesterol induces changes in lipid packaging into the bilayer, promoting the formation of lipid domains. Nucleic acids induce lipid separation from anionic liposomes into neutral lipid-rich areas and anionic lipid-rich areas. The inclusion of cholesterol in the bilayer may stabilize lipid separation and avoid electrostatic repulsion between RNA and anionic lipids in the bilayer.

[0061] Preferably, particles according to the present disclosure comprise up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mol % of cationic molecules, such as cationic lipids, cationic polymers, and multivalent cations, relative to the total lipid molar amount of the particle.

[0062] In one embodiment, the nucleic acid-lipid particle in the context of the present invention is free or substantially free of cationic lipids. In one embodiment, the nucleic acid-lipid particle in the context of the present invention, or one or more of its lipid bilayers, comprises at most 1 mol%, preferably at most 0.1 mol%, more preferably at most 0.01 mol%, more preferably at most 0.001 mol%, and most preferably 0 mol% of (at least one) cationic lipid, cationic polymer, or polyvalent cationic ion relative to the total lipid molar amount of the particle.

[0063] In one embodiment, the nucleic acid-lipid particle in the context of the present invention, or its one or more lipid bilayers, is free of polymers (other than nucleic acids), preferably cationic polymers. In one embodiment, the nucleic acid-lipid particle in the context of the present invention, or its one or more lipid bilayers, contains at most 1 mol%, preferably at most 0.1 mol%, more preferably at most 0.01 mol%, more preferably at most 0.001 mol%, and most preferably 0 mol% of polymers (other than nucleic acids), preferably cationic polymers, relative to the total lipid molar amount of the particle.

[0064] As used herein, the term "absent" in the context of a molecule, substance, or compound in a nucleic acid-lipid particle can mean that the molecule, substance, or compound is essentially absent from the nucleic acid-lipid particle, e.g., the amount is not measurable according to standard analytical techniques in the art. Additionally or alternatively, "(essentially) absent" can mean an amount of less than 0.001% by weight, or less than 0.0001% by weight, or less than 0.00001% by weight, or less than 0.000001% by weight.

[0065] As used herein, the term "cationic polymer" refers to a polymer that carries a net positive charge in an aqueous medium, preferably at pH 7.4 (or pH 3-9, 5-8, or 6.0-7.5). The "cationic polymer" is preferably one or more of poly-L-lysine, polyamidoamine, poly[2-(N,N-dimethylamino)ethyl methacrylate], chitosan, poly-L-ornithine, cyclodextrin, histone, collagen, dextran, and polyethyleneimine (PEI).

[0066] The present inventors have found that fine tuning the lipid composition of anionic liposomes can direct their immune activity to achieve either a predominantly immunostimulatory or immunosilent effect.

[0067] In one embodiment, the particles and / or compositions of the present disclosure are immunostimulatory, meaning they can enable activation of nucleic acid-sensing receptors (e.g., TLRs, RLRs, STING) and induce maturation of immune cells (e.g., macrophages, dendritic cells, T cells).

[0068] In one embodiment, the particles and / or compositions of the present disclosure are immunosilent or immunotolerant, meaning they may reduce activation of nucleic acid-sensing receptors (e.g., TLRs, RLRs, STING) and avoid the maturation of immune cells (e.g., macrophages, dendritic cells, T cells).

[0069] In a preferred embodiment, the nucleic acid-lipid particles in the compositions disclosed herein comprise 0.5-40 mol%, preferably 5-35 mol%, and more preferably 10-30 mol% of at least one anionic lipid relative to the total lipid molar mass of the particle. In this embodiment, the composition is preferably an immunostimulatory composition (e.g., leading to dendritic cell activation, T cell activation, and / or IFN-alpha secretion).

[0070] In a preferred embodiment, the nucleic acid-lipid particles in the compositions disclosed herein comprise 0.5-80 mol%, preferably 3-40 mol%, and more preferably 5-30 mol% of at least one anionic lipid relative to the total lipid molar amount of the particle. In this embodiment, the composition is preferably immunosilent (e.g., does not induce immune stimulation, such as dendritic cell activation, T cell activation, and / or IFN-alpha secretion).

[0071] The nucleic acid-lipid particles of the present invention preferably have a particle size or average particle size of 30 to 500 nm, preferably 50 to 300 nm, more preferably 50 to 250 nm, more preferably 75 to 200 nm, and even more preferably 75 to 190 or 100 to 150 nm. Additionally or alternatively, the nucleic acid-lipid particles of the present invention have a particle size of less than 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 199, 198, 197, 196, 195, 194, 193, 192, 191, 190, 180, 170 nm, and / or greater than 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 nm. Additionally or alternatively, the nucleic acid-lipid particles have a particle size of at least 2, 10, 100, 1000, 10, etc. per ml (or per μl). 4 , 10 5 , 10 6 , 10 7 , 10 8 The composition may contain one or more particles.

[0072] "Particle size" in the context of the present invention is preferably the hydrodynamic diameter, i.e., the diameter of a perfect solid sphere that exhibits the same fluid friction as the particle. (Average) particle size can be measured by dynamic light scattering (DLS), for example, using a Malvern Zetasizer Nano S (Malvern Instruments, Malvern, UK) equipped with a He-Ne 633 nm laser configured at a scattering angle of 173°. "(Average) hydrodynamic particle size" can refer to the diameter of a single particle or the average particle size measured for a collection of particles present in, for example, a composition.

[0073] As used herein, "polydispersity" (or polydispersity index, PDI) is a measure of the heterogeneity of a sample based on particle size. A preferred method for determining hydrodynamic diameter and polydispersity (PDI) is by dynamic light scattering (DLS) using, for example, a Malvern Zetasizer Nano S (Malvern Instruments, Malvern, UK) equipped with a He-Ne 633 nm laser configured at a scattering angle of 173°. A preferred DLS protocol is as follows: Samples are diluted with PBS. DLS is performed using a Malvern Zetasizer Nano S (Malvern Instruments, Malvern, UK) equipped with a He-Ne 633 nm laser configured at a scattering angle of 173°. Measurements are performed using Malvern's Zetasizer v7.13 software (Malvern Instruments, Malvern, UK). A minimum of three measurements are performed per sample, and the measurement duration is automatically set by the software depending on the sample characteristics. A viscosity of 887.2 Pa·s (0.8872 centipoise (cP)) and a refractive index (RI) of 1.330 for the dispersion, and an RI of 0.1590 and an absorbance of 0.010 for the material in suspension are set in the instrument software. Samples are diluted with PBS and measured at 25°C. A PDI of <0.3 is considered acceptable.

[0074] Preferably, the nucleic acid-lipid particles of the present disclosure have a particle size polydispersity index (i.e., PDI) of 0.6, 0.5, 0.4, 0.35, 0.30, 0.25, 0.2, or 0.15 or less. In one embodiment, the nucleic acid-lipid particles of the present disclosure have a polydispersity index (i.e., PDI) of 0.01 to 0.5, preferably 0.01 to 0.5, more preferably 0.01 to 0.4, and more preferably 0.01 to 0.25.

[0075] In one embodiment, the nucleic acid-lipid particles of the present disclosure have a zeta potential of between -100 and -10 mV, preferably between -200 and -5 mV, or between -150 and -10 mV, or between -100 and -15 mV, or between -50 and -20 mV. Exemplary measurement conditions are described below.

[0076] The term "zeta potential" as used herein is a measure of the total charge that one or more particles acquire in a particular medium. "Zeta potential" can refer to the charge of a single particle determined on a particle population or the (average) total charge of a particle population. Generally, a larger zeta potential predicts a more stable dispersion, meaning that all particles in the suspension tend to repel each other, thereby preventing and tending to aggregate. Zeta potential is preferably expressed in mV and can be determined by electrophoretic light scattering (ELS). A preferred protocol for measuring zeta potential by ELS is as follows: Samples are diluted with 0.1x PBS (1 volume PBS and 9 volumes purified water). Zeta potential is determined using a Zetasizer Nano Z (Malvern Instruments, Malvern, UK) and Malvern's Zetasizer v7.13 software. A minimum of three measurements are performed per sample, and the measurement duration is automatically set by the software depending on the sample characteristics. Samples are measured at 25°C. A viscosity of 887.2 Pa·s (0.8872 cP), an RI of 1.330, and a dielectric constant of 78.5 for the dispersion, and an RI of 1.590 and an absorbance of 0.010 for the material in suspension are set in the instrument software.

[0077] In a preferred embodiment, the nucleic acid-lipid particles comprise: - average diameter of 50-250 nm; - PDI between 0.01 and 0.3; and / or - Zeta potential between -100 and -10mV It has.

[0078] In preferred embodiments, the nucleic acid-lipid particles are non-inflammatory and / or non-immunogenic, meaning that they do not (substantially) result in dendritic cell activation, T cell activation and / or IFN-alpha secretion.

[0079] In certain embodiments, for example, when the one or more nucleic acids are one or more mRNAs, the nucleic acid-lipid particles comprise an endosomal escape peptide, preferably present on or extending from the surface of the nucleic acid-lipid particle.

[0080] In one embodiment, the composition is administered in combination with one or more selected from the group consisting of an antigen, an antigen derived from a pathogen, a tumor-associated (or specific) antigen, a vaccine, an immunological adjuvant, and an anti-neoplastic agent.

[0081] Antineoplastic agents include - Cell-based antineoplastic agents; - lymphocyte-based anti-neoplastic agents, preferably selected from B cells, αβT cells, γδT cells, NK cells, NKT cells, autologous tumor-infiltrating lymphocytes (TIL), autologous NK cells, CAR-T cells, CAR-B cells, CAR-NK cells, CAR-NKT cells; - a bone marrow-based antineoplastic agent, preferably selected from a dendritic cell-based antineoplastic agent, a macrophage-based antineoplastic agent, or a neutrophil-based antineoplastic agent; - an antibody or immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is preferably an antibody, more preferably an anti-CTLA4 antibody, an anti-PD1 antibody and / or an anti-PD-L1 antibody; - small molecule drugs, preferably selected from alkylating agents, antibiotics, antimetabolites, hormone antagonists, photosensitizers, protein kinase inhibitors, poly(ADP-ribose) polymerase inhibitors, taxanes and / or topoisomerase inhibitors; - Radiation therapy; - cytokines, preferably selected from IL-2, IL-12, IL-15 and IL-21; growth factors, preferably selected from the CSF family and Flt3L; and / or - steroidal or nonsteroidal anti-inflammatory drugs It may be one or more of the following.

[0082] As used herein, the term "in combination with" does not exclude the possibility that the individual components of the combination are not a single physical entity; that is, the individual components of the combination may be administered, for example, simultaneously, sequentially, or separately. However, the combination may be encapsulated together in the same particle disclosed herein. In certain embodiments of the present invention, a synergistic effect may be beneficial for the components combined in a single formulation (e.g., a nucleic acid-lipid particle and an anti-neoplastic agent). In other embodiments, a synergistic effect may be beneficial for the components combined in separate formulations. In still other embodiments, a synergistic effect may be beneficial for the components combined at different times and / or by different administration routes. The components of the combination may be provided in parallel (e.g., to a human or animal subject) so that they can be administered to the subject simultaneously, separately, or at intervals. Additionally or alternatively, the components of the combination may be packaged separately in the form of a "kit of parts." Additionally or alternatively, the selective administration scheme of the components of the combination may be determined on a case-by-case basis, for example, depending on the subject being treated, the disease or disorder being treated, the administration route, the formulation, and the dosage.

[0083] The term "vaccine" in the context of the present invention especially means a preparation containing one or more antigens used for the purposes of prevention or treatment of, for example, infections, cancer, autoimmune diseases or allergies.

[0084] The anti-neoplastic agent may refer to a radiotherapeutic agent, a chemotherapeutic agent, an antibody, an immune cell such as a T cell receptor or CAR T cell, or an immune checkpoint inhibitor such as, for example, an anti-CTLA-4, an anti-PD1 antibody, and / or an anti-PD-L1 antibody. The immune cell may be an immune cell administered as part of an immunotherapy. An "immune cell" according to the present disclosure may be any cell belonging to the immune system, preferably selected from lymphocytes, granulocytes, myeloid cells, T cells (e.g., T helper cells, T helper 17 cells, follicular helper T cells, cytotoxic T cells, gamma delta T cells), monocytes, macrophages, NK cells, basophils, dendritic cells (e.g., myeloid dendritic cells, plasmacytoid dendritic cells), neutrophils, eosinophils, basophils, mast cells, B cells, or plasma cells, among others. An "immune cell" according to the present disclosure may also be a genetically engineered immune cell as taught herein. In a preferred embodiment, the immune cells according to the present disclosure are genetically engineered cytotoxic T cells or NK cells, such as chimeric antigen receptor (CAR) T or CAR NK cells.

[0085] Uses of the present invention In a preferred embodiment, the nucleic acid-lipid particle and / or composition disclosed herein is used for preventing and / or treating disease.In other words, the present invention also comprises the method for preventing or treating disease, comprising administering the composition disclosed herein to a subject (animal, preferably human) that requires it.Preferably, administration is by oral, topical (including skin, oral cavity and sublingual), rectal, parenteral (intradermal, intramuscular, subcutaneous or intravenous), nasal and pulmonary, more preferably by parenteral administration, most preferably by intravenous administration.

[0086] The term "preventing" or "prevention" in the context of the present invention means reducing the chance that a subject will develop a disease or disorder. Preventing also encompasses situations in which a disease or disorder is delayed, reduced in severity, and / or reduced in incidence, even if the disease or disorder is not completely avoided. The term "preventing" or "prevention" in the context of the present invention encompasses situations in which a subject has previously experienced a disease or disorder but is kept free of recurrence due to intervention. The term "preventing" or "prevention" may have a therapeutic and / or non-therapeutic effect, but preferably has a therapeutic effect. If "preventing" or "prevention" is therapeutic in nature, it may be directed at the symptoms of the disease or disorder and / or its underlying pathology. The term "preventing" or "prevention" can be defined by any delay, change in severity, and / or change in incidence, such as at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, compared to a control or reference, as measured by any standard technique.

[0087] The term "treating" or "treatment" in the context of the present invention means to reduce and / or cure a disease or disorder by intervention if the disease or disorder is already present. "Treating" or "treatment" may have a therapeutic and / or non-therapeutic effect, but preferably has a therapeutic effect. If "treating" or "treatment" is therapeutic in nature, it may be directed at the symptoms of the disease or disorder and / or its underlying pathology. Treatment can be any reduction in the severity, incidence, and / or frequency of the disease or disorder, such as by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or complete (100%) compared to a control or reference as measured by any standard technique.

[0088] The disease may be one or more of cancer, infection, chronic inflammation, genetic disease, and autoimmune disease. In one embodiment, the disease is cancer, such as bile duct cancer, brain cancer (such as glioblastoma and medulloblastoma), breast cancer (such as inflammatory breast cancer), cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, hematological neoplasms (such as acute lymphocytic and myeloid leukemia), multiple myeloma, AIDS-related leukemia, and adult T-cell leukemia-lymphoma, intraepithelial neoplasia (such as Bowen's disease and Paget's disease), liver cancer (hepatocellular carcinoma), lung cancer, lymphoma (such as Hodgkin's disease and lymphocytic lymphoma), neuroblastoma, oral cancer (such as squamous cell carcinoma, leukemia, and lymphoma), and the like. Cancer, etc.), ovarian cancer (e.g., those arising from epithelial cells, stromal cells, germ cells, and mesenchymal cells), pancreatic cancer, prostate cancer, rectal cancer, sarcoma (e.g., leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma), skin cancer (e.g., melanoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma), testicular cancer (e.g., germ cell carcinoma, seminoma, non-seminoma [teratoma, choriocarcinoma], etc.), stromal tumor and germ cell tumor, thyroid cancer (e.g., thyroid adenocarcinoma and medullary carcinoma), and kidney cancer (e.g., adenocarcinoma and Wilms' tumor). In a preferred embodiment, the composition is administered to a patient who has been previously diagnosed with a cancer that has been removed. In many cases, such as melanoma, a portion of the tumor tissue may be removed.

[0089] In one embodiment, the disease is a hyperproliferative or differentiative disorder such as fibrosis or hypertrophy, such as pulmonary fibrosis or hypertrophy (eg, benign prostatic hypertrophy), cardiac fibrosis, or hepatic fibrosis.

[0090] In one embodiment, the disease is an infection, such as, but not limited to, a bacterial infection, a viral infection, or a fungal infection, such as human immunodeficiency virus (HIV) infection, hepatitis C virus (HCV) infection, hepatitis B virus (HBV) infection, or cytomegalovirus (CMV) infection. The use of the composition in preventing or treating infection includes the use of the composition as a monotherapy and / or in combination with a pathogen-derived antigen or vaccine. For example, the composition can be administered sequentially or simultaneously with a vaccine in preventing or treating infection.

[0091] In one embodiment, the disease is (chronic) inflammation, such as prostatitis, vernal conjunctivitis, atherosclerosis, or idiopathic pneumonia.

[0092] In one embodiment, the disease is an autoimmune disease or a (chronic) inflammatory condition, such as systemic lupus erythematosus (SLE), multiple sclerosis (MS), pemphigus vulgaris (PV), or myasthenia gravis.

[0093] In a preferred embodiment, the use of the disclosed nucleic acid-lipid particles and / or compositions (e.g., in cancer) is in combination with immunotherapy, preferably the immunotherapy comprises the administration of an antibody, such as an immune checkpoint inhibitor, or an immune cell-based immunotherapy, such as DC cell therapy, CAR T cell therapy, or NK cell therapy.

[0094] The immune checkpoint inhibitor may be one or more inhibitors of CTLA-4, LAG-3, Tim3 VISTA, CD137, 0X40, ID01 PD-1, and PD-L1, preferably PD-1 and PD-L1. Additionally or alternatively, the immune checkpoint inhibitor may be one or more selected from the group consisting of ipilimumab, pembrolizumab, nivolumab, cemiplimab, pidilizumab, atezolizumab, avelumab, durvalumab, BMS936559, JNJ61610588, urelumab, 9B12, PF-04518600, BMS-986016, TSR-022, MBG453, MEDI6469, MEDI6383, epacadostat, anti-PD1, and anti-PDL1, preferably anti-PD1 and / or anti-PDL1.

[0095] Methods of the Invention The present invention also relates to a method for preparing a composition comprising the nucleic acid-lipid particles disclosed herein, the method comprising: a) providing one or more organic solutions comprising one or more lipids, preferably at least one anionic lipid and / or at least one neutral lipid; b) providing one or more aqueous solutions containing one or more nucleic acids; c) combining one or more organic solutions with one or more aqueous solutions, thereby producing a composition comprising the nucleic acid-lipid particles disclosed herein; Includes.

[0096] Preferably, combining in step c) refers to mixing one or more organic solutions with one or more aqueous solutions in a (substantially) laminar flow, preferably in a microfluidics device. The combining step may also be enabled by other methods such as, but not limited to, solvent jet, pulse jet, expanded solution reduced pressure crystallization (DELOS), supercritical antisolvent (SAS), or thin film hydration.

[0097] In a preferred embodiment, one or more of steps a) to c) of the method are performed by a microfluidics device. As used herein, the term "microfluidics device" refers to a device that includes a combination of two or more (micro)channels, preferably etched and / or molded into a material (e.g., glass, silicon, or polymer), where at least two (micro)channels are connected to each other to achieve a desired feature (e.g., mixing, pumping, sorting), preferably to achieve laminar flow and fluid mixing. Preferably, the method for preparing a composition comprising nucleic acid-lipid particles according to the present disclosure does not employ dialysis-based purification.

[0098] In one embodiment, the combining or mixing in step c) is in a (substantially) laminar flow, and / or the mixing of the aqueous and organic solutions in step c) of the method is laminar mixing. The mixing is preferably performed by passive diffusion of molecules from a domain of higher concentration to a domain of lower concentration in a laminar flow. As used herein, the term "laminar flow" means that the fluid follows a smooth path through multiple layers, with each layer passing smoothly through adjacent layers. "Laminar flow" is preferably defined by a Reynolds number of 2000 or less (e.g., 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000 or less), and more preferably cooperatively defined as smooth flow (e.g., not inducing chaotic advection, chaotic mixing, and / or laminar mixing). The Reynolds number is preferably determined by considering the flow as flow in a pipe, where the Reynolds number is:

number

[0099] The inventors have found that better encapsulation efficiency and better PDI can be achieved with laminar mixing compared to the turbulent flow of chaotic mixing. Higher encapsulation efficiency and lower PDI are particularly achieved with laminar mixing and the combination of at least one organic solution and two consecutive aqueous solutions.

[0100] In one embodiment, the present disclosure does not exclude that step c) utilizes chaotic flow mixing and / or that the aqueous solution and the organic solution are mixed in step c) of the method under chaotic flow. As used herein, the term "chaotic flow" refers to a flow that is not laminar, is not constant in time, or does not exhibit any regular periodicity. A turbulent flow can be an example of a chaotic flow. As used herein, "chaotic mixing" means that mixing occurs under chaotic flow.

[0101] In one embodiment, the present disclosure does not exclude that the flow in step c) is turbulent, and that the mixing of the aqueous solution and the organic solution in step c) of the method is turbulent mixing. As used herein, the term "turbulent mixing" means that the mixing occurs under turbulent flow. As used herein, the term "turbulent flow" means fluid motion characterized by fluid flow that is not in parallel layers and / or has disruptions between those layers. "Turbulent flow" is preferably dominated by inertial forces, which typically produce chaotic vortices, swirls, and other flow instabilities. "Turbulent flow" preferably means that the flow is characterized by chaotic changes in pressure and flow velocity. In one embodiment, the term "turbulent flow" encompasses any type of flow that is not laminar. In one embodiment, turbulent flow is defined by a Reynolds number greater than 4000, preferably greater than 5000, and more preferably greater than 6000. Turbulent flow can be an example of a chaotic flow.

[0102] Typically, laminar flow is interspersed with turbulence up to Reynolds numbers of 2000-4000, or 2500-3500. Flows between 2000-4000, or between 2500-3500, may be defined herein as laminar and / or turbulent.

[0103] In one embodiment, chaotic and / or turbulent mixing (or chaotic and / or turbulent flow) according to the present invention is achieved by providing a mixing device, preferably a micromixer chip on a microfluidics device. The mixer device may achieve chaotic and / or turbulent flow by mixing laminar flows.

[0104] In one embodiment, the solvent selected for dissolving lipids to produce particles, e.g., liposomes containing one or more nucleic acids, is one or more solvents, preferably selected from the group of water-miscible solvents, including but not limited to methanol, ethanol, isopropanol, butanol, acetonitrile, acetone, and dimethyl sulfoxide. More preferably, the organic solvent is a water-miscible solvent classified as a Class 3 solvent. Class 3 does not include solvents known to pose a human health hazard at levels normally tolerated in pharmaceuticals (acceptable doses of 50 mg or more per day) (Q3C(R6): Impurities: Residual Solvents Guideline EMA / CHMP / ICH / 82260 / 2006). Examples of Class 3 solvents are ethanol, acetone, dimethyl sulfoxide, isopropanol, ethyl ether, methyl acetate, 1-pentanol, and 1-propanol. Most preferably, the organic solvent(s) is ethanol, isopropanol, or a mixture thereof. The ethanol solution may comprise at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% (v / v) ethanol. The ethanol solution may be pure ethanol (100% ethanol). Additionally or alternatively, the ethanol solution may comprise no more than 99, 95, 90, 80, 70, 60, or 50% (v / v) ethanol. The isopropanol solution may comprise at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% (v / v) isopropanol. The isopropanol solution may be pure isopropanol (100% isopropanol). Additionally or alternatively, the isopropanol solution may comprise no more than 99, 95, 90, 80, 70, 60, or 50% (v / v) isopropanol.

[0105] The present inventors have found that using ethanol as an organic solution is beneficial.Furthermore, the present inventors have found that increasing ethanol concentration can lead to larger and more monodisperse particles.Without being bound by theory, hydrophobic interactions may determine the mechanism of nucleic acid encapsulation in nucleic acid-lipid particles.In the presence of ethanol, nucleic acid changes its conformation, exposing hydrophobic nucleotide bases that can interact with lipid tails, allowing the formation of liposomes containing nucleic acid in bilayer(s).

[0106] In one embodiment, the aqueous solution in the context of the present invention is water.In one embodiment, the aqueous solution in the context of the present invention contains an osmotic agent such as NaCl or sucrose and / or a pH buffering group, such as, but not limited to, one or more selected from the group consisting of phosphate, histidine, HEPES, Tris, acetate, carbonate and citrate.In addition or alternatively, the aqueous solution disclosed herein is free of RNase and DNase.

[0107] In a preferred embodiment, the aqueous solutions disclosed herein have a pH between 5.0 and 9.5, preferably between 5.5 and 9.0, more preferably between 6.0 and 8.5, more preferably between 6.0 and 8.0.

[0108] In a preferred embodiment, the flow ratio (A:B) between the one or more aqueous solutions (A) and the one or more organic solutions (B) is between 48:1 and 1:10, preferably between 24:1 and 1:8, more preferably between 18:1 and 1:3, more preferably between 10:1 and 1:1.

[0109] In a preferred embodiment, at least one anionic lipid and / or at least one neutral lipid is provided in the organic solution disclosed herein at a concentration of 0.01 to 500 mM, preferably 0.1 to 50 mM, or even 1 to 20 mM.

[0110] In a preferred embodiment, the one or more nucleic acids are provided in an aqueous solution at a concentration of 5 to 50,000 μg / ml, preferably 10 to 50,000 μg / ml, more preferably 50 to 5,000 μg / ml, more preferably 100 to 2,000 μg / ml, more preferably 200 to 1,000 μg / ml.

[0111] In a preferred embodiment, the organic solution of step a) comprises at least 10 mol%, preferably at least 20 mol%, more preferably at least 30 mol%, more preferably at least 40 mol%, and most preferably at least 50 mol% (e.g., at least 60, 70, 80, 90, 95, or 99 mol%) of at least one anionic lipid and / or at least one neutral lipid relative to the total molar amount of lipid in the organic solution.

[0112] In one embodiment, the organic solution contains 5 to 50 mol %, preferably 10 to 40 mol %, more preferably 20 to 30 mol % of anionic lipids and / or neutral lipids relative to the total lipid molar amount in the organic solution.

[0113] In a preferred embodiment, the organic solution of step a) comprises at most 40 mol%, preferably at most 30 mol%, more preferably at most 20 mol%, more preferably at most 10 mol%, and most preferably at most 5 mol% (e.g., at most 4, 3, 2, 1, 0.5, or 0.1 mol%) of (at least one) cationic lipid relative to the total molar amount of lipid in the organic solution.

[0114] In a preferred embodiment, the organic solution of step a) comprises at least 1 mol%, preferably at least 5 mol%, more preferably at least 10 mol%, more preferably at least 20 mol%, and most preferably at least 30 mol% of at least one sterol relative to the total molar amount of lipids (including sterols) in the organic solution. In one embodiment, the organic solution comprises 5-66 mol%, preferably 10-40 mol%, more preferably 20-35 mol% of sterol relative to the total molar amount of lipids (including sterols) in the organic solution.

[0115] In a preferred embodiment, the organic solution of step a) is free of cationic lipids and / or comprises at most 1 mol % of (at least one) cationic lipid relative to the total lipid molar amount in the organic solution.

[0116] In a preferred embodiment, the organic solution of step a) is free of cationic polymer and / or contains at most 1 mol%, preferably 0.1 mol%, more preferably at most 0.01 mol% of at least one (cationic) polymer, calculated on the total lipid molar amount of the organic solution.

[0117] In a preferred embodiment, the methods and / or particles of the present invention are characterized by the inclusion of multivalent cations (Ca 2+ , Mn 2+ , Mg 2+ The cationic lipids and / or (cationic) polymers of the present invention may be free of, or may comprise, at most 10, 5, 1 mol % of the cationic lipids and / or (cationic) polymers of the present invention relative to the total lipid molar amount of the particle.

[0118] In one embodiment, the methods of the present invention provide (or achieve) a nucleic acid encapsulation efficiency in nucleic acid-lipid particles of at least 40%, preferably at least 60%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95%. Additionally or alternatively, the methods of the present invention provide (or achieve) a nucleic acid encapsulation efficiency in nucleic acid-lipid particles of preferably 60-95%, preferably 70-90%, and more preferably 75-85%. As used herein, encapsulation efficiency (of nucleic acid) refers to the percentage of nucleic acid incorporated into nucleic acid-lipid particles relative to the total amount of nucleic acid present in the formulation, and is calculated as follows:

number

[0119] general definition The reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that one, and only one, of the element is present. Thus, the indefinite article "a" or "an" usually means "at least one."

[0120] As used herein, the terms "comprising" or "comprises" and their conjugations refer to a situation where the term is used in an open-ended sense to mean that the items following the term are included, but terms not specifically stated are not excluded. It also encompasses the more restrictive verbs "consisting essentially of" and "consisting of." [Brief explanation of the drawings]

[0121] [Figure 1A]Microfluidic chip configurations (A and B) used for encapsulating RNA (poly(I:C)) into anionic liposomes. In design A, lipids (yellow) dissolved in ethanol flow through the central channel of the microfluidic chip, and RNA (light blue) dissolved in RNase-free purified water flows through the outer channel. Cocurrent mixing induces the formation of RNA liposomes. In design B, two chips are combined in a two-step encapsulation process. In chip No. 1, RNA (poly(I:C)) (light blue) dissolved in RNase-free purified water flows through the center of the first mixing channel, and lipids (yellow) dissolved in ethanol flows through the outer channel. Cocurrent mixing occurs, and the resulting mixture enters chip No. 2. The flow rate of the additional water stream (dark blue) influences the liposome characteristics. Five layers of flowing particles mix in laminar flow, inducing the formation of RNA liposomes. The sample is then collected from the outlet tube and diluted with RNase-free purified water to an ethanol concentration of at least 20%. Ethanol and free RNA are removed using vertical or centrifugal filtration techniques. [Figure 1B]Microfluidic chip configurations (A and B) used for encapsulating RNA (poly(I:C)) into anionic liposomes. In design A, lipids (yellow) dissolved in ethanol flow through the central channel of the microfluidic chip, and RNA (light blue) dissolved in RNase-free purified water flows through the outer channel. Cocurrent mixing induces the formation of RNA liposomes. In design B, two chips are combined in a two-step encapsulation process. In chip No. 1, RNA (poly(I:C)) (light blue) dissolved in RNase-free purified water flows through the center of the first mixing channel, and lipids (yellow) dissolved in ethanol flows through the outer channel. Cocurrent mixing occurs, and the resulting mixture enters chip No. 2. The flow rate of the additional water stream (dark blue) influences the liposome characteristics. Five layers of flowing particles mix in laminar flow, inducing the formation of RNA liposomes. The sample is then collected from the outlet tube and diluted with RNase-free purified water to an ethanol concentration of at least 20%. Ethanol and free RNA are removed using vertical or centrifugal filtration techniques. [Figure 2] A design of experiments (DoE) approach was applied to develop a microfluidic process for the encapsulation of RNA in anionic liposomes using Design A or B. (I) Flow rate, lipid and RNA concentrations in the solvent were the variables selected in the DoE. (II) The mean particle size, polydispersity index (PDI), and RNA encapsulation efficiency of the RNA-anionic liposomes were tested in this DoE. (III) A central composite design was selected as the experimental design for constructing a response surface model (RSM). (IV) The DoE was designed using appropriate software (Desig-Expert™). (V) The DoE was performed with Design A (n = 30) and Design B (n = 53), and the resulting data were analyzed to allow the construction of an RSM. (VI) The response surface model was used to predict the optimal process parameters. (VII) Five independent batches were generated for model validation. The grey areas are indicators of the predicted intervals by RSM for mean size, PDI and encapsulation efficiency (mean ± SEM of three replicate analyses). [Figure 3A] Effect of mixing geometry and flow rate on average size (A), polydispersity (B), and encapsulation efficiency (C). 500 μg / mL of RNA dissolved in RNase-free purified water was mixed with an organic solution containing lipids (5 mM) [EPC:EPG:cholesterol (3:2:1 molar ratio)] at a flow ratio of 1:1 and a total flow rate of 0.25, 0.75, or 1.5 mL / min using a laminar flow and chaotic mixing microfluidic chip according to the scheme in Design B. Error bars represent the standard deviation calculated from analytical triplicates from one independent experiment. [Figure 3B] Effect of mixing geometry and flow rate on average size (A), polydispersity (B), and encapsulation efficiency (C). 500 μg / mL of RNA dissolved in RNase-free purified water was mixed with an organic solution containing lipids (5 mM) [EPC:EPG:cholesterol (3:2:1 molar ratio)] at a flow ratio of 1:1 and a total flow rate of 0.25, 0.75, or 1.5 mL / min using a laminar flow and chaotic mixing microfluidic chip according to the scheme in Design B. Error bars represent the standard deviation calculated from analytical triplicates from one independent experiment. [Figure 3C] Effect of mixing geometry and flow rate on average size (A), polydispersity (B), and encapsulation efficiency (C). 500 μg / mL of RNA dissolved in RNase-free purified water was mixed with an organic solution containing lipids (5 mM) [EPC:EPG:cholesterol (3:2:1 molar ratio)] at a flow ratio of 1:1 and a total flow rate of 0.25, 0.75, or 1.5 mL / min using a laminar flow and chaotic mixing microfluidic chip according to the scheme in Design B. Error bars represent the standard deviation calculated from analytical triplicates from one independent experiment. [Figure 4A]Physical characterization of encapsulated RNA and size stability of RNA-containing anionic liposomes [EPC:EPG:cholesterol (3:1:2)] upon incubation with human plasma. Encapsulated poly(I:C) was extracted from anionic liposomes by precipitation with 100% isopropanol. The clean RNA pellet was resuspended in PBS and injected onto a size-exclusion chromatography (SEC) column (TSKgel® G-DNA-PW) run in PBS (flow rate = 0.5 mL / min, 25°C). In addition, samples of free poly(I:C) and standard DNA ruler (Gene Ruler 1 kb plus, Thermoscientific) were similarly treated and injected onto the SEC column. A UV detector at λ = 260 nm was used for RNA detection (A), and a multi-angle light scattering detector was used for molecular weight determination (B). Molecular weight analysis confirmed that the poly(I:C) used was a polymer with a broad molecular weight distribution. Encapsulated poly(I:C) had similar retention times (A) and molecular weights compared to free poly(I:C), demonstrating that microfluidics technology can encapsulate nucleic acids without altering the original molecular weight distribution. The stability of empty and RNA-containing anionic liposomes in human plasma was tested using asymmetric field-flow fractionation (AF4). Liposomes (2 mM lipid) were incubated with PBS or 20% human plasma at 37°C for 2 hours and then analyzed by AF4. The particle radius of gyration (Rg) and light scattering intensity were determined using MALS (C), and protein corona formation was determined using a fluorescence detector (λem = 280 nm, λex = 340 nm) (D). Empty and RNA-containing anionic liposomes did not exhibit particle aggregation in human plasma, as required for IV injection. Anionic liposomes protected encapsulated poly(I:C) from RNA degradation. Free and encapsulated poly(I:C) were incubated for 2 hours at 37°C in reaction buffer or reaction buffer supplemented with human plasma (20% v / v) with or without RNase. Samples were then run on a 1.0% agarose gel (E). [Figure 4B]Physical characterization of encapsulated RNA and size stability of RNA-containing anionic liposomes [EPC:EPG:cholesterol (3:1:2)] upon incubation with human plasma. Encapsulated poly(I:C) was extracted from anionic liposomes by precipitation with 100% isopropanol. The clean RNA pellet was resuspended in PBS and injected onto a size-exclusion chromatography (SEC) column (TSKgel® G-DNA-PW) run in PBS (flow rate = 0.5 mL / min, 25°C). In addition, samples of free poly(I:C) and standard DNA ruler (Gene Ruler 1 kb plus, Thermoscientific) were similarly treated and injected onto the SEC column. A UV detector at λ = 260 nm was used for RNA detection (A), and a multi-angle light scattering detector was used for molecular weight determination (B). Molecular weight analysis confirmed that the poly(I:C) used was a polymer with a broad molecular weight distribution. Encapsulated poly(I:C) had similar retention times (A) and molecular weights compared to free poly(I:C), demonstrating that microfluidics technology can encapsulate nucleic acids without altering the original molecular weight distribution. The stability of empty and RNA-containing anionic liposomes in human plasma was tested using asymmetric field-flow fractionation (AF4). Liposomes (2 mM lipid) were incubated with PBS or 20% human plasma at 37°C for 2 hours and then analyzed by AF4. The particle radius of gyration (Rg) and light scattering intensity were determined using MALS (C), and protein corona formation was determined using a fluorescence detector (λem = 280 nm, λex = 340 nm) (D). Empty and RNA-containing anionic liposomes did not exhibit particle aggregation in human plasma, as required for IV injection. Anionic liposomes protected encapsulated poly(I:C) from RNA degradation. Free and encapsulated poly(I:C) were incubated for 2 hours at 37°C in reaction buffer or reaction buffer supplemented with human plasma (20% v / v) with or without RNase. Samples were then run on a 1.0% agarose gel (E). [Figure 4C]Physical characterization of encapsulated RNA and size stability of RNA-containing anionic liposomes [EPC:EPG:cholesterol (3:1:2)] upon incubation with human plasma. Encapsulated poly(I:C) was extracted from anionic liposomes by precipitation with 100% isopropanol. The clean RNA pellet was resuspended in PBS and injected onto a size-exclusion chromatography (SEC) column (TSKgel® G-DNA-PW) run in PBS (flow rate = 0.5 mL / min, 25°C). In addition, samples of free poly(I:C) and standard DNA ruler (Gene Ruler 1 kb plus, Thermoscientific) were similarly treated and injected onto the SEC column. A UV detector at λ = 260 nm was used for RNA detection (A), and a multi-angle light scattering detector was used for molecular weight determination (B). Molecular weight analysis confirmed that the poly(I:C) used was a polymer with a broad molecular weight distribution. Encapsulated poly(I:C) had similar retention times (A) and molecular weights compared to free poly(I:C), demonstrating that microfluidics technology can encapsulate nucleic acids without altering the original molecular weight distribution. The stability of empty and RNA-containing anionic liposomes in human plasma was tested using asymmetric field-flow fractionation (AF4). Liposomes (2 mM lipid) were incubated with PBS or 20% human plasma at 37°C for 2 hours and then analyzed by AF4. The particle radius of gyration (Rg) and light scattering intensity were determined using MALS (C), and protein corona formation was determined using a fluorescence detector (λem = 280 nm, λex = 340 nm) (D). Empty and RNA-containing anionic liposomes did not exhibit particle aggregation in human plasma, as required for IV injection. Anionic liposomes protected encapsulated poly(I:C) from RNA degradation. Free and encapsulated poly(I:C) were incubated for 2 hours at 37°C in reaction buffer or reaction buffer supplemented with human plasma (20% v / v) with or without RNase. Samples were then run on a 1.0% agarose gel (E). [Figure 4D]Physical characterization of encapsulated RNA and size stability of RNA-containing anionic liposomes [EPC:EPG:cholesterol (3:1:2)] upon incubation with human plasma. Encapsulated poly(I:C) was extracted from anionic liposomes by precipitation with 100% isopropanol. The clean RNA pellet was resuspended in PBS and injected onto a size-exclusion chromatography (SEC) column (TSKgel® G-DNA-PW) run in PBS (flow rate = 0.5 mL / min, 25°C). In addition, samples of free poly(I:C) and standard DNA ruler (Gene Ruler 1 kb plus, Thermoscientific) were similarly treated and injected onto the SEC column. A UV detector at λ = 260 nm was used for RNA detection (A), and a multi-angle light scattering detector was used for molecular weight determination (B). Molecular weight analysis confirmed that the poly(I:C) used was a polymer with a broad molecular weight distribution. Encapsulated poly(I:C) had similar retention times (A) and molecular weights compared to free poly(I:C), demonstrating that microfluidics technology can encapsulate nucleic acids without altering the original molecular weight distribution. The stability of empty and RNA-containing anionic liposomes in human plasma was tested using asymmetric field-flow fractionation (AF4). Liposomes (2 mM lipid) were incubated with PBS or 20% human plasma at 37°C for 2 hours and then analyzed by AF4. The particle radius of gyration (Rg) and light scattering intensity were determined using MALS (C), and protein corona formation was determined using a fluorescence detector (λem = 280 nm, λex = 340 nm) (D). Empty and RNA-containing anionic liposomes did not exhibit particle aggregation in human plasma, as required for IV injection. Anionic liposomes protected encapsulated poly(I:C) from RNA degradation. Free and encapsulated poly(I:C) were incubated for 2 hours at 37°C in reaction buffer or reaction buffer supplemented with human plasma (20% v / v) with or without RNase. Samples were then run on a 1.0% agarose gel (E). [Figure 4E]Physical characterization of encapsulated RNA and size stability of RNA-containing anionic liposomes [EPC:EPG:cholesterol (3:1:2)] upon incubation with human plasma. Encapsulated poly(I:C) was extracted from anionic liposomes by precipitation with 100% isopropanol. The clean RNA pellet was resuspended in PBS and injected onto a size-exclusion chromatography (SEC) column (TSKgel® G-DNA-PW) run in PBS (flow rate = 0.5 mL / min, 25°C). In addition, samples of free poly(I:C) and standard DNA ruler (Gene Ruler 1 kb plus, Thermoscientific) were similarly treated and injected onto the SEC column. A UV detector at λ = 260 nm was used for RNA detection (A), and a multi-angle light scattering detector was used for molecular weight determination (B). Molecular weight analysis confirmed that the poly(I:C) used was a polymer with a broad molecular weight distribution. Encapsulated poly(I:C) had similar retention times (A) and molecular weights compared to free poly(I:C), demonstrating that microfluidics technology can encapsulate nucleic acids without altering the original molecular weight distribution. The stability of empty and RNA-containing anionic liposomes in human plasma was tested using asymmetric field-flow fractionation (AF4). Liposomes (2 mM lipid) were incubated with PBS or 20% human plasma at 37°C for 2 hours and then analyzed by AF4. The particle radius of gyration (Rg) and light scattering intensity were determined using MALS (C), and protein corona formation was determined using a fluorescence detector (λem = 280 nm, λex = 340 nm) (D). Empty and RNA-containing anionic liposomes did not exhibit particle aggregation in human plasma, as required for IV injection. Anionic liposomes protected encapsulated poly(I:C) from RNA degradation. Free and encapsulated poly(I:C) were incubated for 2 hours at 37°C in reaction buffer or reaction buffer supplemented with human plasma (20% v / v) with or without RNase. Samples were then run on a 1.0% agarose gel (E). [Figure 5A]TEM images of empty (A) and RNA-containing (B) anionic liposomes [EPC:EPG:cholesterol (3:1:2)] show the preservation of the lipid bilayer after nucleic acid incorporation. However, the contrast of the nanoparticles changed after nucleic acid encapsulation. Uranyl salt was used to stain the phosphate groups of phospholipids and RNA molecules and increase particle contrast. 10 μL of the diluted particle suspension (2 mM phospholipid) was placed into a pre-glow-discharged Formvar / carbon-coated copper grid, contrasted with uranyl oxalate (pH 7), and then contrast-embedded with a mixture of 2% methylcellulose and 4% uranyl acetate (pH 4). Images were then taken using a Tecnai12 TEM microscope. [Figure 5B] TEM images of empty (A) and RNA-containing (B) anionic liposomes [EPC:EPG:cholesterol (3:1:2)] show the preservation of the lipid bilayer after nucleic acid incorporation. However, the contrast of the nanoparticles changed after nucleic acid encapsulation. Uranyl salt was used to stain the phosphate groups of phospholipids and RNA molecules and increase particle contrast. 10 μL of the diluted particle suspension (2 mM phospholipid) was placed into a pre-glow-discharged Formvar / carbon-coated copper grid, contrasted with uranyl oxalate (pH 7), and then contrast-embedded with a mixture of 2% methylcellulose and 4% uranyl acetate (pH 4). Images were then taken using a Tecnai12 TEM microscope. [Figure 6A]Empty and poly(I:C)-containing anionic liposomes (empty-AL and plC-AL) share a shell-core spherical structure (A, B). The shell is composed of lipids, and the core is aqueous. Poly(I:C)-containing anionic liposomes have a higher scattered neutron density (SLD) in the shell than empty liposomes. In addition, the scattering profile of RNA-containing liposomes remains unchanged in 68% DO, a contrast consistent with soluble RNA. This suggests that the RNA is embedded in the liposome bilayer rather than soluble in the aqueous core. Furthermore, poly(I:C) encapsulation alters the bilayer flow properties at temperatures above 40 °C, an indication of the presence of RNA in the hydrophobic bilayer (C). Additionally, the RNA-containing anionic liposomes, unlike the empty liposomes, exhibited an exothermic event at 74 °C, suggesting that poly(I:C) crystallizes upon encapsulation in the liposome bilayer (D). (A) SANS data (symbols) for empty (gray rectangles) and poly(I:C)-containing liposomes (black circles) resuspended in 27%, 50%, 68%, and 100% DO water. The solid line corresponds to the best fit using a spherical core-shell model. (B) Scattering length density (SLD) profiles as a function of distance to the liposome center corresponding to the fit of the data in A. (C) General polarization measurements of empty anionic liposomes, anionic liposomes incubated with free poly(I:C), and poly(I:C)-containing anionic liposomes over a temperature interval of 10 to 80 °C (0, 1 °C change per measurement). (D) Differential scanning calorimetry of free poly(I:C), empty anionic liposomes, empty anionic liposomes incubated with free poly(I:C), and poly(I:C)-containing anionic liposomes at a temperature interval of 10–100 °C (0, 5 °C change per minute). [Figure 6B]Empty and poly(I:C)-containing anionic liposomes (empty-AL and plC-AL) share a shell-core spherical structure (A, B). The shell is composed of lipids, and the core is aqueous. Poly(I:C)-containing anionic liposomes have a higher scattered neutron density (SLD) in the shell than empty liposomes. In addition, the scattering profile of RNA-containing liposomes remains unchanged in 68% DO, a contrast consistent with soluble RNA. This suggests that the RNA is embedded in the liposome bilayer rather than soluble in the aqueous core. Furthermore, poly(I:C) encapsulation alters the bilayer flow properties at temperatures above 40 °C, an indication of the presence of RNA in the hydrophobic bilayer (C). Additionally, the RNA-containing anionic liposomes, unlike the empty liposomes, exhibited an exothermic event at 74 °C, suggesting that poly(I:C) crystallizes upon encapsulation in the liposome bilayer (D). (A) SANS data (symbols) for empty (gray rectangles) and poly(I:C)-containing liposomes (black circles) resuspended in 27%, 50%, 68%, and 100% DO water. The solid line corresponds to the best fit using a spherical core-shell model. (B) Scattering length density (SLD) profiles as a function of distance to the liposome center corresponding to the fit of the data in A. (C) General polarization measurements of empty anionic liposomes, anionic liposomes incubated with free poly(I:C), and poly(I:C)-containing anionic liposomes over a temperature interval of 10 to 80 °C (0, 1 °C change per measurement). (D) Differential scanning calorimetry of free poly(I:C), empty anionic liposomes, empty anionic liposomes incubated with free poly(I:C), and poly(I:C)-containing anionic liposomes at a temperature interval of 10–100 °C (0, 5 °C change per minute). [Figure 6C]Empty and poly(I:C)-containing anionic liposomes (empty-AL and plC-AL) share a shell-core spherical structure (A, B). The shell is composed of lipids, and the core is aqueous. Poly(I:C)-containing anionic liposomes have a higher scattered neutron density (SLD) in the shell than empty liposomes. In addition, the scattering profile of RNA-containing liposomes remains unchanged in 68% DO, a contrast consistent with soluble RNA. This suggests that the RNA is embedded in the liposome bilayer rather than soluble in the aqueous core. Furthermore, poly(I:C) encapsulation alters the bilayer flow properties at temperatures above 40 °C, an indication of the presence of RNA in the hydrophobic bilayer (C). Additionally, the RNA-containing anionic liposomes, unlike the empty liposomes, exhibited an exothermic event at 74 °C, suggesting that poly(I:C) crystallizes upon encapsulation in the liposome bilayer (D). (A) SANS data (symbols) for empty (gray rectangles) and poly(I:C)-containing liposomes (black circles) resuspended in 27%, 50%, 68%, and 100% DO water. The solid line corresponds to the best fit using a spherical core-shell model. (B) Scattering length density (SLD) profiles as a function of distance to the liposome center corresponding to the fit of the data in A. (C) General polarization measurements of empty anionic liposomes, anionic liposomes incubated with free poly(I:C), and poly(I:C)-containing anionic liposomes over a temperature interval of 10 to 80 °C (0, 1 °C change per measurement). (D) Differential scanning calorimetry of free poly(I:C), empty anionic liposomes, empty anionic liposomes incubated with free poly(I:C), and poly(I:C)-containing anionic liposomes at a temperature interval of 10–100 °C (0, 5 °C change per minute). [Figure 6D]Empty and poly(I:C)-containing anionic liposomes (empty-AL and plC-AL) share a shell-core spherical structure (A, B). The shell is composed of lipids, and the core is aqueous. Poly(I:C)-containing anionic liposomes have a higher scattered neutron density (SLD) in the shell than empty liposomes. In addition, the scattering profile of RNA-containing liposomes remains unchanged in 68% DO, a contrast consistent with soluble RNA. This suggests that the RNA is embedded in the liposome bilayer rather than soluble in the aqueous core. Furthermore, poly(I:C) encapsulation alters the bilayer flow properties at temperatures above 40 °C, an indication of the presence of RNA in the hydrophobic bilayer (C). Additionally, the RNA-containing anionic liposomes, unlike the empty liposomes, exhibited an exothermic event at 74 °C, suggesting that poly(I:C) crystallizes upon encapsulation in the liposome bilayer (D). (A) SANS data (symbols) for empty (gray rectangles) and poly(I:C)-containing liposomes (black circles) resuspended in 27%, 50%, 68%, and 100% DO water. The solid line corresponds to the best fit using a spherical core-shell model. (B) Scattering length density (SLD) profiles as a function of distance to the liposome center corresponding to the fit of the data in A. (C) General polarization measurements of empty anionic liposomes, anionic liposomes incubated with free poly(I:C), and poly(I:C)-containing anionic liposomes over a temperature interval of 10 to 80 °C (0, 1 °C change per measurement). (D) Differential scanning calorimetry of free poly(I:C), empty anionic liposomes, empty anionic liposomes incubated with free poly(I:C), and poly(I:C)-containing anionic liposomes at a temperature interval of 10–100 °C (0, 5 °C change per minute). [Figure 7A]Stability study of empty and poly(I:C)-containing anionic liposomes [EPC:EPG:cholesterol (3:1:2)]. Anionic liposomes in 10% sucrose, 20 mM citrate buffer (pH 6.5) were stored at -20°C and 5°C for 5 months. The mean particle size (A), polydispersity (B), zeta potential (C), poly(I:C) content (D), and % poly(I:C) released from liposomes (E), as well as the in vitro activity of poly(I:C) (F) were determined at time 0 (a common characterization for both storage conditions) and at 1 and 5 months (mean ± SEM of three replicate analyses). [Figure 7B] Stability study of empty and poly(I:C)-containing anionic liposomes [EPC:EPG:cholesterol (3:1:2)]. Anionic liposomes in 10% sucrose, 20 mM citrate buffer (pH 6.5) were stored at -20°C and 5°C for 5 months. The mean particle size (A), polydispersity (B), zeta potential (C), poly(I:C) content (D), and % poly(I:C) released from liposomes (E), as well as the in vitro activity of poly(I:C) (F) were determined at time 0 (a common characterization for both storage conditions) and at 1 and 5 months (mean ± SEM of three replicate analyses). [Figure 7C] Stability study of empty and poly(I:C)-containing anionic liposomes [EPC:EPG:cholesterol (3:1:2)]. Anionic liposomes in 10% sucrose, 20 mM citrate buffer (pH 6.5) were stored at -20°C and 5°C for 5 months. The mean particle size (A), polydispersity (B), zeta potential (C), poly(I:C) content (D), and % poly(I:C) released from liposomes (E), as well as the in vitro activity of poly(I:C) (F) were determined at time 0 (a common characterization for both storage conditions) and at 1 and 5 months (mean ± SEM of three replicate analyses). [Figure 7D]Stability study of empty and poly(I:C)-containing anionic liposomes [EPC:EPG:cholesterol (3:1:2)]. Anionic liposomes in 10% sucrose, 20 mM citrate buffer (pH 6.5) were stored at -20°C and 5°C for 5 months. The mean particle size (A), polydispersity (B), zeta potential (C), poly(I:C) content (D), and % poly(I:C) released from liposomes (E), as well as the in vitro activity of poly(I:C) (F) were determined at time 0 (a common characterization for both storage conditions) and at 1 and 5 months (mean ± SEM of three replicate analyses). [Figure 7E] Stability study of empty and poly(I:C)-containing anionic liposomes [EPC:EPG:cholesterol (3:1:2)]. Anionic liposomes in 10% sucrose, 20 mM citrate buffer (pH 6.5) were stored at -20°C and 5°C for 5 months. The mean particle size (A), polydispersity (B), zeta potential (C), poly(I:C) content (D), and % poly(I:C) released from liposomes (E), as well as the in vitro activity of poly(I:C) (F) were determined at time 0 (a common characterization for both storage conditions) and at 1 and 5 months (mean ± SEM of three replicate analyses). [Figure 7F] Stability study of empty and poly(I:C)-containing anionic liposomes [EPC:EPG:cholesterol (3:1:2)]. Anionic liposomes in 10% sucrose, 20 mM citrate buffer (pH 6.5) were stored at -20°C and 5°C for 5 months. The mean particle size (A), polydispersity (B), zeta potential (C), poly(I:C) content (D), and % poly(I:C) released from liposomes (E), as well as the in vitro activity of poly(I:C) (F) were determined at time 0 (a common characterization for both storage conditions) and at 1 and 5 months (mean ± SEM of three replicate analyses). [Figure 8A]Encapsulation of poly(I:C) in anionic liposomes [EPC:EPG:cholesterol (3:1:2)] induces superior maturation of splenic conventional type 1 dendritic cells (cDC1) ex vivo compared with soluble poly(I:C). Single-cell suspensions of splenocytes were incubated with 0.5 μg of liposomally encapsulated poly(I:C) or increasing amounts of soluble poly(I:C) (1, 10, and 100 μg) (100 μL / well). After 45 min of incubation at 37°C, splenocytes were extensively washed and incubated for 15 h at 37°C before staining. Expression of CD80, CD86, and MHC II was determined by flow cytometry analysis. Shown are the mean ± SEM (n=5) geometric mean fluorescence intensity (GMFI) of CD80 (A), CD86 (B), and MHC-II (C) expression by conventional type 1 dendritic cells. *p<0.05, **p<0.01, ****p<0.0001. [Figure 8B] Encapsulation of poly(I:C) in anionic liposomes [EPC:EPG:cholesterol (3:1:2)] induces superior maturation of splenic conventional type 1 dendritic cells (cDC1) ex vivo compared with soluble poly(I:C). Single-cell suspensions of splenocytes were incubated with 0.5 μg of liposomally encapsulated poly(I:C) or increasing amounts of soluble poly(I:C) (1, 10, and 100 μg) (100 μL / well). After 45 min of incubation at 37°C, splenocytes were extensively washed and incubated for 15 h at 37°C before staining. Expression of CD80, CD86, and MHC II was determined by flow cytometry analysis. Shown are the mean ± SEM (n=5) geometric mean fluorescence intensity (GMFI) of CD80 (A), CD86 (B), and MHC-II (C) expression by conventional type 1 dendritic cells. *p<0.05, **p<0.01, ****p<0.0001. [Figure 8C]Encapsulation of poly(I:C) in anionic liposomes [EPC:EPG:cholesterol (3:1:2)] induces superior maturation of splenic conventional type 1 dendritic cells (cDC1) ex vivo compared with soluble poly(I:C). Single-cell suspensions of splenocytes were incubated with 0.5 μg of liposomally encapsulated poly(I:C) or increasing amounts of soluble poly(I:C) (1, 10, and 100 μg) (100 μL / well). After 45 min of incubation at 37°C, splenocytes were extensively washed and incubated for 15 h at 37°C before staining. Expression of CD80, CD86, and MHC II was determined by flow cytometry analysis. Shown are the mean ± SEM (n=5) geometric mean fluorescence intensity (GMFI) of CD80 (A), CD86 (B), and MHC-II (C) expression by conventional type 1 dendritic cells. *p<0.05, **p<0.01, ****p<0.0001. [Figure 9A] Intravenous administration of poly(I:C)-containing anionic liposomes (pIC-AL) to mice amplifies the immunostimulatory effects of poly(I:C) in vivo without inducing acute hepatotoxicity. Mice were administered 10 or 50 μg of soluble or anionic liposomal poly(I:C) [EPC:EPG:cholesterol (3:1:2)] intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. After 24 hours, mice were sacrificed, and blood was collected for cytokine and liver enzyme (AST, ALT) activity determinations. In addition, spleens were harvested, and single-cell suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensities (GMFIs) of maturation markers in cDC1 (A, B, C) are shown [n=4]. The fold increase (log-transformed) and concentration of serum antiviral cytokines 3 and 24 hours after IV injection are shown (D, E). Mean serum enzyme activity (UI / L) ± SEM of liver enzymes ALT (F) and AST (G) are shown. *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 9B]Intravenous administration of poly(I:C)-containing anionic liposomes (pIC-AL) to mice amplifies the immunostimulatory effects of poly(I:C) in vivo without inducing acute hepatotoxicity. Mice were administered 10 or 50 μg of soluble or anionic liposomal poly(I:C) [EPC:EPG:cholesterol (3:1:2)] intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. After 24 hours, mice were sacrificed, and blood was collected for cytokine and liver enzyme (AST, ALT) activity determinations. In addition, spleens were harvested, and single-cell suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensities (GMFIs) of maturation markers in cDC1 (A, B, C) are shown [n=4]. The fold increase (log-transformed) and concentration of serum antiviral cytokines 3 and 24 hours after IV injection are shown (D, E). Mean serum enzyme activity (UI / L) ± SEM of liver enzymes ALT (F) and AST (G) are shown. *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 9C]Intravenous administration of poly(I:C)-containing anionic liposomes (pIC-AL) to mice amplifies the immunostimulatory effects of poly(I:C) in vivo without inducing acute hepatotoxicity. Mice were administered 10 or 50 μg of soluble or anionic liposomal poly(I:C) [EPC:EPG:cholesterol (3:1:2)] intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. After 24 hours, mice were sacrificed, and blood was collected for cytokine and liver enzyme (AST, ALT) activity determinations. In addition, spleens were harvested, and single-cell suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensities (GMFIs) of maturation markers in cDC1 (A, B, C) are shown [n=4]. The fold increase (log-transformed) and concentration of serum antiviral cytokines 3 and 24 hours after IV injection are shown (D, E). Mean serum enzyme activity (UI / L) ± SEM of liver enzymes ALT (F) and AST (G) are shown. *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 9D]Intravenous administration of poly(I:C)-containing anionic liposomes (pIC-AL) to mice amplifies the immunostimulatory effects of poly(I:C) in vivo without inducing acute hepatotoxicity. Mice were administered 10 or 50 μg of soluble or anionic liposomal poly(I:C) [EPC:EPG:cholesterol (3:1:2)] intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. After 24 hours, mice were sacrificed, and blood was collected for cytokine and liver enzyme (AST, ALT) activity determinations. In addition, spleens were harvested, and single-cell suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensities (GMFIs) of maturation markers in cDC1 (A, B, C) are shown [n=4]. The fold increase (log-transformed) and concentration of serum antiviral cytokines 3 and 24 hours after IV injection are shown (D, E). Mean serum enzyme activity (UI / L) ± SEM of liver enzymes ALT (F) and AST (G) are shown. *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 9E]Intravenous administration of poly(I:C)-containing anionic liposomes (pIC-AL) to mice amplifies the immunostimulatory effects of poly(I:C) in vivo without inducing acute hepatotoxicity. Mice were administered 10 or 50 μg of soluble or anionic liposomal poly(I:C) [EPC:EPG:cholesterol (3:1:2)] intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. After 24 hours, mice were sacrificed, and blood was collected for cytokine and liver enzyme (AST, ALT) activity determinations. In addition, spleens were harvested, and single-cell suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensities (GMFIs) of maturation markers in cDC1 (A, B, C) are shown [n=4]. The fold increase (log-transformed) and concentration of serum antiviral cytokines 3 and 24 hours after IV injection are shown (D, E). Mean serum enzyme activity (UI / L) ± SEM of liver enzymes ALT (F) and AST (G) are shown. *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 9F]Intravenous administration of poly(I:C)-containing anionic liposomes (pIC-AL) to mice amplifies the immunostimulatory effects of poly(I:C) in vivo without inducing acute hepatotoxicity. Mice were administered 10 or 50 μg of soluble or anionic liposomal poly(I:C) [EPC:EPG:cholesterol (3:1:2)] intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. After 24 hours, mice were sacrificed, and blood was collected for cytokine and liver enzyme (AST, ALT) activity determinations. In addition, spleens were harvested, and single-cell suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensities (GMFIs) of maturation markers in cDC1 (A, B, C) are shown [n=4]. The fold increase (log-transformed) and concentration of serum antiviral cytokines 3 and 24 hours after IV injection are shown (D, E). Mean serum enzyme activity (UI / L) ± SEM of liver enzymes ALT (F) and AST (G) are shown. *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 9G]Intravenous administration of poly(I:C)-containing anionic liposomes (pIC-AL) to mice amplifies the immunostimulatory effects of poly(I:C) in vivo without inducing acute hepatotoxicity. Mice were administered 10 or 50 μg of soluble or anionic liposomal poly(I:C) [EPC:EPG:cholesterol (3:1:2)] intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. After 24 hours, mice were sacrificed, and blood was collected for cytokine and liver enzyme (AST, ALT) activity determinations. In addition, spleens were harvested, and single-cell suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensities (GMFIs) of maturation markers in cDC1 (A, B, C) are shown [n=4]. The fold increase (log-transformed) and concentration of serum antiviral cytokines 3 and 24 hours after IV injection are shown (D, E). Mean serum enzyme activity (UI / L) ± SEM of liver enzymes ALT (F) and AST (G) are shown. *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 10A]Intravenous administration of poly(I:C)-containing anionic liposomes (pIC-AL), unlike free poly(I:C), significantly enhanced vaccine efficacy against the OVA247-279 model antigen. Anionic liposomes [EPC:EPG:cholesterol (3:1:2)] containing OVA247-279 long peptide alone or supplemented with soluble poly(I:C) (10 or 100 μg) or encapsulated poly(I:C) (10 μg) in anionic liposomes [EPC:EPG:cholesterol (3:1:2)] were administered intravenously to mice (22.5 nmol phospholipid, 1.25 μg peptide). On day 7, H-2Kb / SIINFEKL tetramer-binding T cells were identified (A). Spleen cells were restimulated ex vivo with SIINFEKL peptide for 5 or 25 hours to induce CD8+ or CD4+ T cell responses, respectively. IFNγ-producing CD8+ (B) or CD4+ (C) T cells were then detected by intracellular flow cytometry staining. GMFI ± SEM is shown (n=4). ns: not significant, * p<0.05, ** p<0.01, *** p<0.005, and **** p<0.0001; ns: not significant. [Figure 10B]Intravenous administration of poly(I:C)-containing anionic liposomes (pIC-AL), unlike free poly(I:C), significantly enhanced vaccine efficacy against the OVA247-279 model antigen. Anionic liposomes [EPC:EPG:cholesterol (3:1:2)] containing OVA247-279 long peptide alone or supplemented with soluble poly(I:C) (10 or 100 μg) or encapsulated poly(I:C) (10 μg) in anionic liposomes [EPC:EPG:cholesterol (3:1:2)] were administered intravenously to mice (22.5 nmol phospholipid, 1.25 μg peptide). On day 7, H-2Kb / SIINFEKL tetramer-binding T cells were identified (A). Spleen cells were restimulated ex vivo with SIINFEKL peptide for 5 or 25 hours to induce CD8+ or CD4+ T cell responses, respectively. IFNγ-producing CD8+ (B) or CD4+ (C) T cells were then detected by intracellular flow cytometry staining. GMFI ± SEM is shown (n=4). ns: not significant, * p<0.05, ** p<0.01, *** p<0.005, and **** p<0.0001; ns: not significant. [Figure 10C]Intravenous administration of poly(I:C)-containing anionic liposomes (pIC-AL), unlike free poly(I:C), significantly enhanced vaccine efficacy against the OVA247-279 model antigen. Anionic liposomes [EPC:EPG:cholesterol (3:1:2)] containing OVA247-279 long peptide alone or supplemented with soluble poly(I:C) (10 or 100 μg) or encapsulated poly(I:C) (10 μg) in anionic liposomes [EPC:EPG:cholesterol (3:1:2)] were administered intravenously to mice (22.5 nmol phospholipid, 1.25 μg peptide). On day 7, H-2Kb / SIINFEKL tetramer-binding T cells were identified (A). Spleen cells were restimulated ex vivo with SIINFEKL peptide for 5 or 25 hours to induce CD8+ or CD4+ T cell responses, respectively. IFNγ-producing CD8+ (B) or CD4+ (C) T cells were then detected by intracellular flow cytometry staining. GMFI ± SEM is shown (n=4). ns: not significant, * p<0.05, ** p<0.01, *** p<0.005, and **** p<0.0001; ns: not significant. [Figure 11A]Intravenous administration of poly(I:C)-containing liposomes potently activates CD8+ T cells to produce interferon-γ in an antigen-independent manner. Poly(I:C)-containing anionic liposomes [EPC:EPG:cholesterol (3:1:2)] were administered IV to mice alone (10 μg) or in combination with low- or high-dose liposomes containing an OTI / III model ovalbumin peptide (low dose: 22.5 nmol phospholipid and 1.25 μg peptide; high dose: 200 nmol phospholipid and 11.1 μg peptide) (n = 4 / group, except for naive controls, where n = 3). On day 7, H-2Kb / SIINFEKL tetramer-binding T cells were identified (A). Spleen cells were restimulated ex vivo with SIINFEKL peptide for 5 or 25 hours for CD8+ or CD4+ T cell responses, respectively. IFNγ-producing CD8+ (B) or CD4+ (C) T cells were then detected by intracellular flow cytometry staining. GMFI ± SEM is shown (n=4). ns: not significant, * p<0.05, ** p<0.01, *** p<0.005, and **** p<0.0001. [Figure 11B]Intravenous administration of poly(I:C)-containing liposomes potently activates CD8+ T cells to produce interferon-γ in an antigen-independent manner. Poly(I:C)-containing anionic liposomes [EPC:EPG:cholesterol (3:1:2)] were administered IV to mice alone (10 μg) or in combination with low- or high-dose liposomes containing an OTI / III model ovalbumin peptide (low dose: 22.5 nmol phospholipid and 1.25 μg peptide; high dose: 200 nmol phospholipid and 11.1 μg peptide) (n = 4 / group, except for naive controls, where n = 3). On day 7, H-2Kb / SIINFEKL tetramer-binding T cells were identified (A). Spleen cells were restimulated ex vivo with SIINFEKL peptide for 5 or 25 hours for CD8+ or CD4+ T cell responses, respectively. IFNγ-producing CD8+ (B) or CD4+ (C) T cells were then detected by intracellular flow cytometry staining. GMFI ± SEM is shown (n=4). ns: not significant, * p<0.05, ** p<0.01, *** p<0.005, and **** p<0.0001. [Figure 11C]Intravenous administration of poly(I:C)-containing liposomes potently activates CD8+ T cells to produce interferon-γ in an antigen-independent manner. Poly(I:C)-containing anionic liposomes [EPC:EPG:cholesterol (3:1:2)] were administered IV to mice alone (10 μg) or in combination with low- or high-dose liposomes containing an OTI / III model ovalbumin peptide (low dose: 22.5 nmol phospholipid and 1.25 μg peptide; high dose: 200 nmol phospholipid and 11.1 μg peptide) (n = 4 / group, except for naive controls, where n = 3). On day 7, H-2Kb / SIINFEKL tetramer-binding T cells were identified (A). Spleen cells were restimulated ex vivo with SIINFEKL peptide for 5 or 25 hours for CD8+ or CD4+ T cell responses, respectively. IFNγ-producing CD8+ (B) or CD4+ (C) T cells were then detected by intracellular flow cytometry staining. GMFI ± SEM is shown (n=4). ns: not significant, * p<0.05, ** p<0.01, *** p<0.005, and **** p<0.0001. [Figure 12A] The effect of the relative molar concentration of the anionic phospholipid EPG in poly(I:C) liposomes on uptake and maturation by cDC1 cells. Single-cell suspensions of splenocytes were incubated with 0.5 μg of liposomally encapsulated poly(I:C). The EPG molar ratio in the liposomes was varied between samples (0, 4, 17, 33, and 67%), with cholesterol replacing EPC and kept constant (33% molar ratio). After 45 min of incubation at 37°C, splenocytes were extensively washed and incubated for 15 h at 37°C before staining. Uptake of liposomes (DiD) by cDC1 cells and expression of CD80 and CD86 were determined by flow cytometry analysis. The mean ± SEM (n = 3) of geometric mean fluorescence intensity (GMFI) for DiD (A), CD80 (B), and CD86 (C) by cDC1 cells is shown. [Figure 12B]The effect of the relative molar concentration of the anionic phospholipid EPG in poly(I:C) liposomes on uptake and maturation by cDC1 cells. Single-cell suspensions of splenocytes were incubated with 0.5 μg of liposomally encapsulated poly(I:C). The EPG molar ratio in the liposomes was varied between samples (0, 4, 17, 33, and 67%), with cholesterol replacing EPC and kept constant (33% molar ratio). After 45 min of incubation at 37°C, splenocytes were extensively washed and incubated for 15 h at 37°C before staining. Uptake of liposomes (DiD) by cDC1 cells and expression of CD80 and CD86 were determined by flow cytometry analysis. The mean ± SEM (n = 3) of geometric mean fluorescence intensity (GMFI) for DiD (A), CD80 (B), and CD86 (C) by cDC1 cells is shown. [Figure 12C] The effect of the relative molar concentration of the anionic phospholipid EPG in poly(I:C) liposomes on uptake and maturation by cDC1 cells. Single-cell suspensions of splenocytes were incubated with 0.5 μg of liposomally encapsulated poly(I:C). The EPG molar ratio in the liposomes was varied between samples (0, 4, 17, 33, and 67%), with cholesterol replacing EPC and kept constant (33% molar ratio). After 45 min of incubation at 37°C, splenocytes were extensively washed and incubated for 15 h at 37°C before staining. Uptake of liposomes (DiD) by cDC1 cells and expression of CD80 and CD86 were determined by flow cytometry analysis. The mean ± SEM (n = 3) of geometric mean fluorescence intensity (GMFI) for DiD (A), CD80 (B), and CD86 (C) by cDC1 cells is shown. [Figure 13A]Uptake of anionic liposomes containing poly(I:C) by splenic cDC1 cells (A) and cDC2 cells (B) and red pulp macrophages (C). Mice were intravenously administered 10 or 50 μg of soluble or anionic liposomal poly(I:C) [EPC:EPG:cholesterol (3:1:2)]. 24 hours later, spleens were harvested, and single-cell suspensions were used for flow cytometry staining and analysis. The mean ± SEM of the representative percentage of DiD-positive cells as an indicator of liposome uptake by cDC1, cDC2, and red pulp macrophages is shown (n=4). *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001). [Figure 13B] Uptake of anionic liposomes containing poly(I:C) by splenic cDC1 cells (A) and cDC2 cells (B) and red pulp macrophages (C). Mice were intravenously administered 10 or 50 μg of soluble or anionic liposomal poly(I:C) [EPC:EPG:cholesterol (3:1:2)]. 24 hours later, spleens were harvested, and single-cell suspensions were used for flow cytometry staining and analysis. The mean ± SEM of the representative percentage of DiD-positive cells as an indicator of liposome uptake by cDC1, cDC2, and red pulp macrophages is shown (n=4). *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001). [Figure 13C] Uptake of anionic liposomes containing poly(I:C) by splenic cDC1 cells (A) and cDC2 cells (B) and red pulp macrophages (C). Mice were intravenously administered 10 or 50 μg of soluble or anionic liposomal poly(I:C) [EPC:EPG:cholesterol (3:1:2)]. 24 hours later, spleens were harvested, and single-cell suspensions were used for flow cytometry staining and analysis. The mean ± SEM of the representative percentage of DiD-positive cells as an indicator of liposome uptake by cDC1, cDC2, and red pulp macrophages is shown (n=4). *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001). [Figure 14A]Enhanced therapeutic efficacy as a result of combination therapy of a-PD-L1 antibody and anionic liposomes containing poly(I:C). (A) Schematic experimental design of mice bearing B16F10 syngeneic tumors implanted into the right flank after subcutaneous inoculation of 5x105 B16F10 cells. Seven days after inoculation, when tumor size reached an average of 100mm3, mice were randomized into different groups and treatment was initiated. Mice were treated intravenously with free poly(I:C) (10 μg or 50 μg), anionic liposomes containing poly(I:C) (10 μg) [EPC:EPG:cholesterol (3:1:2)], or PBS (control) on days 0, 3, 6, 9, and 12 after the start of treatment, and intraperitoneally with a-PD-L1 (100 μg) or rat IgG antibody (100 μg) (control) on days 1, 4, 7, 10, and 13 after the start of treatment. (B) Mean tumor growth curves; (C) tumor growth inhibition in the a-PD-L1 monotherapy and combination poly(I:C) groups compared to the IgG control group; and (D) normalized body weight curves from the start of treatment through day 5 when the first mouse reached a tumor size >1500 mm3 (humane endpoint (HEP)). (D) Survival curves of treated mice whose tumors reached the humane endpoint (HEP) (tumor size >1500 mm3) over time from the start of treatment to 1 day after the end of treatment (day 14). Animals that reached another HEP (e.g., tumor ulceration) but did not reach the tumor size HEP were not included in the survival analysis. Black arrows indicate poly(I:C) injection, and gray arrows indicate a-PD-L1 injection. Mean ± SEM is shown (n=10 except for the IgG group, n=8, and a-PD-L1 / poly(I:C) 10 μg, n=9). *p<0.05, **p<0.01, ***p<0.005. [Figure 14B]Enhanced therapeutic efficacy as a result of combination therapy of a-PD-L1 antibody and anionic liposomes containing poly(I:C). (A) Schematic experimental design of mice bearing B16F10 syngeneic tumors implanted into the right flank after subcutaneous inoculation of 5x105 B16F10 cells. Seven days after inoculation, when tumor size reached an average of 100mm3, mice were randomized into different groups and treatment was initiated. Mice were treated intravenously with free poly(I:C) (10 μg or 50 μg), anionic liposomes containing poly(I:C) (10 μg) [EPC:EPG:cholesterol (3:1:2)], or PBS (control) on days 0, 3, 6, 9, and 12 after the start of treatment, and intraperitoneally with a-PD-L1 (100 μg) or rat IgG antibody (100 μg) (control) on days 1, 4, 7, 10, and 13 after the start of treatment. (B) Mean tumor growth curves; (C) tumor growth inhibition in the a-PD-L1 monotherapy and combination poly(I:C) groups compared to the IgG control group; and (D) normalized body weight curves from the start of treatment through day 5 when the first mouse reached a tumor size >1500 mm3 (humane endpoint (HEP)). (D) Survival curves of treated mice whose tumors reached the humane endpoint (HEP) (tumor size >1500 mm3) over time from the start of treatment to 1 day after the end of treatment (day 14). Animals that reached another HEP (e.g., tumor ulceration) but did not reach the tumor size HEP were not included in the survival analysis. Black arrows indicate poly(I:C) injection, and gray arrows indicate a-PD-L1 injection. Mean ± SEM is shown (n=10 except for the IgG group, n=8, and a-PD-L1 / poly(I:C) 10 μg, n=9). *p<0.05, **p<0.01, ***p<0.005. [Figure 14C]Enhanced therapeutic efficacy as a result of combination therapy of a-PD-L1 antibody and anionic liposomes containing poly(I:C). (A) Schematic experimental design of mice bearing B16F10 syngeneic tumors implanted into the right flank after subcutaneous inoculation of 5x105 B16F10 cells. Seven days after inoculation, when tumor size reached an average of 100mm3, mice were randomized into different groups and treatment was initiated. Mice were treated intravenously with free poly(I:C) (10 μg or 50 μg), anionic liposomes containing poly(I:C) (10 μg) [EPC:EPG:cholesterol (3:1:2)], or PBS (control) on days 0, 3, 6, 9, and 12 after the start of treatment, and intraperitoneally with a-PD-L1 (100 μg) or rat IgG antibody (100 μg) (control) on days 1, 4, 7, 10, and 13 after the start of treatment. (B) Mean tumor growth curves; (C) tumor growth inhibition in the a-PD-L1 monotherapy and combination poly(I:C) groups compared to the IgG control group; and (D) normalized body weight curves from the start of treatment through day 5 when the first mouse reached a tumor size >1500 mm3 (humane endpoint (HEP)). (D) Survival curves of treated mice whose tumors reached the humane endpoint (HEP) (tumor size >1500 mm3) over time from the start of treatment to 1 day after the end of treatment (day 14). Animals that reached another HEP (e.g., tumor ulceration) but did not reach the tumor size HEP were not included in the survival analysis. Black arrows indicate poly(I:C) injection, and gray arrows indicate a-PD-L1 injection. Mean ± SEM is shown (n=10 except for the IgG group, n=8, and a-PD-L1 / poly(I:C) 10 μg, n=9). *p<0.05, **p<0.01, ***p<0.005. [Figure 14D]Enhanced therapeutic efficacy as a result of combination therapy of a-PD-L1 antibody and anionic liposomes containing poly(I:C). (A) Schematic experimental design of mice bearing B16F10 syngeneic tumors implanted into the right flank after subcutaneous inoculation of 5x105 B16F10 cells. Seven days after inoculation, when tumor size reached an average of 100mm3, mice were randomized into different groups and treatment was initiated. Mice were treated intravenously with free poly(I:C) (10 μg or 50 μg), anionic liposomes containing poly(I:C) (10 μg) [EPC:EPG:cholesterol (3:1:2)], or PBS (control) on days 0, 3, 6, 9, and 12 after the start of treatment, and intraperitoneally with a-PD-L1 (100 μg) or rat IgG antibody (100 μg) (control) on days 1, 4, 7, 10, and 13 after the start of treatment. (B) Mean tumor growth curves; (C) tumor growth inhibition in the a-PD-L1 monotherapy and combination poly(I:C) groups compared to the IgG control group; and (D) normalized body weight curves from the start of treatment through day 5 when the first mouse reached a tumor size >1500 mm3 (humane endpoint (HEP)). (D) Survival curves of treated mice whose tumors reached the humane endpoint (HEP) (tumor size >1500 mm3) over time from the start of treatment to 1 day after the end of treatment (day 14). Animals that reached another HEP (e.g., tumor ulceration) but did not reach the tumor size HEP were not included in the survival analysis. Black arrows indicate poly(I:C) injection, and gray arrows indicate a-PD-L1 injection. Mean ± SEM is shown (n=10 except for the IgG group, n=8, and a-PD-L1 / poly(I:C) 10 μg, n=9). *p<0.05, **p<0.01, ***p<0.005. [Figure 14E]Enhanced therapeutic efficacy as a result of combination therapy of a-PD-L1 antibody and anionic liposomes containing poly(I:C). (A) Schematic experimental design of mice bearing B16F10 syngeneic tumors implanted into the right flank after subcutaneous inoculation of 5x105 B16F10 cells. Seven days after inoculation, when tumor size reached an average of 100mm3, mice were randomized into different groups and treatment was initiated. Mice were treated intravenously with free poly(I:C) (10 μg or 50 μg), anionic liposomes containing poly(I:C) (10 μg) [EPC:EPG:cholesterol (3:1:2)], or PBS (control) on days 0, 3, 6, 9, and 12 after the start of treatment, and intraperitoneally with a-PD-L1 (100 μg) or rat IgG antibody (100 μg) (control) on days 1, 4, 7, 10, and 13 after the start of treatment. (B) Mean tumor growth curves; (C) tumor growth inhibition in the a-PD-L1 monotherapy and combination poly(I:C) groups compared to the IgG control group; and (D) normalized body weight curves from the start of treatment through day 5 when the first mouse reached a tumor size >1500 mm3 (humane endpoint (HEP)). (D) Survival curves of treated mice whose tumors reached the humane endpoint (HEP) (tumor size >1500 mm3) over time from the start of treatment to 1 day after the end of treatment (day 14). Animals that reached another HEP (e.g., tumor ulceration) but did not reach the tumor size HEP were not included in the survival analysis. Black arrows indicate poly(I:C) injection, and gray arrows indicate a-PD-L1 injection. Mean ± SEM is shown (n=10 except for the IgG group, n=8, and a-PD-L1 / poly(I:C) 10 μg, n=9). *p<0.05, **p<0.01, ***p<0.005. [Figure 15A] Enhanced therapeutic efficacy by combining a-PD-L1 antibody with anionic liposomes containing poly(I:C). Individual tumor volume (length x width (with)²) / ² over time after initiation of treatment. Mean ± SEM is shown (n=10 except for the IgG group, n=8, and a-PD-L1 / poly(I:C) 10μg, n=9). *p<0.05, **p<0.01, ***p<0.005. [Figure 15B]Enhanced therapeutic efficacy by combining a-PD-L1 antibody with anionic liposomes containing poly(I:C). Individual tumor volume (length x width (with)²) / ² over time after initiation of treatment. Mean ± SEM is shown (n=10 except for the IgG group, n=8, and a-PD-L1 / poly(I:C) 10μg, n=9). *p<0.05, **p<0.01, ***p<0.005. [Figure 15C] Enhanced therapeutic efficacy by combining a-PD-L1 antibody with anionic liposomes containing poly(I:C). Individual tumor volume (length x width (with)²) / ² over time after initiation of treatment. Mean ± SEM is shown (n=10 except for the IgG group, n=8, and a-PD-L1 / poly(I:C) 10μg, n=9). *p<0.05, **p<0.01, ***p<0.005. [Figure 15D] Enhanced therapeutic efficacy by combining a-PD-L1 antibody with anionic liposomes containing poly(I:C). Individual tumor volume (length x width (with)²) / ² over time after initiation of treatment. Mean ± SEM is shown (n=10 except for the IgG group, n=8, and a-PD-L1 / poly(I:C) 10μg, n=9). *p<0.05, **p<0.01, ***p<0.005. [Figure 15E] Enhanced therapeutic efficacy by combining a-PD-L1 antibody with anionic liposomes containing poly(I:C). Individual tumor volume (length x width (with)²) / ² over time after initiation of treatment. Mean ± SEM is shown (n=10 except for the IgG group, n=8, and a-PD-L1 / poly(I:C) 10μg, n=9). *p<0.05, **p<0.01, ***p<0.005. [Figure 16A]Effect of lipid composition on the characteristics of anionic liposomes containing RNA. RNA-anionic liposome formulations with different molar ratios of EPC, EPG, and cholesterol were prepared in a design mixture experiment (n=17) (F). Particle mean size (A), PDI (B), zeta potential (C), RNA encapsulation efficiency (D), and TLR3 activation (%) by HEK293T TLR3+ cells versus free poly(I:C) (E) were analyzed for each formulation. RSMs were constructed for each parameter. Mean ± SEM from replicate analyses (n=3) is shown. [Figure 16B] Effect of lipid composition on the characteristics of anionic liposomes containing RNA. RNA-anionic liposome formulations with different molar ratios of EPC, EPG, and cholesterol were prepared in a design mixture experiment (n=17) (F). Particle mean size (A), PDI (B), zeta potential (C), RNA encapsulation efficiency (D), and TLR3 activation (%) by HEK293T TLR3+ cells versus free poly(I:C) (E) were analyzed for each formulation. RSMs were constructed for each parameter. Mean ± SEM from replicate analyses (n=3) is shown. [Figure 16C] Effect of lipid composition on the characteristics of anionic liposomes containing RNA. RNA-anionic liposome formulations with different molar ratios of EPC, EPG, and cholesterol were prepared in a design mixture experiment (n=17) (F). Particle mean size (A), PDI (B), zeta potential (C), RNA encapsulation efficiency (D), and TLR3 activation (%) by HEK293T TLR3+ cells versus free poly(I:C) (E) were analyzed for each formulation. RSMs were constructed for each parameter. Mean ± SEM from replicate analyses (n=3) is shown. [Figure 16D]Effect of lipid composition on the characteristics of anionic liposomes containing RNA. RNA-anionic liposome formulations with different molar ratios of EPC, EPG, and cholesterol were prepared in a design mixture experiment (n=17) (F). Particle mean size (A), PDI (B), zeta potential (C), RNA encapsulation efficiency (D), and TLR3 activation (%) by HEK293T TLR3+ cells versus free poly(I:C) (E) were analyzed for each formulation. RSMs were constructed for each parameter. Mean ± SEM from replicate analyses (n=3) is shown. [Figure 16E] Effect of lipid composition on the characteristics of anionic liposomes containing RNA. RNA-anionic liposome formulations with different molar ratios of EPC, EPG, and cholesterol were prepared in a design mixture experiment (n=17) (F). Particle mean size (A), PDI (B), zeta potential (C), RNA encapsulation efficiency (D), and TLR3 activation (%) by HEK293T TLR3+ cells versus free poly(I:C) (E) were analyzed for each formulation. RSMs were constructed for each parameter. Mean ± SEM from replicate analyses (n=3) is shown. [Figure 16F] Effect of lipid composition on the characteristics of anionic liposomes containing RNA. RNA-anionic liposome formulations with different molar ratios of EPC, EPG, and cholesterol were prepared in a design mixture experiment (n=17) (F). Particle mean size (A), PDI (B), zeta potential (C), RNA encapsulation efficiency (D), and TLR3 activation (%) by HEK293T TLR3+ cells versus free poly(I:C) (E) were analyzed for each formulation. RSMs were constructed for each parameter. Mean ± SEM from replicate analyses (n=3) is shown. [Figure 17A]Effect of microfluidics process parameters of design A on particle properties of anionic liposomes containing poly(I:C). RNA anionic liposome formulations were prepared at different aqueous RNA phase flow rates, ethanolic lipid phase flow rates, lipid concentrations, and RNA concentrations in response-phase designed experiments (n=30) (D). Particle mean size (A), PDI (B), and RNA encapsulation efficiency (C) were analyzed for each formulation. RSM was constructed for each parameter. Mean ± SEM (n=3) of replicate analyses is shown. ND=not detectable. NM=not measurable. [Figure 17B] Effect of microfluidics process parameters of design A on particle properties of anionic liposomes containing poly(I:C). RNA anionic liposome formulations were prepared at different aqueous RNA phase flow rates, ethanolic lipid phase flow rates, lipid concentrations, and RNA concentrations in response-phase designed experiments (n=30) (D). Particle mean size (A), PDI (B), and RNA encapsulation efficiency (C) were analyzed for each formulation. RSM was constructed for each parameter. Mean ± SEM (n=3) of replicate analyses is shown. ND=not detectable. NM=not measurable. [Figure 17C] Effect of microfluidics process parameters of design A on particle properties of anionic liposomes containing poly(I:C). RNA anionic liposome formulations were prepared at different aqueous RNA phase flow rates, ethanolic lipid phase flow rates, lipid concentrations, and RNA concentrations in response-phase designed experiments (n=30) (D). Particle mean size (A), PDI (B), and RNA encapsulation efficiency (C) were analyzed for each formulation. RSM was constructed for each parameter. Mean ± SEM (n=3) of replicate analyses is shown. ND=not detectable. NM=not measurable. [Figure 17D]Effect of microfluidics process parameters of design A on particle properties of anionic liposomes containing poly(I:C). RNA anionic liposome formulations were prepared at different aqueous RNA phase flow rates, ethanolic lipid phase flow rates, lipid concentrations, and RNA concentrations in response-phase designed experiments (n=30) (D). Particle mean size (A), PDI (B), and RNA encapsulation efficiency (C) were analyzed for each formulation. RSM was constructed for each parameter. Mean ± SEM (n=3) of replicate analyses is shown. ND=not detectable. NM=not measurable. [Figure 18A] Effect of microfluidics process parameters of Design B on particle properties of anionic liposomes containing poly(I:C). RNA anionic liposome formulations were prepared at different aqueous RNA phase flow rates, ethanolic lipid phase flow rates, aqueous phase flow rates, lipid concentrations, and RNA concentrations in response-phase designed experiments (n=53) (D). Particle mean size (A), PDI (B), and RNA encapsulation efficiency (C) were analyzed for each formulation. RSMs were constructed for each parameter. Mean ± SEM (n=3) of replicate analyses is shown. [Figure 18B] Effect of microfluidics process parameters of Design B on particle properties of anionic liposomes containing poly(I:C). RNA anionic liposome formulations were prepared at different aqueous RNA phase flow rates, ethanolic lipid phase flow rates, aqueous phase flow rates, lipid concentrations, and RNA concentrations in response-phase designed experiments (n=53) (D). Particle mean size (A), PDI (B), and RNA encapsulation efficiency (C) were analyzed for each formulation. RSMs were constructed for each parameter. Mean ± SEM (n=3) of replicate analyses is shown. [Figure 18C]Effect of microfluidics process parameters of Design B on particle properties of anionic liposomes containing poly(I:C). RNA anionic liposome formulations were prepared at different aqueous RNA phase flow rates, ethanolic lipid phase flow rates, aqueous phase flow rates, lipid concentrations, and RNA concentrations in response-phase designed experiments (n=53) (D). Particle mean size (A), PDI (B), and RNA encapsulation efficiency (C) were analyzed for each formulation. RSMs were constructed for each parameter. Mean ± SEM (n=3) of replicate analyses is shown. [Figure 18D] Effect of microfluidics process parameters of Design B on particle properties of anionic liposomes containing poly(I:C). RNA anionic liposome formulations were prepared at different aqueous RNA phase flow rates, ethanolic lipid phase flow rates, aqueous phase flow rates, lipid concentrations, and RNA concentrations in response-phase designed experiments (n=53) (D). Particle mean size (A), PDI (B), and RNA encapsulation efficiency (C) were analyzed for each formulation. RSMs were constructed for each parameter. Mean ± SEM (n=3) of replicate analyses is shown. [Figure 19A]Increasing the poly(I:C) (pIC) / lipid weight ratio decreases the scattering intensity of anionic liposomes (AL) in 100% DO (A) and increases the scattered neutron density within the shell (B). The scattering patterns of pIC encapsulated in nanoparticles and pIC added externally to preformed empty nanoparticles are different (C). When pIC is added externally to preformed empty anionic liposomes and incubated, there is an increase in scattering at low Q (0.1-1 Å), indicating an increase in background. The increase in background may be due to more hydrogen in the DO solvent after the addition of pIC, suggesting that pIC is localized in the solvent rather than the nanoparticles. No clear increase in background is observed in the case of nanoparticles with encapsulated pIC, suggesting that pIC is localized in the nanoparticles rather than the solvent. Comparison of the scattering patterns of anionic liposomes (AL) and LNPs before and after pIC encapsulation (empty nanoparticles) (D). Encapsulation of pIC into LNPs, unlike anionic liposomes, modified the scattering profile, suggesting a rearrangement of the internal LNP structure. The rearrangement observed in LNPs can be explained by electrostatic interactions between the ionizable cationic lipids and anionic pIC, which does not occur in pIC-AL. In addition, pIC-AL has a distinctly different structure compared to pIC-LNP. Symbols represent SANS data, and solid lines correspond to the best fit using a spherical core-shell model (A), (B), and (D). [Figure 19B]Increasing the poly(I:C) (pIC) / lipid weight ratio decreases the scattering intensity of anionic liposomes (AL) in 100% DO (A) and increases the scattered neutron density within the shell (B). The scattering patterns of pIC encapsulated in nanoparticles and pIC added externally to preformed empty nanoparticles are different (C). When pIC is added externally to preformed empty anionic liposomes and incubated, there is an increase in scattering at low Q (0.1-1 Å), indicating an increase in background. The increase in background may be due to more hydrogen in the DO solvent after the addition of pIC, suggesting that pIC is localized in the solvent rather than the nanoparticles. No clear increase in background is observed in the case of nanoparticles with encapsulated pIC, suggesting that pIC is localized in the nanoparticles rather than the solvent. Comparison of the scattering patterns of anionic liposomes (AL) and LNPs before and after pIC encapsulation (empty nanoparticles) (D). Encapsulation of pIC into LNPs, unlike anionic liposomes, modified the scattering profile, suggesting a rearrangement of the internal LNP structure. The rearrangement observed in LNPs can be explained by electrostatic interactions between the ionizable cationic lipids and anionic pIC, which does not occur in pIC-AL. In addition, pIC-AL has a distinctly different structure compared to pIC-LNP. Symbols represent SANS data, and solid lines correspond to the best fit using a spherical core-shell model (A), (B), and (D). [Figure 19C]Increasing the poly(I:C) (pIC) / lipid weight ratio decreases the scattering intensity of anionic liposomes (AL) in 100% DO (A) and increases the scattered neutron density within the shell (B). The scattering patterns of pIC encapsulated in nanoparticles and pIC added externally to preformed empty nanoparticles are different (C). When pIC is added externally to preformed empty anionic liposomes and incubated, there is an increase in scattering at low Q (0.1-1 Å), indicating an increase in background. The increase in background may be due to more hydrogen in the DO solvent after the addition of pIC, suggesting that pIC is localized in the solvent rather than the nanoparticles. No clear increase in background is observed in the case of nanoparticles with encapsulated pIC, suggesting that pIC is localized in the nanoparticles rather than the solvent. Comparison of the scattering patterns of anionic liposomes (AL) and LNPs before and after pIC encapsulation (empty nanoparticles) (D). Encapsulation of pIC into LNPs, unlike anionic liposomes, modified the scattering profile, suggesting a rearrangement of the internal LNP structure. The rearrangement observed in LNPs can be explained by electrostatic interactions between the ionizable cationic lipids and anionic pIC, which does not occur in pIC-AL. In addition, pIC-AL has a distinctly different structure compared to pIC-LNP. Symbols represent SANS data, and solid lines correspond to the best fit using a spherical core-shell model (A), (B), and (D). [Figure 19D]Increasing the poly(I:C) (pIC) / lipid weight ratio decreases the scattering intensity of anionic liposomes (AL) in 100% DO (A) and increases the scattered neutron density within the shell (B). The scattering patterns of pIC encapsulated in nanoparticles and pIC added externally to preformed empty nanoparticles are different (C). When pIC is added externally to preformed empty anionic liposomes and incubated, there is an increase in scattering at low Q (0.1-1 Å), indicating an increase in background. The increase in background may be due to more hydrogen in the DO solvent after the addition of pIC, suggesting that pIC is localized in the solvent rather than the nanoparticles. No clear increase in background is observed in the case of nanoparticles with encapsulated pIC, suggesting that pIC is localized in the nanoparticles rather than the solvent. Comparison of the scattering patterns of anionic liposomes (AL) and LNPs before and after pIC encapsulation (empty nanoparticles) (D). Encapsulation of pIC into LNPs, unlike anionic liposomes, modified the scattering profile, suggesting a rearrangement of the internal LNP structure. The rearrangement observed in LNPs can be explained by electrostatic interactions between the ionizable cationic lipids and anionic pIC, which does not occur in pIC-AL. In addition, pIC-AL has a distinctly different structure compared to pIC-LNP. Symbols represent SANS data, and solid lines correspond to the best fit using a spherical core-shell model (A), (B), and (D). [Figure 20A]The strength of the interaction between RNA incubated with preformed empty DMPC vesicles and RNA encapsulated in DMPC vesicles was examined using asymmetric field-flow fractionation (AF4). RNA encapsulation allows for a strong interaction between RNA and DMPC vesicles, but the interaction between RNA incubated with DMPC vesicles is weak and reversible. DMPC vesicles and / or poly(I:C) (pIC) were incubated with RNase-free water. Afterward, RiboGreen dye was added for fluorescent detection of RNA, and the samples were analyzed by AF4. RNA was determined using a fluorescence detector (λem = 495 nm, λex = 520 nm) (left axis), and particle light scattering intensity was determined using MALS (right axis). (A) Soluble pIC, (B) preformed empty DMPC vesicles, (C) preformed empty DMPC vesicles incubated with exogenously added RNA, and (D) DMPC vesicles with encapsulated RNA are shown. The melting temperature of DMPC vesicles was obtained by differential scanning calorimetry and showed a significant decrease in melting temperature only when RNA was encapsulated in DMPC vesicles, suggesting interaction of the RNA within the hydrophobic regions of the DMPC vesicles. n=3 replicate analyses. [Figure 20B]The strength of the interaction between RNA incubated with preformed empty DMPC vesicles and RNA encapsulated in DMPC vesicles was examined using asymmetric field-flow fractionation (AF4). RNA encapsulation allows for a strong interaction between RNA and DMPC vesicles, but the interaction between RNA incubated with DMPC vesicles is weak and reversible. DMPC vesicles and / or poly(I:C) (pIC) were incubated with RNase-free water. Afterward, RiboGreen dye was added for fluorescent detection of RNA, and the samples were analyzed by AF4. RNA was determined using a fluorescence detector (λem = 495 nm, λex = 520 nm) (left axis), and particle light scattering intensity was determined using MALS (right axis). (A) Soluble pIC, (B) preformed empty DMPC vesicles, (C) preformed empty DMPC vesicles incubated with exogenously added RNA, and (D) DMPC vesicles with encapsulated RNA are shown. The melting temperature of DMPC vesicles was obtained by differential scanning calorimetry and showed a significant decrease in melting temperature only when RNA was encapsulated in DMPC vesicles, suggesting interaction of the RNA within the hydrophobic regions of the DMPC vesicles. n=3 replicate analyses. [Figure 20C]The strength of the interaction between RNA incubated with preformed empty DMPC vesicles and RNA encapsulated in DMPC vesicles was examined using asymmetric field-flow fractionation (AF4). RNA encapsulation allows for a strong interaction between RNA and DMPC vesicles, but the interaction between RNA incubated with DMPC vesicles is weak and reversible. DMPC vesicles and / or poly(I:C) (pIC) were incubated with RNase-free water. Afterward, RiboGreen dye was added for fluorescent detection of RNA, and the samples were analyzed by AF4. RNA was determined using a fluorescence detector (λem = 495 nm, λex = 520 nm) (left axis), and particle light scattering intensity was determined using MALS (right axis). (A) Soluble pIC, (B) preformed empty DMPC vesicles, (C) preformed empty DMPC vesicles incubated with exogenously added RNA, and (D) DMPC vesicles with encapsulated RNA are shown. The melting temperature of DMPC vesicles was obtained by differential scanning calorimetry and showed a significant decrease in melting temperature only when RNA was encapsulated in DMPC vesicles, suggesting interaction of the RNA within the hydrophobic regions of the DMPC vesicles. n=3 replicate analyses. [Figure 20D]The strength of the interaction between RNA incubated with preformed empty DMPC vesicles and RNA encapsulated in DMPC vesicles was examined using asymmetric field-flow fractionation (AF4). RNA encapsulation allows for a strong interaction between RNA and DMPC vesicles, but the interaction between RNA incubated with DMPC vesicles is weak and reversible. DMPC vesicles and / or poly(I:C) (pIC) were incubated with RNase-free water. Afterward, RiboGreen dye was added for fluorescent detection of RNA, and the samples were analyzed by AF4. RNA was determined using a fluorescence detector (λem = 495 nm, λex = 520 nm) (left axis), and particle light scattering intensity was determined using MALS (right axis). (A) Soluble pIC, (B) preformed empty DMPC vesicles, (C) preformed empty DMPC vesicles incubated with exogenously added RNA, and (D) DMPC vesicles with encapsulated RNA are shown. The melting temperature of DMPC vesicles was obtained by differential scanning calorimetry and showed a significant decrease in melting temperature only when RNA was encapsulated in DMPC vesicles, suggesting interaction of the RNA within the hydrophobic regions of the DMPC vesicles. n=3 replicate analyses. [Figure 20E]The strength of the interaction between RNA incubated with preformed empty DMPC vesicles and RNA encapsulated in DMPC vesicles was examined using asymmetric field-flow fractionation (AF4). RNA encapsulation allows for a strong interaction between RNA and DMPC vesicles, but the interaction between RNA incubated with DMPC vesicles is weak and reversible. DMPC vesicles and / or poly(I:C) (pIC) were incubated with RNase-free water. Afterward, RiboGreen dye was added for fluorescent detection of RNA, and the samples were analyzed by AF4. RNA was determined using a fluorescence detector (λem = 495 nm, λex = 520 nm) (left axis), and particle light scattering intensity was determined using MALS (right axis). (A) Soluble pIC, (B) preformed empty DMPC vesicles, (C) preformed empty DMPC vesicles incubated with exogenously added RNA, and (D) DMPC vesicles with encapsulated RNA are shown. The melting temperature of DMPC vesicles was obtained by differential scanning calorimetry and showed a significant decrease in melting temperature only when RNA was encapsulated in DMPC vesicles, suggesting interaction of the RNA within the hydrophobic regions of the DMPC vesicles. n=3 replicate analyses. [Figure 21A]Anionic liposomes containing poly(I:C) (pIC-AL) showed potent antitumor effects when administered intravenously in a colon cancer model, both as monotherapy and in combination with an a-PD-L1 antibody. Mice were subcutaneously inoculated with 5x105 MC38 cells into the right flank and then implanted with murine colon cancer syngeneic tumors. Seven days after inoculation, when tumor size reached an average of approximately 100mm3, mice were randomized into different groups and treatment began (day 0). Mice were treated intravenously with pIC-AL (10μg) or PBS (control) on days 0, 3, 6, 9, and 12 after the start of treatment, and intraperitoneally with a-PD-L1 (100μg) or a rat IgG antibody (100μg) (control) on days 1, 4, 7, 10, and 13 after the start of treatment. (A) Average tumor growth curves from the start of treatment (day 0) to day 7, when the first mouse reached a tumor size >1500 mm3 (humane endpoint (HEP)). Mean ± SEM (n = 8) is shown. (B) Survival curves of treated mice over time, from day 0 to day 60 after the start of treatment. (C) Cured mice (n = 3) and naive control mice (n = 3) were re-challenged with 5 × 105 MC38 cells subcutaneously in the left flank on day 60 after the start of treatment. Average tumor growth curves from the MC38 cell re-challenge until all control mice reached tumor size HEP. Mean ± SEM is shown. (D) Characterization of immune cells within the colon cancer tumor microenvironment after treatment with pIC-AL. Mice were implanted with murine colon cancer syngeneic tumors after inoculation of 5 × 105 MC38-CEA cells into the subcutaneous mammary fat pad. Six days after inoculation, when tumor size reached an average of approximately 100 mm3, mice were randomized into different groups and treatment was initiated. Mice were intravenously treated with pIC-AL (10 μg) or PBS (control) on days 0 and 3 after the start of treatment. Five days after the start of treatment, tumors were harvested, and single-cell suspensions were used for flow cytometry staining and analysis of tumor-infiltrating lymphocytes (TILs) and myeloid-derived immune suppressor cells (MSDCs). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 21B]Anionic liposomes containing poly(I:C) (pIC-AL) showed potent antitumor effects when administered intravenously in a colon cancer model, both as monotherapy and in combination with an a-PD-L1 antibody. Mice were subcutaneously inoculated with 5x105 MC38 cells into the right flank and then implanted with murine colon cancer syngeneic tumors. Seven days after inoculation, when tumor size reached an average of approximately 100mm3, mice were randomized into different groups and treatment began (day 0). Mice were treated intravenously with pIC-AL (10μg) or PBS (control) on days 0, 3, 6, 9, and 12 after the start of treatment, and intraperitoneally with a-PD-L1 (100μg) or a rat IgG antibody (100μg) (control) on days 1, 4, 7, 10, and 13 after the start of treatment. (A) Average tumor growth curves from the start of treatment (day 0) to day 7, when the first mouse reached a tumor size >1500 mm3 (humane endpoint (HEP)). Mean ± SEM (n = 8) is shown. (B) Survival curves of treated mice over time, from day 0 to day 60 after the start of treatment. (C) Cured mice (n = 3) and naive control mice (n = 3) were re-challenged with 5 × 105 MC38 cells subcutaneously in the left flank on day 60 after the start of treatment. Average tumor growth curves from the MC38 cell re-challenge until all control mice reached tumor size HEP. Mean ± SEM is shown. (D) Characterization of immune cells within the colon cancer tumor microenvironment after treatment with pIC-AL. Mice were implanted with murine colon cancer syngeneic tumors after inoculation of 5 × 105 MC38-CEA cells into the subcutaneous mammary fat pad. Six days after inoculation, when tumor size reached an average of approximately 100 mm3, mice were randomized into different groups and treatment was initiated. Mice were intravenously treated with pIC-AL (10 μg) or PBS (control) on days 0 and 3 after the start of treatment. Five days after the start of treatment, tumors were harvested, and single-cell suspensions were used for flow cytometry staining and analysis of tumor-infiltrating lymphocytes (TILs) and myeloid-derived immune suppressor cells (MSDCs). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 21C]Anionic liposomes containing poly(I:C) (pIC-AL) showed potent antitumor effects when administered intravenously in a colon cancer model, both as monotherapy and in combination with an a-PD-L1 antibody. Mice were subcutaneously inoculated with 5x105 MC38 cells into the right flank and then implanted with murine colon cancer syngeneic tumors. Seven days after inoculation, when tumor size reached an average of approximately 100mm3, mice were randomized into different groups and treatment began (day 0). Mice were treated intravenously with pIC-AL (10μg) or PBS (control) on days 0, 3, 6, 9, and 12 after the start of treatment, and intraperitoneally with a-PD-L1 (100μg) or a rat IgG antibody (100μg) (control) on days 1, 4, 7, 10, and 13 after the start of treatment. (A) Average tumor growth curves from the start of treatment (day 0) to day 7, when the first mouse reached a tumor size >1500 mm3 (humane endpoint (HEP)). Mean ± SEM (n = 8) is shown. (B) Survival curves of treated mice over time, from day 0 to day 60 after the start of treatment. (C) Cured mice (n = 3) and naive control mice (n = 3) were re-challenged with 5 × 105 MC38 cells subcutaneously in the left flank on day 60 after the start of treatment. Average tumor growth curves from the MC38 cell re-challenge until all control mice reached tumor size HEP. Mean ± SEM is shown. (D) Characterization of immune cells within the colon cancer tumor microenvironment after treatment with pIC-AL. Mice were implanted with murine colon cancer syngeneic tumors after inoculation of 5 × 105 MC38-CEA cells into the subcutaneous mammary fat pad. Six days after inoculation, when tumor size reached an average of approximately 100 mm3, mice were randomized into different groups and treatment was initiated. Mice were intravenously treated with pIC-AL (10 μg) or PBS (control) on days 0 and 3 after the start of treatment. Five days after the start of treatment, tumors were harvested, and single-cell suspensions were used for flow cytometry staining and analysis of tumor-infiltrating lymphocytes (TILs) and myeloid-derived immune suppressor cells (MSDCs). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 21D]Anionic liposomes containing poly(I:C) (pIC-AL) showed potent antitumor effects when administered intravenously in a colon cancer model, both as monotherapy and in combination with an a-PD-L1 antibody. Mice were subcutaneously inoculated with 5x105 MC38 cells into the right flank and then implanted with murine colon cancer syngeneic tumors. Seven days after inoculation, when tumor size reached an average of approximately 100mm3, mice were randomized into different groups and treatment began (day 0). Mice were treated intravenously with pIC-AL (10μg) or PBS (control) on days 0, 3, 6, 9, and 12 after the start of treatment, and intraperitoneally with a-PD-L1 (100μg) or a rat IgG antibody (100μg) (control) on days 1, 4, 7, 10, and 13 after the start of treatment. (A) Average tumor growth curves from the start of treatment (day 0) to day 7, when the first mouse reached a tumor size >1500 mm3 (humane endpoint (HEP)). Mean ± SEM (n = 8) is shown. (B) Survival curves of treated mice over time, from day 0 to day 60 after the start of treatment. (C) Cured mice (n = 3) and naive control mice (n = 3) were re-challenged with 5 × 105 MC38 cells subcutaneously in the left flank on day 60 after the start of treatment. Average tumor growth curves from the MC38 cell re-challenge until all control mice reached tumor size HEP. Mean ± SEM is shown. (D) Characterization of immune cells within the colon cancer tumor microenvironment after treatment with pIC-AL. Mice were implanted with murine colon cancer syngeneic tumors after inoculation of 5 × 105 MC38-CEA cells into the subcutaneous mammary fat pad. Six days after inoculation, when tumor size reached an average of approximately 100 mm3, mice were randomized into different groups and treatment was initiated. Mice were intravenously treated with pIC-AL (10 μg) or PBS (control) on days 0 and 3 after the start of treatment. Five days after the start of treatment, tumors were harvested, and single-cell suspensions were used for flow cytometry staining and analysis of tumor-infiltrating lymphocytes (TILs) and myeloid-derived immune suppressor cells (MSDCs). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 22A]Anionic liposomes containing poly(I:C) (pIC-AL) have potent antitumor effects as monotherapy in an orthotopic liver cancer model. Hepatocellular carcinoma tumor cells (Hepa1-6: 5 x 10 cells / mouse) in 100 μl of PBS were injected into the mouse liver via the mesenteric vein. Tumor growth was monitored weekly by MRI imaging. Mice with detectable tumors (n = 5) were divided into two groups: 1) control (n = 2) and 10 μg pIC-AL (n = 3). Treatment (PBS or pIC-AL) was administered twice weekly for 4 weeks or until tumor size reached HEP. Tumor growth was tracked up to 8 weeks after treatment initiation until the humane endpoint was reached. Representative MRI images of PBS-treated mice taken 1 day before treatment initiation (A) and 13 days after treatment initiation (B) are shown. C–E show representative MRI images of mice treated with pIC-AL 1 day before the start of treatment (C), 13 days after the start of treatment (D), and 27 days after the start of treatment (E). [Figure 22B] Anionic liposomes containing poly(I:C) (pIC-AL) have potent antitumor effects as monotherapy in an orthotopic liver cancer model. Hepatocellular carcinoma tumor cells (Hepa1-6: 5 x 10 cells / mouse) in 100 μl of PBS were injected into the mouse liver via the mesenteric vein. Tumor growth was monitored weekly by MRI imaging. Mice with detectable tumors (n = 5) were divided into two groups: 1) control (n = 2) and 10 μg pIC-AL (n = 3). Treatment (PBS or pIC-AL) was administered twice weekly for 4 weeks or until tumor size reached HEP. Tumor growth was tracked up to 8 weeks after treatment initiation until the humane endpoint was reached. Representative MRI images of PBS-treated mice taken 1 day before treatment initiation (A) and 13 days after treatment initiation (B) are shown. C–E show representative MRI images of mice treated with pIC-AL 1 day before the start of treatment (C), 13 days after the start of treatment (D), and 27 days after the start of treatment (E). [Figure 22C]Anionic liposomes containing poly(I:C) (pIC-AL) have potent antitumor effects as monotherapy in an orthotopic liver cancer model. Hepatocellular carcinoma tumor cells (Hepa1-6: 5 x 10 cells / mouse) in 100 μl of PBS were injected into the mouse liver via the mesenteric vein. Tumor growth was monitored weekly by MRI imaging. Mice with detectable tumors (n = 5) were divided into two groups: 1) control (n = 2) and 10 μg pIC-AL (n = 3). Treatment (PBS or pIC-AL) was administered twice weekly for 4 weeks or until tumor size reached HEP. Tumor growth was tracked up to 8 weeks after treatment initiation until the humane endpoint was reached. Representative MRI images of PBS-treated mice taken 1 day before treatment initiation (A) and 13 days after treatment initiation (B) are shown. C–E show representative MRI images of mice treated with pIC-AL 1 day before the start of treatment (C), 13 days after the start of treatment (D), and 27 days after the start of treatment (E). [Figure 22D] Anionic liposomes containing poly(I:C) (pIC-AL) have potent antitumor effects as monotherapy in an orthotopic liver cancer model. Hepatocellular carcinoma tumor cells (Hepa1-6: 5 x 10 cells / mouse) in 100 μl of PBS were injected into the mouse liver via the mesenteric vein. Tumor growth was monitored weekly by MRI imaging. Mice with detectable tumors (n = 5) were divided into two groups: 1) control (n = 2) and 10 μg pIC-AL (n = 3). Treatment (PBS or pIC-AL) was administered twice weekly for 4 weeks or until tumor size reached HEP. Tumor growth was tracked up to 8 weeks after treatment initiation until the humane endpoint was reached. Representative MRI images of PBS-treated mice taken 1 day before treatment initiation (A) and 13 days after treatment initiation (B) are shown. C–E show representative MRI images of mice treated with pIC-AL 1 day before the start of treatment (C), 13 days after the start of treatment (D), and 27 days after the start of treatment (E). [Figure 22E]Anionic liposomes containing poly(I:C) (pIC-AL) have potent antitumor effects as monotherapy in an orthotopic liver cancer model. Hepatocellular carcinoma tumor cells (Hepa1-6: 5 x 10 cells / mouse) in 100 μl of PBS were injected into the mouse liver via the mesenteric vein. Tumor growth was monitored weekly by MRI imaging. Mice with detectable tumors (n = 5) were divided into two groups: 1) control (n = 2) and 10 μg pIC-AL (n = 3). Treatment (PBS or pIC-AL) was administered twice weekly for 4 weeks or until tumor size reached HEP. Tumor growth was tracked up to 8 weeks after treatment initiation until the humane endpoint was reached. Representative MRI images of PBS-treated mice taken 1 day before treatment initiation (A) and 13 days after treatment initiation (B) are shown. C–E show representative MRI images of mice treated with pIC-AL 1 day before the start of treatment (C), 13 days after the start of treatment (D), and 27 days after the start of treatment (E). [Figure 23A]Poly(I:C)-containing anionic liposomes (pIC-AL) induce potent antitumor activity without the acute toxicity associated with pIC-LNP: Antitumor activity, acute toxicity, and cytokine analysis following intravenous administration of pIC-AL or pIC-LNP, both as monotherapy and in combination with an a-PD-L1 antibody. Mice were implanted with murine colon cancer syngeneic tumors after subcutaneous inoculation of 5 x 10 MC38 cells into the right flank. Seven days after inoculation, when tumor size averaged approximately 100 mm, mice were randomized into different groups and treatment was initiated. Mice were treated intravenously with pIC-AL (10 μg), pIC-LNP (10 μg), or PBS (control) on day 0, and intraperitoneally with a-PD-L1 (100 μg) or PBS (control) on day 1. (A) Mortality (%) and (B) body weight loss after 3 days of treatment initiation, and (C) mean tumor growth curves from day 0 to day 3 after treatment initiation are shown. Mean ± SEM (n = 6) for all groups except pIC-AL (n = 4) are shown. Intravenous injection of pIC-LNP, unlike pIC-AL, induces a dramatically potent pro-inflammatory cytokine response. Plasma concentrations (left Y-axis) and fold changes (or fold increases) compared to the PBS group (right Y-axis) for the following pro-inflammatory cytokines are shown 3 hours after administration of pIC-AL or pIC-LNP nanoparticles or PBS (n = 6 for PBS, n = 10 for pIC-AL, and n = 12 for pIC-LNP): IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I). a-PD-L1 was not administered at the time of blood collection 3 hours after the start of treatment. ns: not significant, *p<0.05, **p<0.01, ***p<0.005 and ****p<0.0001. [Figure 23B]Poly(I:C)-containing anionic liposomes (pIC-AL) induce potent antitumor activity without the acute toxicity associated with pIC-LNP: Antitumor activity, acute toxicity, and cytokine analysis following intravenous administration of pIC-AL or pIC-LNP, both as monotherapy and in combination with an a-PD-L1 antibody. Mice were implanted with murine colon cancer syngeneic tumors after subcutaneous inoculation of 5 x 10 MC38 cells into the right flank. Seven days after inoculation, when tumor size averaged approximately 100 mm, mice were randomized into different groups and treatment was initiated. Mice were treated intravenously with pIC-AL (10 μg), pIC-LNP (10 μg), or PBS (control) on day 0, and intraperitoneally with a-PD-L1 (100 μg) or PBS (control) on day 1. (A) Mortality (%) and (B) body weight loss after 3 days of treatment initiation, and (C) mean tumor growth curves from day 0 to day 3 after treatment initiation are shown. Mean ± SEM (n = 6) for all groups except pIC-AL (n = 4) are shown. Intravenous injection of pIC-LNP, unlike pIC-AL, induces a dramatically potent pro-inflammatory cytokine response. Plasma concentrations (left Y-axis) and fold changes (or fold increases) compared to the PBS group (right Y-axis) for the following pro-inflammatory cytokines are shown 3 hours after administration of pIC-AL or pIC-LNP nanoparticles or PBS (n = 6 for PBS, n = 10 for pIC-AL, and n = 12 for pIC-LNP): IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I). a-PD-L1 was not administered at the time of blood collection 3 hours after the start of treatment. ns: not significant, *p<0.05, **p<0.01, ***p<0.005 and ****p<0.0001. [Figure 23C]Poly(I:C)-containing anionic liposomes (pIC-AL) induce potent antitumor activity without the acute toxicity associated with pIC-LNP: Antitumor activity, acute toxicity, and cytokine analysis following intravenous administration of pIC-AL or pIC-LNP, both as monotherapy and in combination with an a-PD-L1 antibody. Mice were implanted with murine colon cancer syngeneic tumors after subcutaneous inoculation of 5 x 10 MC38 cells into the right flank. Seven days after inoculation, when tumor size averaged approximately 100 mm, mice were randomized into different groups and treatment was initiated. Mice were treated intravenously with pIC-AL (10 μg), pIC-LNP (10 μg), or PBS (control) on day 0, and intraperitoneally with a-PD-L1 (100 μg) or PBS (control) on day 1. (A) Mortality (%) and (B) body weight loss after 3 days of treatment initiation, and (C) mean tumor growth curves from day 0 to day 3 after treatment initiation are shown. Mean ± SEM (n = 6) for all groups except pIC-AL (n = 4) are shown. Intravenous injection of pIC-LNP, unlike pIC-AL, induces a dramatically potent pro-inflammatory cytokine response. Plasma concentrations (left Y-axis) and fold changes (or fold increases) compared to the PBS group (right Y-axis) for the following pro-inflammatory cytokines are shown 3 hours after administration of pIC-AL or pIC-LNP nanoparticles or PBS (n = 6 for PBS, n = 10 for pIC-AL, and n = 12 for pIC-LNP): IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I). a-PD-L1 was not administered at the time of blood collection 3 hours after the start of treatment. ns: not significant, *p<0.05, **p<0.01, ***p<0.005 and ****p<0.0001. [Figure 23D]Poly(I:C)-containing anionic liposomes (pIC-AL) induce potent antitumor activity without the acute toxicity associated with pIC-LNP: Antitumor activity, acute toxicity, and cytokine analysis following intravenous administration of pIC-AL or pIC-LNP, both as monotherapy and in combination with an a-PD-L1 antibody. Mice were implanted with murine colon cancer syngeneic tumors after subcutaneous inoculation of 5 x 10 MC38 cells into the right flank. Seven days after inoculation, when tumor size averaged approximately 100 mm, mice were randomized into different groups and treatment was initiated. Mice were treated intravenously with pIC-AL (10 μg), pIC-LNP (10 μg), or PBS (control) on day 0, and intraperitoneally with a-PD-L1 (100 μg) or PBS (control) on day 1. (A) Mortality (%) and (B) body weight loss after 3 days of treatment initiation, and (C) mean tumor growth curves from day 0 to day 3 after treatment initiation are shown. Mean ± SEM (n = 6) for all groups except pIC-AL (n = 4) are shown. Intravenous injection of pIC-LNP, unlike pIC-AL, induces a dramatically potent pro-inflammatory cytokine response. Plasma concentrations (left Y-axis) and fold changes (or fold increases) compared to the PBS group (right Y-axis) for the following pro-inflammatory cytokines are shown 3 hours after administration of pIC-AL or pIC-LNP nanoparticles or PBS (n = 6 for PBS, n = 10 for pIC-AL, and n = 12 for pIC-LNP): IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I). a-PD-L1 was not administered at the time of blood collection 3 hours after the start of treatment. ns: not significant, *p<0.05, **p<0.01, ***p<0.005 and ****p<0.0001. [Figure 23E]Poly(I:C)-containing anionic liposomes (pIC-AL) induce potent antitumor activity without the acute toxicity associated with pIC-LNP: Antitumor activity, acute toxicity, and cytokine analysis following intravenous administration of pIC-AL or pIC-LNP, both as monotherapy and in combination with an a-PD-L1 antibody. Mice were implanted with murine colon cancer syngeneic tumors after subcutaneous inoculation of 5 x 10 MC38 cells into the right flank. Seven days after inoculation, when tumor size averaged approximately 100 mm, mice were randomized into different groups and treatment was initiated. Mice were treated intravenously with pIC-AL (10 μg), pIC-LNP (10 μg), or PBS (control) on day 0, and intraperitoneally with a-PD-L1 (100 μg) or PBS (control) on day 1. (A) Mortality (%) and (B) body weight loss after 3 days of treatment initiation, and (C) mean tumor growth curves from day 0 to day 3 after treatment initiation are shown. Mean ± SEM (n = 6) for all groups except pIC-AL (n = 4) are shown. Intravenous injection of pIC-LNP, unlike pIC-AL, induces a dramatically potent pro-inflammatory cytokine response. Plasma concentrations (left Y-axis) and fold changes (or fold increases) compared to the PBS group (right Y-axis) for the following pro-inflammatory cytokines are shown 3 hours after administration of pIC-AL or pIC-LNP nanoparticles or PBS (n = 6 for PBS, n = 10 for pIC-AL, and n = 12 for pIC-LNP): IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I). a-PD-L1 was not administered at the time of blood collection 3 hours after the start of treatment. ns: not significant, *p<0.05, **p<0.01, ***p<0.005 and ****p<0.0001. [Figure 23F]Poly(I:C)-containing anionic liposomes (pIC-AL) induce potent antitumor activity without the acute toxicity associated with pIC-LNP: Antitumor activity, acute toxicity, and cytokine analysis following intravenous administration of pIC-AL or pIC-LNP, both as monotherapy and in combination with an a-PD-L1 antibody. Mice were implanted with murine colon cancer syngeneic tumors after subcutaneous inoculation of 5 x 10 MC38 cells into the right flank. Seven days after inoculation, when tumor size averaged approximately 100 mm, mice were randomized into different groups and treatment was initiated. Mice were treated intravenously with pIC-AL (10 μg), pIC-LNP (10 μg), or PBS (control) on day 0, and intraperitoneally with a-PD-L1 (100 μg) or PBS (control) on day 1. (A) Mortality (%) and (B) body weight loss after 3 days of treatment initiation, and (C) mean tumor growth curves from day 0 to day 3 after treatment initiation are shown. Mean ± SEM (n = 6) for all groups except pIC-AL (n = 4) are shown. Intravenous injection of pIC-LNP, unlike pIC-AL, induces a dramatically potent pro-inflammatory cytokine response. Plasma concentrations (left Y-axis) and fold changes (or fold increases) compared to the PBS group (right Y-axis) for the following pro-inflammatory cytokines are shown 3 hours after administration of pIC-AL or pIC-LNP nanoparticles or PBS (n = 6 for PBS, n = 10 for pIC-AL, and n = 12 for pIC-LNP): IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I). a-PD-L1 was not administered at the time of blood collection 3 hours after the start of treatment. ns: not significant, *p<0.05, **p<0.01, ***p<0.005 and ****p<0.0001. [Figure 23G]Poly(I:C)-containing anionic liposomes (pIC-AL) induce potent antitumor activity without the acute toxicity associated with pIC-LNP: Antitumor activity, acute toxicity, and cytokine analysis following intravenous administration of pIC-AL or pIC-LNP, both as monotherapy and in combination with an a-PD-L1 antibody. Mice were implanted with murine colon cancer syngeneic tumors after subcutaneous inoculation of 5 x 10 MC38 cells into the right flank. Seven days after inoculation, when tumor size averaged approximately 100 mm, mice were randomized into different groups and treatment was initiated. Mice were treated intravenously with pIC-AL (10 μg), pIC-LNP (10 μg), or PBS (control) on day 0, and intraperitoneally with a-PD-L1 (100 μg) or PBS (control) on day 1. (A) Mortality (%) and (B) body weight loss after 3 days of treatment initiation, and (C) mean tumor growth curves from day 0 to day 3 after treatment initiation are shown. Mean ± SEM (n = 6) for all groups except pIC-AL (n = 4) are shown. Intravenous injection of pIC-LNP, unlike pIC-AL, induces a dramatically potent pro-inflammatory cytokine response. Plasma concentrations (left Y-axis) and fold changes (or fold increases) compared to the PBS group (right Y-axis) for the following pro-inflammatory cytokines are shown 3 hours after administration of pIC-AL or pIC-LNP nanoparticles or PBS (n = 6 for PBS, n = 10 for pIC-AL, and n = 12 for pIC-LNP): IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I). a-PD-L1 was not administered at the time of blood collection 3 hours after the start of treatment. ns: not significant, *p<0.05, **p<0.01, ***p<0.005 and ****p<0.0001. [Figure 23H]Poly(I:C)-containing anionic liposomes (pIC-AL) induce potent antitumor activity without the acute toxicity associated with pIC-LNP: Antitumor activity, acute toxicity, and cytokine analysis following intravenous administration of pIC-AL or pIC-LNP, both as monotherapy and in combination with an a-PD-L1 antibody. Mice were implanted with murine colon cancer syngeneic tumors after subcutaneous inoculation of 5 x 10 MC38 cells into the right flank. Seven days after inoculation, when tumor size averaged approximately 100 mm, mice were randomized into different groups and treatment was initiated. Mice were treated intravenously with pIC-AL (10 μg), pIC-LNP (10 μg), or PBS (control) on day 0, and intraperitoneally with a-PD-L1 (100 μg) or PBS (control) on day 1. (A) Mortality (%) and (B) body weight loss after 3 days of treatment initiation, and (C) mean tumor growth curves from day 0 to day 3 after treatment initiation are shown. Mean ± SEM (n = 6) for all groups except pIC-AL (n = 4) are shown. Intravenous injection of pIC-LNP, unlike pIC-AL, induces a dramatically potent pro-inflammatory cytokine response. Plasma concentrations (left Y-axis) and fold changes (or fold increases) compared to the PBS group (right Y-axis) for the following pro-inflammatory cytokines are shown 3 hours after administration of pIC-AL or pIC-LNP nanoparticles or PBS (n = 6 for PBS, n = 10 for pIC-AL, and n = 12 for pIC-LNP): IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I). a-PD-L1 was not administered at the time of blood collection 3 hours after the start of treatment. ns: not significant, *p<0.05, **p<0.01, ***p<0.005 and ****p<0.0001. [Figure 23I]Poly(I:C)-containing anionic liposomes (pIC-AL) induce potent antitumor activity without the acute toxicity associated with pIC-LNP: Antitumor activity, acute toxicity, and cytokine analysis following intravenous administration of pIC-AL or pIC-LNP, both as monotherapy and in combination with an a-PD-L1 antibody. Mice were implanted with murine colon cancer syngeneic tumors after subcutaneous inoculation of 5 x 10 MC38 cells into the right flank. Seven days after inoculation, when tumor size averaged approximately 100 mm, mice were randomized into different groups and treatment was initiated. Mice were treated intravenously with pIC-AL (10 μg), pIC-LNP (10 μg), or PBS (control) on day 0, and intraperitoneally with a-PD-L1 (100 μg) or PBS (control) on day 1. (A) Mortality (%) and (B) body weight loss after 3 days of treatment initiation, and (C) mean tumor growth curves from day 0 to day 3 after treatment initiation are shown. Mean ± SEM (n = 6) for all groups except pIC-AL (n = 4) are shown. Intravenous injection of pIC-LNP, unlike pIC-AL, induces a dramatically potent pro-inflammatory cytokine response. Plasma concentrations (left Y-axis) and fold changes (or fold increases) compared to the PBS group (right Y-axis) for the following pro-inflammatory cytokines are shown 3 hours after administration of pIC-AL or pIC-LNP nanoparticles or PBS (n = 6 for PBS, n = 10 for pIC-AL, and n = 12 for pIC-LNP): IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I). a-PD-L1 was not administered at the time of blood collection 3 hours after the start of treatment. ns: not significant, *p<0.05, **p<0.01, ***p<0.005 and ****p<0.0001. [Figure 24A]Histopathological examination and plasma biochemical analysis demonstrated potent liver toxicity in mice treated with poly(I:C)-containing LNP (pIC-LNP), but not with anionic liposomes containing poly(I:C) (pIC-AL). Mice implanted with MC38 tumors (continued from Figure 23) were intravenously treated with pIC-AL (10 μg), pIC-LNP (10 μg), or PBS (control) at the start of treatment (day 0), and then intraperitoneally treated with a-PD-L1 (100 μg) or PBS (control) on day 1 after treatment initiation. Three days after treatment initiation, surviving mice were euthanized, and vital organs and blood samples were collected. Representative hematoxylin and eosin (H&E) staining of liver slides 3 days after treatment initiation is shown; 20x magnification. (A) Control, (B) pIC-AL, (C) pIC-AL + a-PD-L1 antibody, (D) pIC-LNP, and (E) pIC-LNP + a-PD-L1 antibody are shown, demonstrating that the strong liver damage in the pIC-LNP group was further exacerbated when a-PD-L1 antibody was administered one day later. No signs of liver toxicity were observed in mice treated with pIC-AL monotherapy or pIC-AL + a-PD-L1 antibody. (F) Mean enzyme activity (UI / L) ± SEM (F) of the liver enzyme AST. Elevated AST activity was observed only in mice treated with pIC-LNP (only surviving mice were determined in the acute hepatotoxicity analysis: n=6 in the PBS group, n=5 in the pIC-LNP group, n=2 in the pIC-LNP + a-PD-L1 group, n=4 in the pIC-AL group, and n=6 in the pIC-AL + a-PD-L1 group). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 24B]Histopathological examination and plasma biochemical analysis demonstrated potent liver toxicity in mice treated with poly(I:C)-containing LNP (pIC-LNP), but not with anionic liposomes containing poly(I:C) (pIC-AL). Mice implanted with MC38 tumors (continued from Figure 23) were intravenously treated with pIC-AL (10 μg), pIC-LNP (10 μg), or PBS (control) at the start of treatment (day 0), and then intraperitoneally treated with a-PD-L1 (100 μg) or PBS (control) on day 1 after treatment initiation. Three days after treatment initiation, surviving mice were euthanized, and vital organs and blood samples were collected. Representative hematoxylin and eosin (H&E) staining of liver slides 3 days after treatment initiation is shown; 20x magnification. (A) Control, (B) pIC-AL, (C) pIC-AL + a-PD-L1 antibody, (D) pIC-LNP, and (E) pIC-LNP + a-PD-L1 antibody are shown, demonstrating that the strong liver damage in the pIC-LNP group was further exacerbated when a-PD-L1 antibody was administered one day later. No signs of liver toxicity were observed in mice treated with pIC-AL monotherapy or pIC-AL + a-PD-L1 antibody. (F) Mean enzyme activity (UI / L) ± SEM (F) of the liver enzyme AST. Elevated AST activity was observed only in mice treated with pIC-LNP (only surviving mice were determined in the acute hepatotoxicity analysis: n=6 in the PBS group, n=5 in the pIC-LNP group, n=2 in the pIC-LNP + a-PD-L1 group, n=4 in the pIC-AL group, and n=6 in the pIC-AL + a-PD-L1 group). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 24C]Histopathological examination and plasma biochemical analysis demonstrated potent liver toxicity in mice treated with poly(I:C)-containing LNP (pIC-LNP), but not with anionic liposomes containing poly(I:C) (pIC-AL). Mice implanted with MC38 tumors (continued from Figure 23) were intravenously treated with pIC-AL (10 μg), pIC-LNP (10 μg), or PBS (control) at the start of treatment (day 0), and then intraperitoneally treated with a-PD-L1 (100 μg) or PBS (control) on day 1 after treatment initiation. Three days after treatment initiation, surviving mice were euthanized, and vital organs and blood samples were collected. Representative hematoxylin and eosin (H&E) staining of liver slides 3 days after treatment initiation is shown; 20x magnification. (A) Control, (B) pIC-AL, (C) pIC-AL + a-PD-L1 antibody, (D) pIC-LNP, and (E) pIC-LNP + a-PD-L1 antibody are shown, demonstrating that the strong liver damage in the pIC-LNP group was further exacerbated when a-PD-L1 antibody was administered one day later. No signs of liver toxicity were observed in mice treated with pIC-AL monotherapy or pIC-AL + a-PD-L1 antibody. (F) Mean enzyme activity (UI / L) ± SEM (F) of the liver enzyme AST. Elevated AST activity was observed only in mice treated with pIC-LNP (only surviving mice were determined in the acute hepatotoxicity analysis: n=6 in the PBS group, n=5 in the pIC-LNP group, n=2 in the pIC-LNP + a-PD-L1 group, n=4 in the pIC-AL group, and n=6 in the pIC-AL + a-PD-L1 group). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 24D]Histopathological examination and plasma biochemical analysis demonstrated potent liver toxicity in mice treated with poly(I:C)-containing LNP (pIC-LNP), but not with anionic liposomes containing poly(I:C) (pIC-AL). Mice implanted with MC38 tumors (continued from Figure 23) were intravenously treated with pIC-AL (10 μg), pIC-LNP (10 μg), or PBS (control) at the start of treatment (day 0), and then intraperitoneally treated with a-PD-L1 (100 μg) or PBS (control) on day 1 after treatment initiation. Three days after treatment initiation, surviving mice were euthanized, and vital organs and blood samples were collected. Representative hematoxylin and eosin (H&E) staining of liver slides 3 days after treatment initiation is shown; 20x magnification. (A) Control, (B) pIC-AL, (C) pIC-AL + a-PD-L1 antibody, (D) pIC-LNP, and (E) pIC-LNP + a-PD-L1 antibody are shown, demonstrating that the strong liver damage in the pIC-LNP group was further exacerbated when a-PD-L1 antibody was administered one day later. No signs of liver toxicity were observed in mice treated with pIC-AL monotherapy or pIC-AL + a-PD-L1 antibody. (F) Mean enzyme activity (UI / L) ± SEM (F) of the liver enzyme AST. Elevated AST activity was observed only in mice treated with pIC-LNP (only surviving mice were determined in the acute hepatotoxicity analysis: n=6 in the PBS group, n=5 in the pIC-LNP group, n=2 in the pIC-LNP + a-PD-L1 group, n=4 in the pIC-AL group, and n=6 in the pIC-AL + a-PD-L1 group). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 24E]Histopathological examination and plasma biochemical analysis demonstrated potent liver toxicity in mice treated with poly(I:C)-containing LNP (pIC-LNP), but not with anionic liposomes containing poly(I:C) (pIC-AL). Mice implanted with MC38 tumors (continued from Figure 23) were intravenously treated with pIC-AL (10 μg), pIC-LNP (10 μg), or PBS (control) at the start of treatment (day 0), and then intraperitoneally treated with a-PD-L1 (100 μg) or PBS (control) on day 1 after treatment initiation. Three days after treatment initiation, surviving mice were euthanized, and vital organs and blood samples were collected. Representative hematoxylin and eosin (H&E) staining of liver slides 3 days after treatment initiation is shown; 20x magnification. (A) Control, (B) pIC-AL, (C) pIC-AL + a-PD-L1 antibody, (D) pIC-LNP, and (E) pIC-LNP + a-PD-L1 antibody are shown, demonstrating that the strong liver damage in the pIC-LNP group was further exacerbated when a-PD-L1 antibody was administered one day later. No signs of liver toxicity were observed in mice treated with pIC-AL monotherapy or pIC-AL + a-PD-L1 antibody. (F) Mean enzyme activity (UI / L) ± SEM (F) of the liver enzyme AST. Elevated AST activity was observed only in mice treated with pIC-LNP (only surviving mice were determined in the acute hepatotoxicity analysis: n=6 in the PBS group, n=5 in the pIC-LNP group, n=2 in the pIC-LNP + a-PD-L1 group, n=4 in the pIC-AL group, and n=6 in the pIC-AL + a-PD-L1 group). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 24F]Histopathological examination and plasma biochemical analysis demonstrated potent liver toxicity in mice treated with poly(I:C)-containing LNP (pIC-LNP), but not with anionic liposomes containing poly(I:C) (pIC-AL). Mice implanted with MC38 tumors (continued from Figure 23) were intravenously treated with pIC-AL (10 μg), pIC-LNP (10 μg), or PBS (control) at the start of treatment (day 0), and then intraperitoneally treated with a-PD-L1 (100 μg) or PBS (control) on day 1 after treatment initiation. Three days after treatment initiation, surviving mice were euthanized, and vital organs and blood samples were collected. Representative hematoxylin and eosin (H&E) staining of liver slides 3 days after treatment initiation is shown; 20x magnification. (A) Control, (B) pIC-AL, (C) pIC-AL + a-PD-L1 antibody, (D) pIC-LNP, and (E) pIC-LNP + a-PD-L1 antibody are shown, demonstrating that the strong liver damage in the pIC-LNP group was further exacerbated when a-PD-L1 antibody was administered one day later. No signs of liver toxicity were observed in mice treated with pIC-AL monotherapy or pIC-AL + a-PD-L1 antibody. (F) Mean enzyme activity (UI / L) ± SEM (F) of the liver enzyme AST. Elevated AST activity was observed only in mice treated with pIC-LNP (only surviving mice were determined in the acute hepatotoxicity analysis: n=6 in the PBS group, n=5 in the pIC-LNP group, n=2 in the pIC-LNP + a-PD-L1 group, n=4 in the pIC-AL group, and n=6 in the pIC-AL + a-PD-L1 group). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 25A]Anionic liposomes containing non-immunogenic mRNA are not inflammatory and do not induce immune system activation. Mice received 10 μg of non-immunogenic mRNA encapsulated in anionic liposomes (mRNA-AL) or LNPs (mRNA-LNP) intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. 24 hours later, mice were sacrificed, spleens were harvested, and single-cell splenic suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensity (GMFI) ± SEM of the CD80 maturation marker expressed by cDC1 (A) and cDC2 (B), respectively, and the activation marker CD25 expressed by T cells, respectively, are shown (n = 2). Serum inflammatory cytokine concentrations as mean ± SEM (left Y-axis) and fold increase mean ± SEM (right Y-axis) of IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I) 3 hours after IV injection are shown (n=4). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 25B]Anionic liposomes containing non-immunogenic mRNA are not inflammatory and do not induce immune system activation. Mice received 10 μg of non-immunogenic mRNA encapsulated in anionic liposomes (mRNA-AL) or LNPs (mRNA-LNP) intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. 24 hours later, mice were sacrificed, spleens were harvested, and single-cell splenic suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensity (GMFI) ± SEM of the CD80 maturation marker expressed by cDC1 (A) and cDC2 (B), respectively, and the activation marker CD25 expressed by T cells, respectively, are shown (n = 2). Serum inflammatory cytokine concentrations as mean ± SEM (left Y-axis) and fold increase mean ± SEM (right Y-axis) of IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I) 3 hours after IV injection are shown (n=4). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 25C]Anionic liposomes containing non-immunogenic mRNA are not inflammatory and do not induce immune system activation. Mice received 10 μg of non-immunogenic mRNA encapsulated in anionic liposomes (mRNA-AL) or LNPs (mRNA-LNP) intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. 24 hours later, mice were sacrificed, spleens were harvested, and single-cell splenic suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensity (GMFI) ± SEM of the CD80 maturation marker expressed by cDC1 (A) and cDC2 (B), respectively, and the activation marker CD25 expressed by T cells, respectively, are shown (n = 2). Serum inflammatory cytokine concentrations as mean ± SEM (left Y-axis) and fold increase mean ± SEM (right Y-axis) of IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I) 3 hours after IV injection are shown (n=4). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 25D]Anionic liposomes containing non-immunogenic mRNA are not inflammatory and do not induce immune system activation. Mice received 10 μg of non-immunogenic mRNA encapsulated in anionic liposomes (mRNA-AL) or LNPs (mRNA-LNP) intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. 24 hours later, mice were sacrificed, spleens were harvested, and single-cell splenic suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensity (GMFI) ± SEM of the CD80 maturation marker expressed by cDC1 (A) and cDC2 (B), respectively, and the activation marker CD25 expressed by T cells, respectively, are shown (n = 2). Serum inflammatory cytokine concentrations as mean ± SEM (left Y-axis) and fold increase mean ± SEM (right Y-axis) of IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I) 3 hours after IV injection are shown (n=4). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 25E]Anionic liposomes containing non-immunogenic mRNA are not inflammatory and do not induce immune system activation. Mice received 10 μg of non-immunogenic mRNA encapsulated in anionic liposomes (mRNA-AL) or LNPs (mRNA-LNP) intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. 24 hours later, mice were sacrificed, spleens were harvested, and single-cell splenic suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensity (GMFI) ± SEM of the CD80 maturation marker expressed by cDC1 (A) and cDC2 (B), respectively, and the activation marker CD25 expressed by T cells, respectively, are shown (n = 2). Serum inflammatory cytokine concentrations as mean ± SEM (left Y-axis) and fold increase mean ± SEM (right Y-axis) of IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I) 3 hours after IV injection are shown (n=4). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 25F]Anionic liposomes containing non-immunogenic mRNA are not inflammatory and do not induce immune system activation. Mice received 10 μg of non-immunogenic mRNA encapsulated in anionic liposomes (mRNA-AL) or LNPs (mRNA-LNP) intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. 24 hours later, mice were sacrificed, spleens were harvested, and single-cell splenic suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensity (GMFI) ± SEM of the CD80 maturation marker expressed by cDC1 (A) and cDC2 (B), respectively, and the activation marker CD25 expressed by T cells, respectively, are shown (n = 2). Serum inflammatory cytokine concentrations as mean ± SEM (left Y-axis) and fold increase mean ± SEM (right Y-axis) of IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I) 3 hours after IV injection are shown (n=4). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 25G]Anionic liposomes containing non-immunogenic mRNA are not inflammatory and do not induce immune system activation. Mice received 10 μg of non-immunogenic mRNA encapsulated in anionic liposomes (mRNA-AL) or LNPs (mRNA-LNP) intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. 24 hours later, mice were sacrificed, spleens were harvested, and single-cell splenic suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensity (GMFI) ± SEM of the CD80 maturation marker expressed by cDC1 (A) and cDC2 (B), respectively, and the activation marker CD25 expressed by T cells, respectively, are shown (n = 2). Serum inflammatory cytokine concentrations as mean ± SEM (left Y-axis) and fold increase mean ± SEM (right Y-axis) of IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I) 3 hours after IV injection are shown (n=4). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 25H]Anionic liposomes containing non-immunogenic mRNA are not inflammatory and do not induce immune system activation. Mice received 10 μg of non-immunogenic mRNA encapsulated in anionic liposomes (mRNA-AL) or LNPs (mRNA-LNP) intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. 24 hours later, mice were sacrificed, spleens were harvested, and single-cell splenic suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensity (GMFI) ± SEM of the CD80 maturation marker expressed by cDC1 (A) and cDC2 (B), respectively, and the activation marker CD25 expressed by T cells, respectively, are shown (n = 2). Serum inflammatory cytokine concentrations as mean ± SEM (left Y-axis) and fold increase mean ± SEM (right Y-axis) of IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I) 3 hours after IV injection are shown (n=4). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001. [Figure 25I]Anionic liposomes containing non-immunogenic mRNA are not inflammatory and do not induce immune system activation. Mice received 10 μg of non-immunogenic mRNA encapsulated in anionic liposomes (mRNA-AL) or LNPs (mRNA-LNP) intravenously. Blood samples were collected 3 hours after IV injection for cytokine measurements. 24 hours later, mice were sacrificed, spleens were harvested, and single-cell splenic suspensions were used for flow cytometry staining and analysis. Representative geometric mean fluorescence intensity (GMFI) ± SEM of the CD80 maturation marker expressed by cDC1 (A) and cDC2 (B), respectively, and the activation marker CD25 expressed by T cells, respectively, are shown (n = 2). Serum inflammatory cytokine concentrations as mean ± SEM (left Y-axis) and fold increase mean ± SEM (right Y-axis) of IL-6 (D), INF-α (E), INF-β (F), INF-γ (G), TNF-α (H), and IL-1β (I) 3 hours after IV injection are shown (n=4). ns: not significant, *p<0.05, **p<0.01, ***p<0.005, and ****p<0.0001.

[0122] The following examples illustrate different embodiments of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0123] Experimental Section Materials and Methods Preparation of liposomes Liposomes containing poly(I:C) were prepared using single or double microfluidics micromixers, which enable lipid nanoassembly through flow-directed flow. A mixture of egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG) (Lipoid GmbH, Ludwigshafen, Germany), and cholesterol (Sigma-Aldrich, Darmstadt, Germany) was dissolved in ethanol. Lipid concentrations and molar ratios were tested during experimental design, resulting in a final lipid concentration of 5 mM and a 3:1:2 (EPC:EPG:cholesterol) molar ratio for in vivo experiments. Where indicated, the ethanol phase was supplemented with GM3 (3 mol%) (Avanti Polar, Alabaster, AL, USA) and / or 0.1 mol% lipophilic fluorescent tracer DiD (1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, Life Technologies, Frederick, MD, USA). Poly(I:C) (high molecular weight (HMW), InvivoGen, San Diego, USA) was dissolved in RNase-free water (Qiagen, Hilden, Germany). Lipids and poly(I:C) were mixed using a Dolomite microfluidics system (Royston, UK). Two system variants were explored: in design A, one large droplet junction chip (part number 3200130) for laminar flow or a micromixer chip (part number 3200401) for chaotic mixing was used and held within an interface H (part number 3000155). In design B, two large droplet junction chips (part number 3200130) were used, held, and connected by a double H interface (part number 3200088). In design A, two fluids (lipids dissolved in ethanol and RNA dissolved in water) were delivered to the inlet of the chip by two pressure pumps using 1.6 mm OD FEP tubing and a 4-way linear connector (part number 3000024).In design A, two fluids (lipids dissolved in ethanol and RNA dissolved in water) were delivered to the chip inlet by two pressure pumps using 1.6 mm OD FEP tubing and a 4-way linear connector (part number 3000024). In design B, three fluids (lipids dissolved in ethanol, RNA dissolved in water, and an additional water stream in the current chip) were delivered to the chip inlet by three pressure pumps using 1.6 mm OD FEP tubing and a 4-way linear connector (part number 3000024). The total flow rate was controlled using two (design A) or three (design B) Mitos flow sensors (part numbers 32000097 and 3200096). After microfluidic mixing, the sample was recollected and further diluted with RNase-free water to reach an ethanol concentration lower than 10%. Next, the sample was concentrated using a Vivaspin 20 centrifugal concentrator (300 kDa MWCO) (Sartorius Stedim Biotech GmbH, Gottingen, Germany) with a spin at 1000 g. Once concentrated (approximately 200 μL), the sample was diluted to 200 mL with RNase-free water and further concentrated using a Vivaspin 20 centrifugal concentrator (300 kDa MWCO). Finally, the sample was resuspended in sterile PBS (Sigma Aldrich, Darmstadt, Germany).

[0124] For SANS experiments, synthetic anionic liposomes were prepared using commercially available deuterated versions of DPPC, POPC, DPPG, POPG (Lipoid), and cholesterol (Sigma-Aldrich). DPPC and POPC are the major saturated and unsaturated lipid components of egg PC, while DPPG and POPG are the major saturated and unsaturated lipid components of egg PG. In some samples, non-deuterated lipids were replaced with deuterated versions: DPPC-d62 (860355P), POPC-d31 (860399P), DPPG-d62 (860355P), POPG-d31 (860384P), and cholesterol-d7 (700041P) (Avanti Polar). Lipids were dissolved in ethanol at 5 mM lipid concentration and a 3:3:1:1:4 molar ratio (DPPC:POPC:DPPG:POPG:cholesterol) and mixed with a 500 μg / mL solution of poly(I:C) and subsequently DO at a flow ratio of 1:3:9 utilizing the microfluidics configuration of Design B described previously.

[0125] For SANS experiments involving anionic liposomes with increasing RNA:lipid weight ratios (DPPC:POPC:DPPG:POPG:cholesterol), preparations were performed as follows: for 0% and 5% weight ratios, formulations were prepared as previously described; for 0% weight ratio, a 5 mM lipid ethanol solution was mixed with a 500 μg / mL poly(I:C) solution followed by the addition of DO at a flow ratio of 2:3:9; for 15% weight ratio, a 5 mM lipid ethanol solution was mixed with a 500 μg / mL poly(I:C) solution followed by the addition of DO at a flow ratio of 1:1:3; and for 30% weight ratio, a 5 mM lipid ethanol solution was mixed with a 1000 μg / mL poly(I:C) solution followed by the addition of DO at a flow ratio of 1:1:3 using the microfluidics setup of Design B described previously.

[0126] For DMPC DSC and AF4 experiments, DMPC was dissolved in ethanol at a 5 mM lipid concentration and mixed with a 500 μg / mL solution of poly(I:C) and later with water at a flow ratio of 3:3:9 utilizing the microfluidics configuration of Design B described previously.

[0127] For SANS and in vivo experiments, lipid nanoparticles (LNPs) containing poly(I:C) and Fluc mRNA (Ribopro, The Netherlands) were prepared with D-lin-MCR3-DMA (Medchem), cholesterol (Sigma), DSPC (Lipoid), and DMG-PEG2000 (Avanti Polar) in a molar ratio of 50:38.5:10:1.5. The lipids were dissolved in ethanol at a 5 mM lipid concentration and mixed with a 500 μg / mL solution of poly(I:C) and subsequently with DO (SANS) or water at a flow ratio of 1:3:9 using the microfluidics configuration of Design B described previously.

[0128] Liposome characterization The phosphate content of liposome preparations was determined via acid digestion according to Rouser et al. (Lipids 5, 494-496 (1970)). Dilutions of liposome samples and known amounts of 0.5 mM KH2PO4 standard solution were transferred to clean glass tubes, and the solvent was completely evaporated using a heat block at 220 °C. Next, 0.3 mL of perchloric acid was added to each tube, which was then placed in the heat block for 60 minutes or until the yellow color disappeared. Once cooled, 1 mL of water, 0.5 mL of molybdate solution, and then 0.5 mL of ascorbic acid solution were added and vortexed. The tubes were placed in a heated water bath for 5 minutes and then allowed to cool. The absorbance of the samples and standards was then measured at 797 nm using a spectrophotometer microplate reader (BMG SPECTROstar Nano, De Meern, The Netherlands).

[0129] Hydrodynamic diameter and polydispersity index (PDI) were measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano S (Malvern Instruments, Malvern, UK) equipped with a He-Ne 633 nm laser configured at a scattering angle of 173°. Measurements were performed using Malvern's Zetasizer v7.13 software (Malvern Instruments, Malvern, UK). A minimum of three measurements were performed per sample, and the measurement duration was automatically set by the software according to the sample characteristics. The software set a viscosity of 887.2 Pa·s (0.8872 centipoise (cP)) and a refractive index (RI) of 1.330 for the dispersion, and an RI of 0.1590 and an absorbance of 0.010 for the material in suspension. Samples were diluted in PBS and measured at 25°C. A PDI<0.3, indicating a homogeneous size distribution of sufficient homogeneity, was considered acceptable.

[0130] Zeta potential was determined using a Zetasizer Nano Z (Malvern Instruments, Malvern, UK) and Malvern's Zetasizer v7.13 software. A minimum of three measurements were performed per sample, and the measurement duration was automatically set by the software depending on the sample characteristics. Samples were diluted with 0.1× PBS and measured at 25°C. The software set a viscosity of 887.2 Pa·s (0.8872 cP), an RI of 1.330, and a dielectric constant of 78.5 for the dispersion, and an RI of 1.590 and an absorbance of 0.010 for the material in suspension.

[0131] Poly(I:C) content was determined using RiboGreen RNA quantification reagent (Jones et al. Anal Biochem 265, 368-374 (1998)) (Thermofisher). Quantification of poly(I:C) in liposome formulations was performed using a standard curve generated from a dilution series of the corresponding poly(I:C) stock solution. Both samples and standards were diluted with 0.5% Triton X-100 (Sigma) in RNase-free water (Qiagen). Samples were diluted to reach a theoretical concentration of 1 μg / mL. Samples were placed in a black polystyrene 96-well plate and diluted 1:1 with RiboGreen reagent (pre-diluted 1 / 200 in RNase-free water). Fluorescence was measured at λ excitation = 485 nm, λ emission The fluorescence intensity was measured using a Jasco FP-8300 fluorescence spectrophotometer set at λ = 520 nm. The concentration of the samples was obtained through a standard curve calculated by linear regression analysis of the fluorescence intensity plotted against the concentration of standard poly(I:C). The encapsulation efficiency was calculated by dividing the actual poly(I:C) concentration by the theoretical poly(I:C) concentration (total poly(I:C) added divided by the final formulation volume) as follows:

number

[0132] HEK-Blue(TM)-hTLR3 + TLR3 activation and liposome uptake in cells HEK-Blue(TM)-hTLR3 +A reporter cell line (Invitrogen) was stably transfected with the TLR3 gene and an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene linked to the NF-κB / AP-1 promoter. The HEK-Blue™-hTLR3 reporter cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma) with high glucose (4.5 g / L), L-glutamine (2 mM), and fetal bovine serum (FBS). To avoid nonspecific TLR3 activation due to the presence of RNA when supplementing the medium with FBS, cells were seeded in 96-well plates in DMEM without FBS (40,000 cells per well in 100 μL). After 24 hours, 22 ng of free or encapsulated poly(I:C) resuspended in 20 μL of PBS was added to the wells. PBS was used as a negative control. Additionally, 100 μL of QUANTI-Blue™ solution (Invitrogen, San Diego, USA) was added to quantify SEAP (final poly(I:C) concentration = 0.1 μg / mL). After 12 hours, SEAP activity (absorbance), representing TLR3 / NF-κB / AP-1 TLR3 activation, was measured at 650 nm using a Bio-Rad microplate reader (Hercules, California, USA). TLR3 activation was compared to free poly(I:C) using the following equation:

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[0133] For liposome uptake experiments, cells were seeded in DMEM without FBS in 96-well plates (40,000 cells in 100 μL per well). After 24 h, 22 ng of free or encapsulated poly(I:C) resuspended in 20 μL of PBS was added to the wells. Empty anionic liposomes (EPC:EPG:cholesterol 3:1:2) and PBS were used as controls. After 4 h, the cell culture medium was removed, and the cells were purified with 1% FBS in PBS, incubated with Zombie NIR viability dye (Biolegend, USA), and then fixed with 1% PFA. DiD geometric mean fluorescence intensity (GMFI) was then measured using a BD FACSCanto flow cytometer (BD, East Rutherford, NJ, USA), and data analysis was performed using Flowlogic software (Inivai, Mentone, Australia).

[0134] Molecular weight analysis of encapsulated RNA and plasma stability of anionic liposomes containing RNA. Separation of encapsulated and adsorbed RNA in DMPC vesicles using AF4. The molecular weight of encapsulated RNA and the plasma stability of RNA-containing anionic liposomes were analyzed using an AF2000 Asymmetrical Flow Field-Flow Fractionation System (Postnova, Landsberg am Lech, Germany). This system consists of a solvent degasser (Postnova, PN7520), focus and tip pumps (Postnova, PN1130), cross-flow pumps (Postnova, AF2000, AF2000-MF), an autosampler (Postnova, PN5300), an asymmetric AF4 channel (Postnova, PN4020), a multi-angle light scattering (MALS) detector (Postnova, PN3621), a radioisotope detector (Postnova, PN3150), a dual-wavelength absorbance detector (Waters, 2487), a multi-wavelength fluorescence detector (Waters, 2475), and a Zetasizer Nano ZS detector (Malvern).

[0135] Encapsulated poly(I:C) was extracted from anionic liposomes by precipitation with 100% isopropanol. The pure RNA pellet was resuspended in PBS and injected into a size-exclusion chromatography (SEC) column (TSKgel® G-DNA-PW) run in PBS (flow rate = 0.5 mL / min, 25 °C). In addition, free poly(I:C) and standard DNA Ruler (SM1331, Thermoscientific, USA) samples were similarly treated and injected into the SEC column. A UV detector at λ = 260 nm was used for RNA detection, and a multi-angle light scattering (MALS) detector was used for molecular weight determination using NovaFFF Suite software (Postnova).

[0136] The stability of empty and RNA-containing anionic liposomes in human plasma was tested using asymmetric field-flow fractionation (AF4). Liposomes (2 mM lipid) were incubated with PBS or 20% human plasma (Sera Laboratories, UK) for 2 h at 37 °C and then analyzed by AF4. The particle radius of gyration (Rg) and light scattering intensity were determined using MALS, and protein corona formation was determined using a fluorescence detector (λem = 280 nm, λex = 340 nm). A 350 μm spacer and a regenerated cellulose membrane with a 10 kDa molecular weight cutoff (Z-AF4-MEM-612-10 kDa, Postnova) were used in the AF4 channel. Samples were fractionated using a multi-step linear and power-field decay program. The detector flow was maintained at 0.5 mL / min. PBS was filtered through a 0.1 μm filter before use.

[0137] The strength of interaction between RNA incubated with preformed empty DMPC vesicles and RNA encapsulated in DMPC vesicles was tested using the previously described AF2000 asymmetric field-flow fractionation system (Postnova, Landsberg am Lech, Germany). DMPC vesicles and / or poly(I:C) (pIC) were incubated in RNase-free water. Afterwards, RiboGreen stain was added for fluorescent detection of RNA, and the samples were analyzed by AF4. RNA was detected using a fluorescence detector (λ em = 495 nm, λ ex = 520 nm), and the light scattering intensity of the particles was determined using MALS. In the AF4 channel, a 350 μm spacer and a regenerated cellulose membrane with a 10 kDa molecular weight cutoff (Z-AF4-MEM-612-10 kDa, Postnova) were used. Samples were fractionated using a multistage linear force field decay program. The detector flow was maintained at 0.5 mL / min. Purified water was filtered using a 0.1 μm filter before use. In this experiment, purified water was used instead of PBS to avoid aggregation of DMPC vesicles.

[0138] RNase protection assay The protection offered by anionic liposomes against nuclease degradation was evaluated using RNase III, which converts long double-stranded RNA into a heterogeneous mixture of short (18-25 bp) RNAs. Ten micrograms of free and encapsulated poly(I:C) were incubated with ShortCut® RNase III (M0245, New England Biolabs, USA) in reaction buffer or reaction buffer supplemented with human plasma (20% v / v) for 2 hours at 37°C according to the manufacturer's instructions. The samples were then separated by agarose gel electrophoresis (1%) and visualized with 5 μL of Midori Green (Nippon Genetics, Germany) stain per 100 mL of agarose. Additionally, 5 μL of standard DNA ruler (SM1331, Thermo Scientific, USA) was added to the first well of the agarose gel.

[0139] Transmission electron microscope measurements The size and shape of empty and RNA-containing anionic liposome particles were analyzed by taking transmission electron microscopy (TEM) photographs using a Tecnai12 TEM microscope (FEI Company, The Netherlands) operated at 80 kV. Ten microliters of the diluted particle suspension (2 mM phospholipid) was placed on a pre-glow-discharged Formvar / carbon-coated copper grid, contrasted with uranyl oxalate (pH 7), and then contrast-embedded in a mixture of 2% methylcellulose / 4% uranyl acetate (pH 4).

[0140] Small-angle neutron scattering measurements Small-angle neutron scattering (SANS) measurements were performed at the Larmor beamline of the ISIS pulsed neutron source at Rutherford Appleton Laboratory (Didcot, UK). Two types of anionic liposomes were prepared: a concentrated suspension of anionic liposomes was prepared as previously described, replacing RNase-free water with deuterium oxide (DO) (158122, Sigma-Aldrich). Empty and RNA-containing anionic liposomes (4 mg / mL concentration) were loaded into a 1 mm pathlength, 1 cm wide quartz Hellma cell and placed in a temperature-controlled sample holder maintained at 20 °C. A concentrated suspension of anionic liposomes was prepared as previously described, replacing RNase-free water with deuterium oxide (DO) (158122, Sigma-Aldrich). Prior to measurement, samples were resuspended in 100 / 0, 68 / 32, 50 / 50, and 27 / 63 DO / HO (% / %) water. Time-of-flight scattering measurements were collected for 30 min per sample. The wavelength and Q ranges for this experiment were 0.9–12.5 Å and 0.004–0.8 Å, respectively. Data were normalized to the sample transmittance and corrected for detection efficiency and scattering from empty cells. Data reduction was performed using Mantid and scattering simulations fitted using SasView v5.0.2 (www.sasview.org). All measurements were fitted using a spherical core-shell model.

[0141] DSC measurement DSC measurements were performed on a TA Instruments Discovery DSC (TA Instruments, Etten-Leur, The Netherlands). For poly(I:C) melting temperature analysis, samples were placed in DSC hermetic aluminum pans, conditioned at 10°C for 10 minutes, and then heated to 100°C at 0.5°C / min and then cooled. For DMPC vesicle melting temperature analysis, samples were placed in DSC hermetic aluminum pans. Three heating cycles (1°C / min) were performed, starting at 10°C and ending at 50°C, 90°C, and 50°C, respectively. Data were analyzed using Trios v3.3.0.4055 software.

[0142] Membrane fluidity measurement by GP Laurdan assay Empty anionic liposomes, anionic liposomes containing RNA, or empty liposomes plus free RNA (50 μM lipid, 2 μg / mL RNA) were incubated with 2.5 μM Laurdan in RNase-free water. The liposome suspension was transferred to a quartz cuvette and measured with an FP-8300 fluorescence spectrophotometer (Jasco) equipped with a temperature-controlled sample holder. The temperature was increased from 10 to 80°C. The temperature was set to 24 or 37°C. The sample was excited at 356 nm, and the emission intensity was recorded at 440 and 490 nm. The Laurdan general polarization (GP) was

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[0143] Animal testing: C57BL / 6 WT mice were obtained from Charles River or bred in-house. Male and female mice between 8 and 24 weeks of age were used for maturation studies. Female mice between 8 and 12 weeks of age were used for T cell priming experiments. Female mice between 8 and 10 weeks of age were used for tumor animal experiments.

[0144] Enzymatic spleen digestion C57BL / 6 wild-type (WT) spleens were mechanically dissociated and then digested with a mixture of 3 mg / ml lidocaine, 2 Wunsch units / mL Liberase TL (Roche, Mannheim, Germany), and 50 mg / mL DNase I (Roche) for 12 minutes with continuous agitation at 37°C

[38] . Cold medium (RPMI-1640 (Gibco, Life Technologies) supplemented with 10% heat-inactivated FCS (Biowest), 10 mM ethylenediaminetetraacetic acid (EDTA), 20 mM HEPES, and 50 μM 2-mercaptoethanol) was then added, and digestion continued for another 10 minutes with continuous agitation at 4°C. After digestion, red blood cells were lysed using ammonium chloride-potassium lysis buffer, and spleen cells were filtered through a 70-100 μm filter.

[0145] In vitro liposome uptake and dendritic cell maturation Enzyme-digested splenocytes were seeded and washed once. Free or encapsulated poly(I:C) in liposomes was diluted in PBS to 3 × 10 cells at the concentrations indicated. 6 The cells were added and incubated for 45 minutes at 37° C. DiD signals in various immune cell populations were assessed after flow cytometry staining as described below.

[0146] In vivo liposome uptake, dendritic cell maturation, serum cytokines and chemokines, and liver enzyme quantification Mice were injected IV with free or encapsulated poly(I:C) in anionic liposomes (as indicated in the figure). Blood samples were collected 3 hours after IV injection for cytokine and chemokine measurements. 24 hours later, mice were sacrificed, and blood was collected and allowed to clot. The clot was removed by centrifugation, and serum was used to quantify serum antiviral cytokines and chemokines, as well as liver aspartate transaminase (AST) and alanine transaminase (ALT), as described below. Additionally, spleens were collected, and single-cell suspensions were used for flow cytometry staining and analysis, as described below.

[0147] Antiviral cytokine and chemokine quantification Cytokine and chemokine secretion was analyzed using the LEGENDplex™ Mouse Antiviral Response Panel (13-plex) Multiplex Assay (740621, BioLegend®) in combination with a flow cytometer FACSCanto™ II (BD) according to the manufacturer's protocol.

[0148] Serum ALT and AST activity assays ALT activity was determined using an alanine aminotransferase kit (MAK052, Sigma Aldrich) in combination with an FP-8300 fluorescence spectrophotometer (Jasco) according to the manufacturer's instructions.

[0149] AST activity was determined by using an aspartate aminotransferase kit (MAK055, Sigma Aldrich) according to the manufacturer's instructions with a spectrophotometer microplate reader (BMG SPECTROstar Nano or Spectramax ID3 (Molecular Devices)).

[0150] In vivo T cell priming Mice were injected IV with free or encapsulated poly(I:C) in liposomes (as indicated). On day 7, mice were sacrificed and spleens were isolated. Blood was collected via cardiac puncture, if indicated. Spleens were mashed through a 70-100 μm filter to obtain a single-cell suspension. Red blood cells were lysed using ammonium chloride-potassium lysis buffer. Cells were then used for peptide restimulation and flow cytometry analysis.

[0151] Flow cytometry and antibodies for T cell priming experiments To block nonspecific Fc receptor binding, splenocytes were incubated with 10 μg / mL anti-CD16 / 32 (clone 2.4G2, produced in-house) for 15 min at 4°C and stained for 30 min with the following antibodies: anti-CD169 (clone SER-4, produced in-house), anti-B220 (clone RA3-6B2, BD Biosciences), anti-F4 / 80 (clone T45-2342, BD Biosciences), anti-CD8a (clone 53-6.7, BD Biosciences), anti-CD11c (clone HL3, BD Biosciences), anti-IA / IE (clone M5 / 114.15.2, BD Biosciences), and Fixable Viability Dye eFluor 780 (eBioscience, San Diego, CA, USA) diluted in PBS / 0.5% bovine serum albumin (BSA).

[0152] Alternatively, spleen cells were incubated with antibodies against anti-XCR1 (clone ZET, BioLegend, San Diego, CA, USA), anti-CD11c (clone HL3, BD Biosciences), anti-IA / IE (clone M5 / 114.15.2, BioLegend), anti-Ly6G (clone 1A8, BioLegend), anti-BST2 (clone 129C1, BioLegend), anti-CD11b (clone M1 / 70, BioLegend), anti-CD169 (clone SER-4, generated in-house), anti-Siglec-H (clone 551, BioLegend), anti-F4 / 80 (clone T45-2342, BD Biosciences), and anti-CD8a (clone 53-6.7, BD Biosciences). Biosciences), anti-Sirpα (clone P84, BioLegend); the lineage markers anti-CD3e (clone 145-2C11, BioLegend), anti-CD19 (clone 6D5, BioLegend), and anti-NK1.1 (clone PK136, BioLegend); and Fixable Viability Dye eFluor 780 (eBioscience) diluted in PBS / 0.5% BSA.

[0153] OVA-specific CD8+ T cells were identified by staining with anti-CD8a (clone 53-5.6, BD Biosciences), anti-CD44 (clone KM81, ImmunoTools, Friezuide, Germany), and PE-labeled H-2Kb / SIINFEKL tetramer (LUMC, Leiden, The Netherlands) for 60 minutes at 37°C. Spleen cells were restimulated with OVA257-264 for 5 hours or OVA262-276 for 20 hours in the presence of Golgiplug (BD Bioscience), followed by incubation with Golgiplug for 5 hours. Intracellular IFNγ was detected by staining with anti-CD11a (clone M17 / 4, eBioscience) and anti-CD8 (clone 53-5.6, BD Biosciences), or anti-CD4 (clone GK1.5, eBioscience). After surface staining, cells were fixed with 2% PFA (Electron Microscopy Sciences, Hatfield, PA, USA). For intracellular IFNγ detection, cells were permeabilized with 0.5% saponin buffer and stained with anti-IFNγ antibody (clone XMG1.2, eBioscience) for 30 min at 4°C.

[0154] Tumor-bearing animal experiments B16F10 model The B16F10 melanoma cell line was kindly provided by Dr. Ingrid Molema (University Medical Center Groningen (UMCG), The Netherlands). B16F10 cells were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum and dissociated when they reached 85% confluence.

[0155] 5 x 10 B16F10 tumor cells (in 100 μL of DMEM without FBS) 5were initially injected subcutaneously into the right flank of C57BL / 6 mice. Tumors were detected 5-7 days after implementation. Tumor size was monitored daily using a digital caliper and the following formula: volume (mm 3 )=(length x width 2 ) / 2. Seven days after tumor administration (day 0 of treatment), tumors were 100 mm 3 After tumors reached a mean tumor volume of 10 μg, 57 mice were randomized into six treatment groups: PBS / IgG (n=8), PBS / a-PD-L1 (n=10), free (I:C) (10 μg) / a-PD-L1 (n=9), free (I:C) (50 μg) / a-PD-L1 (n=10), and anionic liposome-encapsulated poly(I:C) (10 μg) / a-PD-L1 (n=10). PBS or free or encapsulated poly(I:C) was administered intravenously on days 0, 3, 6, 9, and 12 after treatment initiation. 100 μg of rat IgG2b isotype control (clone LTF-2, BioXcell, USA) or 100 μg of anti-mouse PD-L1 (clone 10F.9G2, BioXcell, USA) was administered intraperitoneally (ip) on days 1, 4, 7, 10, and 13 after treatment initiation. Mice were euthanized upon reaching the following humane endpoint criteria: passivity and other behavioral irregularities, ≥15% weight loss within 2 days, tumor size exceeding 1500 mm3, or tumor ulceration or infection. 3 Mice that reached a tumor size greater than 1000 served as the mortality endpoint for survival analysis. Tumor growth inhibition was

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[0156] MC38 model MC38 colon cancer cell line (catalog number ENH204-FP) was purchased from Kerafast (USA). MC38 cells were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich), and detached with EDTA / trypsin when they reached 85% confluence.

[0157] MC38 model: pIC-AL and ICI combination study: 5 x 10 MC38 tumor cells (in 100 μL of DMEM without FBS) 5 The cells were first injected subcutaneously into the right flank of female C57BL / 6 mice. Tumors were detected 5-7 days after the procedure. Tumor size was monitored daily using a digital caliper and the following formula: volume (mm 3 )=(length x width 2 ) / 2. Seven days after tumor administration (day 0 of treatment), tumors were 100 mm 3 A mean tumor volume of 1000 was reached.

[0158] Mice were randomized into four treatment groups: PBS / IgG (n=8), PBS / α-PD-L1 (n=8), anionic liposomal poly(I:C) (10μg) (pIC-AL) (n=8), and anionic liposomal poly(I:C) (10μg) (pIC-AL) / α-PD-L1 (n=8). PBS or encapsulated poly(I:C) was administered intravenously on days 0, 3, 6, 9, and 12 after treatment initiation. 100μg of rat IgG2b isotype control (clone LTF-2, BioXcell, USA) or 100μg of anti-mouse PD-L1 (clone 10F.9G2, BioXcell, USA) was administered intraperitoneally (i.p.) on days 1, 4, 7, 10, and 13 after treatment initiation. Mice were euthanized upon reaching the following predetermined humane endpoint criteria: passivity and other behavioral irregularities, weight loss of 15% or more within 2 days, tumor size exceeding 1500 mm3, or tumor ulceration or infection. 3Mice exceeding this age served as the mortality endpoint for survival analysis.

[0159] MC38 model: Comparison between pIC-AL and pIC-LNP 5 x 10 MC38 tumor cells (in 100 μL of DMEM without FBS) 5 The cells were first injected subcutaneously into the right flank of C57BL / 6 mice. Tumors were detected 5-7 days after the procedure. Tumor size was monitored daily using a digital caliper and the following formula: volume (mm 3 )=(length x width 2 ) / 2. Seven days after tumor administration (day 0 of treatment), tumors were 100 mm 3 A mean tumor volume of 1000 was reached.

[0160] Mice were randomized into five treatment groups: PBS / IgG (n=5), anionic liposomal poly(I:C) (10 μg) (pIC-AL) / IgG (n=4), LNP-encapsulated poly(I:C) (10 μg) (pIC-LNP) / IgG (n=6), anionic liposomal poly(I:C) (10 μg) (pIC-AL) / α-PD-L1 (n=6), and LNP-encapsulated poly(I:C) (10 μg) (RNA-LNP) / α-PD-L1 (n=6). PBS or encapsulated poly(I:C) was administered intravenously on day 0 after treatment initiation. Blood samples were collected 3 hours after IV injection for cytokine and chemokine measurements. One hundred micrograms of rat IgG2b isotype control (clone LTF-2, BioXcell, USA) or 100 μg of anti-mouse PD-L1 (clone 10F.9G2, BioXcell, USA) was administered intraperitoneally (i.p.) on day 1 after the start of treatment. One of six mice in the RNA-LNP group and four of six mice in the RNA-LNP / ICI group died in their cages due to high toxicity, so all mice were euthanized three days after the start of treatment. All mice in the RNA-LNP and RNA-LNP / ICI groups reached the predetermined humane endpoint criterion (≥15% weight loss within 2 days). After mice were killed by cervical dislocation, blood was collected by cardiac puncture and collected into EDTA tubes. The liver, spleen, kidneys, lungs, and heart were then collected, immediately frozen in liquid nitrogen, and stored at -80°C. Frozen organs were sectioned (5 μm) using a cryostat (Leica CM1950) and mounted on white gelatinized glass slides (StarFrost) coated with adhesive poly-l-lysine. Slides were fixed in formalin, stained with hematoxylin (Gills no. 2) and eosin Y solution (H&E staining), and analyzed using an Olympus BX50 microscope.

[0161] Flow cytometry and antibodies for MC38-CEA tumor microenvironment experiments MC38-CEA cells were harvested in DMEM-High Glutamax 1 with 10% FCS, 100 units penicillin / ml, 100 μg streptomycin, and 1% sodium pyruvate. Growth: 70%–90% confluent cultures were routinely detached using trypsin / EDTA. 1.0–106 MC38-CEA tumor cells in 100 μl of PBS were implanted into the left mammary fat pad of each mouse. Tumor growth was monitored by caliper measurement throughout the study. Treatment began on the same day after randomization on day 6. Anionic liposomes containing poly(I:C) (pIC-AL) were administered intravenously at a dose of 10 μg once on days 0 and 2 after the start of treatment (n=4) and compared with a PBS control (n=5), also given intravenously on days 0 and 2 after the start of treatment. Five days after the start of treatment, the study was terminated, all animals were euthanized, and necropsies were performed. During necropsies, tumor tissues were collected, and wet weights and tumor volumes were determined. In addition, a portion of each tumor tissue was processed for flow cytometry analysis. Primary tumor tissues were collected, and wet weights and tumor volumes were determined. 200-300 mg of tumor tissue samples were selected and processed for flow cytometry. Primary tumor material (approximately 200 mg) was disrupted using gentleMACS™ C tubes containing the enzyme mix from the Tumor Dissociation Kit according to the manufacturer's instructions (Miltenyi Biotec, Germany). Following this, red blood cells were removed with Red Blood Cell Lysis Solution (Miltenyi Biotec, Germany). Single-cell suspensions obtained from the tumors were counted, and, if possible, 3 × 10 cells were removed. 6Cells / well were distributed into a 96-well plate. Samples were incubated with 50 μl / well of Fc block (anti-mouse CD16 / CD32, 1:50) in FACS buffer for 15 minutes. Then, a 2x concentrated master antibody mix (fixable viability dye, CD3 (clone: ​​145-2C11, Biolegend), CD4 (clone: ​​GK1.5, BD Biosciences), CD8a (clone: ​​53-6.7, BD Biosciences), CD45 (clone: ​​30-F11, BD Biosciences), CD25 (clone: ​​PC61, BD Biosciences), CD11b (clone: ​​M1 / 70, Biolegend), Ly6C (AL-21, BD Biosciences), Ly6G (clone: ​​1A8, Biolegend), F4 / 80 (clone: ​​T45-2342, BD Biosciences), CD11c (clone: ​​HL3, BD Biosciences), MHC class II (clone: ​​M5 / 114.15.2, Biolegend), CD206 (MR5D3, BD Biosciences)) was added. CD335 (clone: ​​29A1.4, Biolegend), CD49b (clone: ​​HMα2, BD Biosciences), and B220 (clone: ​​RA3-6B2, BD Biosciences) were added to each well (50 μl) and incubated for 30 minutes in the dark. After washing, intracellular staining was primed by adding 100 μl of Fix / Perm buffer (3 parts Fix / Permeabilize Diluent to 1 part Fix / Permeabilize Concentrate) for 30 minutes. After centrifugation at 840 g, the cell pellet was resuspended in 1× Permeabilize Buffer containing anti-FoxP3 antibody and incubated for 30 minutes in the dark. After washing with 1× Permeabilize Buffer, the cells were washed with FACS buffer. The cells were resuspended in FACS buffer containing counting beads and kept at 4°C in the dark until analysis. Samples were analyzed by flow cytometry using an LSR Fortessa (Becton Dickinson).

[0162] Tumor-infiltrating leukocytes (TILs) were quantified as the sum of B cells (CD45+ CD11b- CD3- B220+), CD4+ T cells (CD45+, CD11b-, CD3+, CD4+, CD8-), and CD8 T cells (CD45+, CD11b-, CD3+, CD4-, CD8+). Myeloid-derived suppressor cells (MDSCs) were identified as the sum of monocyte-MDSCs (M-MDSCs) (CD45+, CD11b+, F4 / 80-, Ly6C+, Ly6G-) and granulocytes (CD45+, CD11b+, F4 / 80-, Ly6G+).

[0163] Orthotopic Hepa1-6 hepatocellular carcinoma (HCC) model The Hepa1-6 hepatocellular carcinoma (HCC) cell line was kindly provided by Dr. Piter Bosma (Tygat Institute for Liver and Intestinal Research, AMC, The Netherlands). Hepa1-6 cells were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and detached with EDTA / trypsin (Sigma-Aldrich) when they reached 85% confluence.

[0164] Female C57BL / 6 mice were used to develop an orthotopic HCC tumor model at the NKI in the Netherlands. The procedure was performed under isoflurane anesthesia (3-4% induction, 2% maintenance). A midline abdominal incision was made and the mesenteric vein was exposed. HCC tumor cells (Hepa 1-6: 5 × 10) in 100 μl of PBS were then injected. 6Cells (1000 cells / mouse) were injected into the mouse liver via the mesenteric vein. The injection site was covered with a cotton ball for 2-3 minutes to allow hemostasis before closing and suturing the abdominal cavity. Mice typically recovered immediately after surgery. Tumor growth was monitored weekly by MRI imaging. Mice (n = 5) that had detectable tumors for 4 weeks were divided into two groups: 1) control (n = 2) and 2) pIC-AL (10 μg) (n = 3). Treatment (PBS or pIC-AL) was administered twice weekly for 4 weeks. Tumor growth was tracked until a humane endpoint was reached, up to 8 weeks after the start of treatment.

[0165] MC38 model: Comparison of RNA-AL and mRNA-LNP Female C57BL / 6 mice were intravenously injected with PBS, anionic liposomes containing Fluc-mRNA (mRNA-AL), or lipid nanoparticles containing Fluc-mRNA (mRNA-LNP). Blood samples were collected in EDTA tubes 3 hours after the intravenous injection for cytokine and chemokine measurements. After 24 hours, the mice were sacrificed and the spleens were harvested. The spleens were placed in a 70-micron cell strainer, mashed using a syringe plunger, and washed with ice-cold RPMI medium (Gibco, ThermoFicher Scientific). The resulting single-cell suspension was centrifuged at 300 × g for 5 minutes at 4°C, and the supernatant was discarded. Erythrocytes were lysed by resuspending the pellet in 1 ml of ammonium chloride-potassium (ACK) lysis buffer and incubating for 2–3 minutes. After lysis, 9 ml of ice-cold medium was added to the cell lysis buffer suspension and centrifuged at 300 × g for 5 minutes at 4°C. The pellet was resuspended in 1 ml of medium, the cells were counted with trypan blue, and 600,000 cells were seeded per well of a 96-well V-bottom plate. The cells were washed once with PBS buffer before proceeding to flow cytometry staining. The plate was centrifuged at 300 × g for 5 minutes at 4 °C, and the supernatant was discarded. 25 μl of Fc blocking antibody was added per well and incubated on ice for 5 minutes.Dyes were prepared in 75 μl of FACS buffer (1× DPBS (Gibco) supplemented with 2% FCS) per well containing the following fluorophore-conjugated antibodies and dilutions: Viakrome808 Fixable Viability Dye (1:1000, Beckman Coulter), CD11b 450 (M1 / 70, Invitrogen), CD3e (145-2C11, BioLegend), B220 (RA3-6B2, BioLegend), Ly6G (1A8, BioLegend), XCR1 (ZET, BioLegend), CD25 (PC61, BioLegend), CD86 (GL-1, BD Biosciences), MHC II (M5 / 114.15.2, eBioscience), CD11c (N418, Miletnyi Cells were stained with CD80 (16-10A1, BioLegend), Ly6C (HK1.4, BioLegend) in the dark for 30 minutes at 4°C. Cells were washed three times with PBS before recording with a flow cytometer, Cytoflex (Beckman Coulter).

[0166] statistical analysis Design expert® software (V13.0.12.0, Stat-Ease Inc. Minneapolis, USA) was used to design response surface and mixture experiments, data fitting and analysis. GraphPad Prism software (V.9.4.1, GraphPad Software LLC, California, USA) was used for one-way analysis of variance (ANOVA) tests with Tukey post-hoc test analysis and long-rank test as indicated in the figures. Error bars were expressed as mean ± SEM as indicated in the figures. P values ​​were considered statistically significant when p<0.05.

[0167] result Microfluidic mixing allows the formation of nucleic acid complexes with anionic lipids without the need for cationic molecules. The present inventors have developed a microfluidics process for encapsulating nucleic acid adjuvants into anionic liposomes. Microfluidics is a common technique used to produce liposomes on a laboratory and industrial scale. The microfluidics nanoprecipitation method involves a controlled mixture of an organic-water miscible solvent (containing lipids) and an aqueous solvent. The diffusion of the organic-water miscible solvent, preferably ethanol, into the aqueous phase reduces the solubility limit of the lipids. As a result, the lipids begin to precipitate and grow from an intermediate structure into liposomes. For encapsulating RNA in lipid nanoparticles, prior art formulations generally contain cationic lipids, which facilitate the entrapment of nucleic acids through electrostatic interactions.

[0168] We began by generating anionic liposomes using a fixed lipid composition of EPC:EPG:cholesterol (3:1:2 molar ratio). Surprisingly, during proof-of-concept, we observed that it was possible to encapsulate a double-stranded RNA model adjuvant, poly(I:C) (see further RNA), into anionic liposomes, e.g., via microfluidics. Remarkably, this was possible without the use of positively charged lipids, polymers, or cations. RNA encapsulation was feasible using various microfluidic chip configurations. We focused on two designs, designated Design A and Design B in this work (Figure 1). To understand the mechanism behind this novel process and to obtain formulations, we decided to conduct an experimental design (Figure 2). Following this approach, we identified flow rate, lipid concentration, and RNA concentration as variables that could be related to lipid characteristics: particle mean size, polydispersity index (PdI), and encapsulation efficiency. We utilized a central composite design, which allows for the construction of a response surface model (RSM) for process optimization (Figures 17 and 18). After preparing samples for Design A (n = 30) and Design B (n = 53), we constructed a response surface model (RSM) to analyze which process parameters are important for the formation of RNA-containing anionic liposomes. While the organic and aqueous flow rates can be used to vary particle properties (size and PDI), the RNA encapsulation efficiency was affected by the RNA concentration in Designs A and B.

[0169] We identified critical process parameters for the production of anionic liposomes containing RNA. In both Designs A and B, the ethanol concentration during mixing of RNA and lipids could be used to vary particle size and PDI. Increasing the ethanol concentration led to larger particle sizes and lower PDI. This suggests that hydrophobic interactions may determine the mechanism for encapsulation of RNA within anionic liposomes. At higher ethanol concentrations, the RNA changes conformation, exposing hydrophobic nucleotide bases that can interact with the lipid tails, resulting in fairly monodisperse nanoparticles. Furthermore, we observed two advantages of Design B over Design A. The additional external aqueous phase allows for selective particle size control without altering the RNA / lipid ratio. In addition, the polydispersity index was lower than 0.3 under most conditions tested, as required for pharmaceutical liposomal products. Therefore, we chose Design B to produce anionic liposomes containing RNA.

[0170] Microfluidics process optimization and model validation The RSM used not only allowed us to understand the influence of each process variable on particle characteristics but also to optimize them for the application. Within the space of design B, we screened process parameters with appropriate size, polydispersity, and encapsulation efficiency. Because all intravenously injectable formulations containing heat-labile components are sterilized using filters with an average pore size of 220 nm, we set a target size of 150 nm for optimal spleen targeting and manufacturability. In addition, the target polydispersity was set to the smallest possible value, and the encapsulation efficiency was set to the largest possible value. The RSM suggested a flow ratio of 1:3:4.4, a lipid concentration of 5 mM, and an RNA concentration of 500 μg / mL.

[0171] We performed five independent batches within two days to verify the optimal conditions and validate RSM (Figure 2VII). The experimental and predicted particle sizes were 149 nm and 150 nm, and the polydispersity index was 0.188 and 0.191, respectively. The experimental and predicted encapsulation efficiencies were 69.6% and 65.8%, respectively. Therefore, particle size, polydispersity index, and RNA encapsulation efficiency could be accurately predicted by RSM. Interestingly, the anionic liposomes containing RNA had an average zeta potential of -56.7 mV, indicating a strong negative charge on the particle surface. Finally, the preparation process demonstrated remarkable reproducibility, which is required for clinical production.

[0172] Effect of mixing flow patterns on the encapsulation of RNA in anionic liposomes During the previously described DoE run, organic and aqueous streams containing lipids and RNA, respectively, were mixed using a laminar flow pattern. In this configuration, characterized by a low Reynolds number (Re < 2000), mixing relies on passive molecular diffusion from high-concentration domains to low-concentration domains. The mixing conditions for inter-nucleic acid interactions are strongly dependent on the flow rate and local geometry of the microfluidic mixer. The application of obstacles and baffles within the mixing chamber generates chaotic advection, rapidly mixing fluids and molecules even at Re numbers below 2000. In a staggered herringbone micromixer (chaotic advection micromixer), the mixing rate is positively correlated with the number of obstacles and the total flow rate. It has been reported that a higher mixing rate is beneficial for the formation of RNA-LNPs, leading to smaller and more monodisperse particles. Here, we examined the effect of mixing regimens involving a chaotic micromixer and increasing the total flow rate on the pharmaceutical properties of anionic liposomes containing RNA (Figure 3). The inclusion of a chaotic mixer led to smaller nanoparticles. Surprisingly, the staggered herringbone micromixer did not improve the PDI, while increasing the total flow rate led to an increase in the PDI. These findings contradict what has been observed with RNA-LNPs. Finally, encapsulation efficiency tends to be higher with laminar mixing. Overall, the use of a chaotic mixer allows for the generation of smaller particles, without further improving the PDI and encapsulation efficiency of RNA in anionic liposomes.

[0173] Optimizing formulations using mix design To understand the role of lipid components (egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), cholesterol) on the characteristics and bioactivity of anionic liposomes containing RNA, we performed an I-optimal mixture design experiment (n=17). In this experiment, we varied the relative proportions of lipid components while maintaining the optimized process parameters (flow rate, lipid, and RNA concentration) described under "Microfluidics process optimization and model validation" (Figure 16F). Statistical significance was observed for the model size (p=0.0005, R 2 =0.88), PDI (p<0.0001, R 2 =0.87), zeta potential (p=0.01, R 2 =0.47), encapsulation efficiency (p=0.005, R 2 =0.69) and HEK293T TLR3 + TLR3 activation in cells (p=0.003, R 2 =0.82).

[0174] Effect of lipid components on particle properties The particle size distribution ranged from 50 nm to 200 nm (except for the pure cholesterol-RNA complex, which had an average size of 800 nm (Figure 16A)). These results indicate that neutral (EPC) and / or anionic (EPG) phospholipids were important for assembling RNA nanoparticles. The proportion of anionic lipid EPG played a role in particle size, PDI, and encapsulation efficiency, with an overall trend of smaller particles and larger PDI observed with higher proportions of anionic EPG (Figures 16B and 16C). In addition, encapsulation efficiency was negatively correlated with EPG proportion, indicating a potential incompatibility between anionic lipids and RNA (Figure 16D). Interestingly, a statistically significant interaction was found between cholesterol and EPG. Cholesterol-rich anionic liposomes tend to have lower PDI and higher encapsulation efficiency. This interaction indicates that cholesterol is important for stabilizing anionic liposomes containing RNA. Cholesterol induces changes in lipid packaging in the bilayer, promoting the formation of lipid domains. The inclusion of cholesterol in the bilayer can stabilize lipid separation and avoid electrostatic interactions between RNA and anionic lipids within the bilayer. Surprisingly, the binary combination of EPG and cholesterol resulted in a low PDI and high encapsulation efficiency of RNA.

[0175] With regard to zeta potential, all nanoparticles had a negatively charged surface. Increasing the EPG proportion led to a more anionic surface. The inclusion of EPG was important for colloidal stability. Formulations without EPG tended to aggregate after 2-3 days of storage.

[0176] Effect of lipid compounds on the biological activity of anionic liposomes containing poly(I:C). The biological functions of the formulations prepared in the mixed design experiments were evaluated in vitro using the HEK-Blue™-hTLR3 reporter cell line. Poly(I:C) is an agonist of the TLR3 receptor. The cell line was stably transfected with the human TLR3 gene and an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. TLR3 is a receptor located in the endosomes of immune cells and is responsible for recognizing extracellular dsRNA, such as endocytosed poly(I:C). Its activity elicits an immune response. We wanted to know whether the lipid components of liposomes could affect poly(I:C) recognition by TLR3.

[0177] Reporter gene induction was optimal when the ratio of EPC, EPG, and cholesterol was close to that of the standard formulation (EPC:EPG:cholesterol = 3:1:2 molar ratio). Additionally, lower TLR3 activation was observed in nanoparticles with a higher EPG ratio (Figure 16E). The effect of EPG on encapsulated poly(I:C) bioactivity can be explained by selective nanoparticle uptake or potential interference with the interaction of poly(I:C) with TLR3. We evaluated particle uptake by HEK-Blue™ h-TLR3 by measuring the fluorescence intensity of the fluorescent probe DiD incorporated into the anionic liposome bilayer by flow cytometry. Particle uptake was enhanced at higher EPG concentrations and did not positively correlate with TLR3 activation. Therefore, increasing the EPG ratio may reduce TLR3 activation by poly(I:C). In fact, dsRNA is recognized by the histidine-rich region of TLR3. In late endosomes, the acidic pH and protonated histidine residues promote attractive electrostatic interactions between TLR3 and RNA phosphate groups. EPG can affect this electrostatic interaction when the anionic lipid molar ratio (%) exceeds a certain level.

[0178] Finally, we evaluated anionic liposome uptake and activation ex vivo by splenic conventional type 1 dendritic cells (cDC1), a cell population that strongly expresses TLR3 and upregulates maturation markers in the presence of poly(I:C). In this experiment, we sought to understand the effect of EPG concentration on particle uptake and maturation by cDC1. We selected five formulations containing increasing concentrations of EPG (0, 4, 17, 33, and 67% molar ratio), decreasing concentrations of EPC, and a constant concentration of cholesterol (33% molar ratio). HEK293 TLR3 + As seen in cell lines, increasing molar ratios of EPG enhance nanoparticle uptake by cDC1 cells (Figure 12A). Liposomes containing 0, 4, and 17 mol% EPG enhanced nanoparticle uptake by CD80 cells. + Increasing the EPG percentage to 33 and 66 mol% reduces the immunostimulatory properties of poly(I:C), although it leads to a significant upregulation of costimulatory markers (FIG. 12B).

[0179] Molecular weight characterization of encapsulated dsRNA and particle size distribution after plasma incubation Poly(I:C) is a non-coding synthetic dsRNA polymer. The high-molecular-weight poly(I:C) used in this study has a published average size of 1 kb to 8 kb. Microfluidic encapsulation of dsRNA can selectively enrich specific molecular size fractions. We characterized the molecular weight of dsRNA before and after encapsulation in anionic liposomes using size-exclusion chromatography (SEC) coupled with multi-angle light scattering and ultraviolet detection (SEC / MALS / UV).

[0180] SEC-MALS-UV analysis showed that the initial free poly(I:C) and poly(I:C) encapsulated in anionic liposomes were 10 4 ~10 7 Da (approximately 10-10 4The results show that the soluble and encapsulated poly(I:C) nucleic acids had similar molecular weights within the range of 0.1 base pairs (Figure 4B). A slightly more abundant high molecular weight fraction was observed in the encapsulated poly(I:C). Comparable retention times and UV absorbance profiles were observed for the soluble and encapsulated poly(I:C) (Figure 4A). These results indicate that the molecular weight of nucleic acids is not a limiting factor for encapsulation into anionic liposomes.

[0181] Liposomal drug products must exhibit physical stability in relevant physiological media. Formulations were incubated with PBS or 20% human plasma for 2 hours at 37°C, and samples were subjected to asymmetric field-flow fractionation (AF4) coupled with multi-angle light scattering (MALS), UV, and fluorescence detectors. Empty and poly(I:C)-containing anionic liposomes exhibited similar elution and light scattering profiles, as well as particle size distributions (Figure 4C). Samples incubated with human plasma exhibited overlapping light scattering curves and similar particle size distributions to those of liposomes incubated in PBS. Furthermore, free poly(I:C) and poly(I:C) encapsulated in anionic liposomes were treated with RNase III in reaction buffer or reaction buffer supplemented with 20% human plasma for 2 hours at 37°C and then analyzed by 1% agarose gel electrophoresis (Figure 4E). Free poly(I:C) was completely degraded by RNase III. Encapsulated poly(I:C) exhibited similar electrophoretic patterns in the presence or absence of RNase III and / or human plasma, indicating that anionic liposomes retain and protect nucleic acids.

[0182] These results demonstrate that anionic liposomes containing RNA remain physically stable and protect nucleic acids from RNase degradation when exposed to human plasma at physiological temperatures.

[0183] RNA can be encapsulated in anionic liposomes while maintaining the lipid bilayer structure. We first evaluated the structure of empty and RNA-containing anionic liposomes using negative-stain transmission electron microscopy (TEM). We used uranyl salts, which bind to phosphates on lipids and nucleic acids, to generate electron contrast within the samples. Empty nanoparticles have the typical liposome vesicle structure of one or more lipid bilayers and an empty core (Figure 5A). On the other hand, the incorporation of RNA into anionic nanoparticles altered their contrast density, particularly within the bilayer, due to the additional phosphate groups (Figure 5B). One or more lipid bilayers were observable within the RNA-containing nanoparticles.

[0184] Second, we used isotropic contrast modulation and SANS with deuterated lipids to understand the mass distribution of empty and pIC-containing anionic liposomes (pIC-AL). Contrast modulation is a technique for elucidating the external and internal structure of nanoparticles. In this experiment, samples were analyzed in 100 / 0%, 68 / 32%, 50 / 50%, and 27 / 73% DO / HO (Figure 6A). In the 68% DO / HO solvent, the contrast between the solvent-accessible RNA and the solvent disappeared, and only the scattering length density (SLD) of the lipids was detected.

[0185] A core-shell model was used to fit the measured neutron scattering curves, and similar shell thicknesses of approximately 4.1 nm were obtained for empty and pIC-AL (Figure 6B). This shell thickness is compatible with that of a phospholipid bilayer. The scattering length density (SLD) of the shell was higher for pIC-AL (0.26) than for empty liposomes (0.22), suggesting that RNA resides within the shell. In addition, the SLD of the cores of empty AL and pIC-AL was close to the solvent in the contrast series. This indicates that 1) solvent can freely diffuse through the shell into the core, 2) the core is primarily composed of aqueous solvent, and 3) some residual lipids form internal vesicles. In the case of pIC-AL, some RNA localizes within the core, contributing to a slightly higher core SLD compared to empty AL. Third, we performed a contrast series with samples containing one or more deuterated lipids (DPPCd62, POPCd31, DPPGd62, POPGd31, cholesterol-d7) to understand the lipid distribution within the nanoparticles. All samples containing deuterated lipids had increased SLD in the shell and a slight increase in the core compared to non-deuterated anionic liposomes, confirming the predominant distribution of neutral and anionic lipids, as well as cholesterol, within the lipid bilayer.

[0186] Knowing the scattered light densities of the components and their volume fractions, it is possible to calculate the solvent content inside the core (Equation 1): SLD コア = SLD 脂質 × V 脂質 + SLD RNA × V RNA + SLDs solvent × V solvent (Equation 1)

[0187] Calculations indicate that the cores of empty AL and pIC-AL are primarily aqueous (90 and 91% v / v), consistent with liposome structure. RNA is a hydrophilic molecule and can be dissolved in the aqueous core of liposomes. When dissolved in the aqueous core, pIC-AL resuspended in 68% DO has a different scattering profile than 100%, 50%, or 27% DO. Surprisingly, no difference in scattering profile was observed for RNA-containing liposomes during the contrast series. This suggests that the RNA may not be accessible to aqueous solvent. Therefore, the SANS data suggest that the RNA may associate with lipids within the hydrophobic bilayer between the shell and the inner vesicle rather than in a water-soluble form within the aqueous core of the liposome.

[0188] Third, to confirm the hypothesis that RNA increases the shell SLD, we re-performed SANS experiments, testing anionic liposomes with increasing concentrations of encapsulated RNA: 0 / 100%, 5 / 95%, 10 / 90%, 15 / 85%, and 30 / 70% (encapsulated RNA / lipid weight ratio (w / w%)) in 100% DO. Using a core-shell model to fit the experimental data, we found a direct correlation (R ) between the encapsulated RNA / nanoparticle lipid weight ratio and the shell SLD. 2 = 0.988, p = 0.005) was observed (Figures 19A and 19B), indicating that the RNA is at least partially localized within the lipid shell.

[0189] Fourth, to understand whether the RNA localizes at the surface of the nanoparticles as an adsorbed layer on the nanoparticles or within the hydrophobic regions of the lipid bilayer, we compared the scattering profiles of preformed empty nanoparticles incubated with exogenously added free RNA and nanoparticles with encapsulated RNA in 100% DO. As observed in Figure 19C, the scattering patterns of the encapsulated and co-incubated RNAs are different. A. For preformed nanoparticles incubated with externally added RNA, there is increased scattering at low Q (0.1-1 Å), indicating an increase in background. The increased background may be due to more hydrogen in the DO solvent after addition of RNA, suggesting that the RNA is localized in the solvent rather than in the nanoparticles. B. In the case of nanoparticles with encapsulated RNA, no significant background increase is observed, suggesting that the RNA is localized in the nanoparticles rather than in the solvent.

[0190] Fifth, we compare the scattering profiles of anionic liposomes with lipid nanoparticles (LNPs), the current state-of-the-art technology for RNA delivery, by analyzing empty nanoparticles and nanoparticles containing 5% w / w RNA. As previously described, the process of encapsulating RNA in anionic liposomes does not alter the nanoparticle structure, and comparable scattering profiles were observed (Figure 19D). However, encapsulating RNA in LNPs alters the scattering profile, indicating a rearrangement of the internal structure, as previously reported by Arteta et al. (Proceedings of the National Academy of Sciences 115, E3351-E336 (2018)). The rearrangement observed within LNPs can be explained by electrostatic interactions between ionizable cationic lipids and anionic RNA, which does not occur in anionic liposomes containing RNA. This evidence therefore suggests that anionic liposomes have a distinct structure and interact with RNA through a novel mechanism that is independent of the internal structural rearrangements observed in LNPs.

[0191] If RNA is indeed embedded within the lipid bilayer through hydrophobic interactions, as suggested by SANS experiments, the physical properties of the RNA and the lipids within the anionic liposomes containing the RNA should be altered.

[0192] We examined the changes in lipid packaging in the bilayer of anionic liposomes in the presence of free and encapsulated RNA over a temperature range (10 °C to 80 °C) (Figure 6C). The membrane-embedded fluorescent molecule lauran was used to measure the relative level of membrane lipid packaging, reported as the general polarization (GP) parameter. Encapsulated RNA, unlike soluble RNA, affects the order of lipid packaging in the liposome bilayer, resulting in lower GP values ​​during the heating gradient (Figure 6C). This effect is particularly observable in anionic liposomes with liquid ordered and disordered domains (DPPC:POPC:DPPG:POPG:cholesterol (3:3:1:1:4)). These results confirm that encapsulated RNA interacts with the hydrophobic bilayer.

[0193] Poly(I:C) is a double-stranded RNA that melts (strands separate) at high temperatures. We used differential scanning calorimetry (DSC) to characterize the heat-induced structural transitions of free and encapsulated poly(I:C) (Figure 6D). Free poly(I:C) exhibits a melting (endothermic) event at 65 °C. In contrast, there is no melting event when poly(I:C) is encapsulated in liposomes or mixed with empty liposomes. The binding of poly(I:C) within the bilayer in the former case or to the surface of the liposome in the latter case may inhibit strand separation. Surprisingly, encapsulated poly(I:C) exhibits an exothermic event at 77 °C, which can be explained by the crystallization of RNA within the hydrophobic environment of the liposome bilayer. In fact, hydrophobic organic solvents are commonly used to grow nucleic acid crystals.

[0194] Michanek et al. previously reported that exogenously added nucleic acids to preformed phospholipid bilayers associate with the surface of the bilayer (Michanek et al. (Biochimica et Biophysica Acta 1798 (2010) 829-838). Using a dissipation-measuring quartz crystal microbalance (QCM-D), Michanek et al. demonstrated that tRNA adheres to the surface of a DOPC bilayer (14 mg / m 2) and DPPC bilayer (6 mg / m 2 reported adsorption to DMPC vesicles. However, the degree of adsorption is very low, as the mass difference measured for tRNA adsorption to the bilayer corresponds to one tRNA molecule per 1700 DOPC lipids and one tRNA molecule per 4300 DPPC lipids. In addition, the adsorbed layer of tRNA is readily removed by rinsing with 10 mM NaBr solution, suggesting that the interaction of RNA with the bilayer surface is weak and reversible. Furthermore, Michanek et al. performed DSC experiments on RNA adsorbed to the surface of DMPC vesicles to describe how RNA interacts with the surface of DMPC bilayers. The melting point of DMPC vesicles was unaffected when they were incubated with different types of nucleic acids (tRNA, ssDNA, and dsDNA), indicating no significant penetration of nucleic acids into the hydrophobic regions of the bilayer. As described by Michanek et al., it is highly unlikely that the highly charged phosphate groups of nucleic acids can penetrate the nonpolar regions of the lipid bilayer.

[0195] To further confirm the unexpected novelty of our invention, we utilized DSC techniques to examine the type of association between RNA and the DMPC bilayer, either when adsorbed to a surface or when encapsulated by the method described in this invention. While the association of RNA with the outer surface of empty DMPC vesicles does not induce the melting point depression described in the prior art, encapsulation of RNA according to our invention induces a significant melting point reduction (Figure 20E). This finding, combined with the findings described above, strongly suggests that our RNA encapsulation methodology promotes unexpected RNA association with nonpolar regions of the lipid bilayer. Additionally, we tested whether encapsulating RNA into liposomes could overcome the current limitation of the prior art, which is the weak and reversible interaction of RNA with the outer bilayer surface. We observed that the interaction of adsorbed RNA with the bilayer surface is weak and reversible, and that simple dilution with water is already sufficient to remove the adsorbed RNA from the surface of preformed DMPC vesicles. Surprisingly, when RNA is encapsulated in DMPC liposomes using the method of the present invention, there is no strong fluorescent signal between 5 and 20 minutes after the start of the run, when the RNA is expected to elute from the AF4 channel. This indicates that the interaction between the RNA and the liposomes is strong and irreversible, and dilution has no elimination effect. Furthermore, the fluorescent signal of the RNA weakens once the particles elute from the AF4 channel (30 to 60 minutes into the run), suggesting that the RNA is inside the nanoparticles and not on the outer surface, resulting in suppressed fluorescence (Figure 20A-D).

[0196] In summary, these results suggest that anionic liposomes containing RNA have an unexpected structure: a core-shell structure with RNA embedded in the lipid bilayer and a mostly empty aqueous core. The structure of pIC-AL is distinct from that reported for conventional RNA-LNPs. Arteta et al. recently used SANS and SAXS techniques to characterize RNA-LNPs and showed that they have a distinct core-shell structure (Arteta et al., Proceedings of the National Academy of Sciences 115, E3351-E3360 (2018)). The shell is composed of DSPC and lipidated PEG, but does not contain RNA. The core is composed of RNA and lipids that do not self-assemble into a bilayer (ionizable cationic lipids and cholesterol) but rather self-assemble into a disordered hexagonal internal structure. The water content is significantly lower in RNA-LNPs than in RNA-anionic liposomes (24% vs. 91%), indicating that the core of RNA-LNPs is larger and more rigid.

[0197] Stability of anionic liposomes containing poly(I:C). Cold chain failure is one of the main reasons for vaccine waste. During storage at 2-8°C, certain refrigerators, such as ice-lined refrigerators, pose a risk of overcooling and freezing liquid formulations, leading to vial waste. Most vaccines are freeze-sensitive, and therefore having a freeze-insensitive formulation is desirable from a supply perspective.

[0198] During formulation development, we conducted stability studies of anionic liposomes containing poly(I:C). The lipid composition EPC:EPG:cholesterol (3:1:2) was selected for stability studies. We tested the incorporation of sucrose, a sugar commonly used as a cryoprotectant, to prevent liposome aggregation after freezing and lyophilization. Anionic liposomes containing poly(I:C) were resuspended in RNase-free 10% sucrose 20 mM citrate buffer (pH 6.5) and stored at -20°C and 5°C for 5 months. Particle size, polydispersity, zeta potential, total poly(I:C) content and % of released poly(I:C), as well as the % release of poly(I:C) were analyzed in HEK TLR3 and HEK TLR3 cells. + Poly(I:C) activity was examined (Figures 7A-F). Anionic liposomes containing poly(I:C) were physically stable, showing no signs of particle aggregation over the 5 months they were stored at -20°C and 5°C (Figures 7A-C). The total concentration of poly(I:C) decreased slightly after 5 months, by 5% at -20°C and 9% at 5°C (Figure 7D). Although some released RNA was detected at 0 hours (less than 10%), further release of RNA was detected after 1 month (Figure 7E), indicating a strong interaction between the encapsulated RNA and the anionic liposomes. Furthermore, HEK TLR3 + The bioactivity in cells did not decrease after 5 months of storage at -20°C and 5°C (Figure 7F). These results demonstrate the stability of poly(I:C) encapsulated in anionic liposomes.

[0199] Effects of incorporation of poly(I:C) into anionic liposomes on maturation, antiviral cytokine levels, and liver toxicity of splenic conventional type 1 dendritic cells The spleen is a major secondary lymphoid organ containing multiple subsets of myeloid cells and dendritic cells (DCs). DCs are initiators of adaptive cellular immune responses through antigen capture, processing, and presentation to naive T cells. DCs mature in the presence of adjuvants and antigens to induce potent T cell responses. DCs are a heterogeneous population consisting of multiple cell-type subsets. Among them, conventional type 1 (cDC1) DCs are characterized by high TLR3 expression in endosomes, which allows them to recognize double-stranded RNA molecules, such as poly(I:C) adjuvants, and initiate adaptive immune responses. cDC1 DCs have attracted particular interest in tumor immunology. The number and activation of cDC1 DCs have been shown to strongly and positively correlate with improved survival in multiple human cancer types.

[0200] We tested the immunostimulatory properties of anionic liposomes on splenic cDC1. Incorporation of poly(I:C) into anionic liposomes enhanced the maturation of splenic cDC1 ex vivo, as reflected by increased expression of CD80, CD86, and MHC-II surface markers (Figures 8A, B, and C).

[0201] We evaluated the extent to which poly(I:C)-containing anionic liposomes could mature cDC1 cells in vivo. Poly(I:C)-containing anionic liposomes were found to be highly effective in inducing cDC1 maturation in a dose-dependent manner after IV administration (Figure 9A, B, C). Encapsulated poly(I:C) was superior to free poly(I:C) in inducing cDC1 maturation at 10 and 50 μg doses. Interestingly, 10 μg of encapsulated poly(I:C) induced similar expression levels of surface maturation markers (CD40, CD80, and CD86) as 50 μg of free poly(I:C). At the 50 μg dose of encapsulated poly(I:C), most splenic cDC1 cells, cDC2 cells, and red pulp macrophages captured poly(I:C)-containing anionic liposomes (Figure 13A, B, and C). Additionally, we examined serum cytokine levels 3 and 24 hours after IV administration of soluble and encapsulated poly(I:C) (Figures 9D-E). Cytokines are a family of small proteins essential for coordinating immune responses and are of interest in cancer immunotherapy. Indeed, cytokines such as interferon alpha and IL-2 have been approved for cancer treatment. Anionic liposomes containing poly(I:C) were found to significantly increase cytokine serum levels in a dose-dependent manner compared to free poly(I:C), particularly for interferons (α, β, and γ), chemokines (CCL2, CCL5, CxCL10), and interleukin-6 (Figure 9D). These cytokines have antitumor growth activity, promote antigen priming, increase T cell cytolytic activity (interferon-α), increase endothelial permeability (IL-6), and promote immune cell infiltration into the tumor microenvironment (CCL2, CCL5, CxCL10). In addition, they have antiviral replication activity. We also measured the immunosuppressive cytokine IL-10, which promotes tumor cell growth, but did not observe a significant effect of liposomal poly(I:C) on the cytokine level. Finally, we tested whether the stronger immune response observed with encapsulated poly(I:C) could be correlated with increased toxicity.Poly(I:C) treatment (20 μg / g) has been used to mimic viral hepatitis in mice. Liver inflammation induces liver tissue damage and the release of liver enzymes such as ALT and AST. Notably, encapsulating poly(I:C) within anionic liposomes induces a potent immune response without observable hepatotoxicity (Figure 9F and G).

[0202] Immunization with anionic liposomes containing poly(I:C) induces stronger T cell responses than free poly(I:C). The next goal was to determine whether liposomal poly(I:C) induces stronger T cell responses than free poly(I:C). In a previous study (Nijen Twilhaar, Pharmaceutics, 2020, vol. 12, no. 12, p. 1138), we observed that the incorporation of GM3 ganglioside into anionic liposomes containing antigens enhanced T cell responses after IV immunization. These liposomes were designed to incorporate the model antigen ovalbumin (OVA), including CD4- and CD8-T cell epitopes. 247-279 (Fig. 10A,B) and CD4+ T cell responses (Fig. 10C) were significantly more robust than antigen-containing liposomes alone. The incorporation of GM3 did not significantly affect the activity of anionic liposomes containing poly(I:C). Here, we coadministered the former antigen-containing GM3 liposomes with PBS (control), or free poly(I:C) (10 or 100 μg) or poly(I:C)-containing anionic liposomes (10 μg) with or without GM3 via IV injection. Seven days later, we analyzed T cell responses in the splenic compartment. Despite utilizing a higher dose of free poly(I:C), coadministration of encapsulated poly(I:C) into anionic liposomes, unlike free poly(I:C), induced significantly more robust CD8+ T cell responses (Fig. 10A,B) and CD4+ T cell responses (Fig. 10C) than antigen-containing liposomes alone. The incorporation of GM3 did not significantly affect the activity of anionic liposomes containing poly(I:C). Thus, these results demonstrate that incorporation of poly(I:C) into anionic liposomes enhances vaccine efficacy in generating T cell responses against model ovalbumin peptides.

[0203] OVA 257-264 After restimulation with peptide, all CD8 produced interferon gamma. + The amount of T cells was significantly increased by treatment with anionic liposomes containing poly(I:C) and specific CD8 + T cells (Figures 10A and 10B). + It has been shown that T cells produce interferon gamma upon stimulation with poly(I:C) in an antigen-independent manner. Administration of poly(I:C)-containing anionic liposomes stimulates CD8 T cells in an antigen-independent manner. + To confirm whether T cells could be stimulated, we repeatedly immunized mice with 10 μg of anionic liposomal poly(I:C) alone or in combination with low or high doses of liposomal antibody (Figure 11). Administration of anionic liposomes containing poly(I:C) was sufficient to activate CD4+ and CD8+ T cells. The amount of CD8+ T cells producing interferon-γ was significantly higher and independent of antigen administration (Figures 11A and 11B). These results are relevant for therapies that rely on CD8+ T cell functionality. Administration of free poly(I:C) has been reported to improve CAR-T therapeutic activity, immune checkpoint inhibitor antibody treatment, chemotherapy, and radiation therapy in mouse cancer models. The combined use of anionic liposomes containing poly(I:C) can further improve the efficacy of such fellow therapy.

[0204] Intravenous administration of anionic liposomes containing poly(I:C) has potent antitumor effects and overcomes resistance to immune checkpoint therapy in multiple allogeneic tumor models. Immune checkpoint inhibitors (ICIs) are a novel class of immunotherapy drugs that have transformed the treatment of a wide range of advanced, intractable cancers, including metastatic melanoma, triple-negative breast cancer, colorectal cancer, and non-small cell and small cell lung cancer, among others. They block immune checkpoint proteins that stop the immune system from attacking cancer cells, inducing long-lasting antitumor responses in selected patients. However, clinical reality is that most patients do not benefit from this revolutionary therapeutic approach. Response rates are low across different cancer types. To be effective, ICIs require the presence of sufficient antitumor immune cells in the tumor microenvironment. ICIs are largely ineffective in patients with few antitumor immune cells in the tumor ("cold" tumors). In addition, the accumulation of immune-suppressive cells (known as myeloid-derived suppressor cells (MDSCs)) in the tumor microenvironment contributes to cancer progression and immunotherapy resistance. Nucleic acid adjuvants are immune stimulators that can convert "cold" tumors into "hot" tumors by increasing the infiltration of tumor-infiltrating lymphocytes (TILs), including CD8+ and CD4+ T cells and B cells. Currently, clinical cancer studies are using intratumoral administration of nucleic acid adjuvants encapsulated in cationic nanoparticles in combination with ICIs. However, utilizing this local administration route has major limitations: most tumor tissues are inaccessible by intratumoral injection, and the adjuvant effect remains localized. These cationic delivery systems are avoided for intravenous administration because they are physically unstable and may aggregate in the bloodstream (risk of pulmonary embolism), and cationic molecules can induce a strong inflammatory response.

[0205] We tested the antitumor efficacy of intravenous anionic liposomes containing poly(I:C) in combination with an anti-PD-L1 antibody, an immune checkpoint inhibitor, in an aggressive metastatic melanoma tumor model (B16F10) in mice (Figure 14A). B16F10 melanoma, syngeneic to C57BL / 6 mice, is a poorly immunogenic tumor with an immunosuppressive microenvironment. It exhibits low expression of MHC class I molecules and low T CD8 +This tumor model is highly invasive and one of the most resistant to ICIs. Mice treated with anti-PD-L1 antibodies as monotherapy failed to delay tumor growth or improve overall survival (Figure 14A). Notably, the combination of anti-PD-L1 antibodies with anionic liposomal poly(I:C) (10 μg) resulted in potent and significant tumor growth inhibition (65%), in contrast to the anti-PD-L1 monotherapy group (-17%), already within 5 days after the first intravenous administration, and a 100% increase in mean overall survival without apparent toxicity (Figures 14B, C, D, and E). Importantly, the therapeutic effect of liposomal poly(I:C) was not observed in the initial tumor size (7-238 mm). 3 ) dependent (Figure 5). Free poly(I:C) (i.e., not encapsulated in liposomes), even at a 5-fold higher dose (50 μg), did not significantly inhibit tumor growth or improve overall survival when given in combination with anti-PD-L1.

[0206] Based on the improved therapeutic efficacy observed when poly(I:C) was encapsulated in anionic liposomes compared to free poly(I:C), we tested whether liposomal poly(I:C) had antitumor efficacy as monotherapy in two additional models: MC38 colon cancer and Hepa1-6 hepatocellular carcinoma. The MC38 colon cancer model is highly invasive and widely used to test immunotherapies. Mice treated with anti-PD-L1 antibodies as monotherapy failed to delay tumor growth or improve overall survival (Figures 21A and 21B), whereas mice treated with liposomal poly(I:C) monotherapy exhibited potent antitumor efficacy and a 100% increase in mean overall survival. Impressively, the combination of liposomal poly(I:C) and a-PD-L1 resulted in a more robust and complete response (at least 50% tumor size reduction) in 63% of mice and long-term cures (60 days) in 38% of mice. Sixty days after the start of treatment, cured mice and naive control mice were re-challenged with fresh MC38 cancer cells. While the new MC38 cancer cells grew invasively in naive mice, cured mice in the combination group (liposomal poly(I:C) and a-PD-L1) were able to block tumor growth and did not require additional treatment (Figure 21C). This finding points to the development of immune memory, which is necessary to prevent relapse in cancer patients. Additionally, we examined the effect of liposomal poly(I:C) on the immune cell composition of the tumor microenvironment of established colon cancer tumors. Flow cytometry analysis of tumors 5 days after treatment initiation revealed that treatment with liposomal poly(I:C) dramatically altered the immune cell (CD45+) composition of the tumor microenvironment, resulting in a robust increase in antitumor TILs (from 12% to 50%) and a strong decrease in tumor-promoting MDSCs (from 30% to 10%) (Figure 21D). These changes in immune cell composition may explain the potent antitumor activity of liposomal poly(I:C) and its synergistic effect in combination with ICI. Furthermore, we tested the effects of liposomal poly(I:C) in an orthotopic hepatocellular carcinoma (HCC) model (Hepa1-6).In mice bearing multiple established tumors, it was observed that liposomal poly(I:C) monotherapy could reduce the size and number of tumor nodules, reflecting potent antitumor activity (Figure 22).

[0207] Finally, we tested the benefits of using nanoparticles that do not contain cationic molecules for the delivery of nucleic acid adjuvants. In an MC38 colon cancer model, we compared poly(I:C) encapsulated in anionic liposomes with poly(I:C) encapsulated in LNPs, the only nanoparticle formulation approved by the FDA for intravenous delivery of nucleic acids. Poly(I:C) was successfully encapsulated in anionic liposomes (pIC-AL) and LNPs (pIC-LNP) using the same manufacturing process and conditions. Particle size distribution and RNA encapsulation efficiency were similar for both types of nanoparticles (Table N1). [Table 1]

[0208] We also tested the effects of the two formulations in combination with a-PD-L1 antibodies. Administration of 10 μg of poly(I:C) in LNP (pIC-LNP) induced severe acute toxicity, which was greatly enhanced when combined with a-PD-L1 antibodies. Animals treated with pIC-LNP exhibited the following severe adverse events: 1) a 16% (monotherapy) and 66% (combination therapy) mortality rate 72 hours after the start of treatment (Figure 23A); 2) all animals experienced a loss of at least 15% body weight (Figure 23B); and 3) all animals exhibited elevated AST liver enzyme activity in the blood, suggesting liver damage (Figure 24F). No severe adverse events occurred with liposomal poly(I:C), yet the antitumor efficacy was as potent as that of the poly(I:C) LNP group (Figure 23C). Due to the severe toxicity observed in the pIC-LNP group, we terminated the experiment and isolated the liver, kidney, heart, and spleen for histopathological analysis. pIC-LNP treatment induced severe tissue damage in the liver (Figure 24D) and spleen, which was enhanced when combined with ICI treatment (Figure 24E). Tissue damage was not observed when poly(I:C) was encapsulated in anionic liposomes as monotherapy (Figure 24B) or in combination with ICI (Figure 24C). Analysis of pro-inflammatory cytokine levels in the bloodstream 3 hours after injection of poly(I:C)-nanoparticles (Figures 23D-I) showed dramatic and significant increases in pIC-LNPs compared to reference levels in the PBS group: IL-6, 1185-fold increase (pIC-LNP) vs. 43-fold increase (pIC-AL); INF-α, 1577-fold increase (pIC-LNP) vs. 170-fold increase (pIC-AL); INF-β, 4703-fold increase (pIC-LNP) vs. 107-fold increase (pIC-AL); INF-γ, 1216-fold increase (pIC-LNP) vs. 16-fold increase (pIC-AL); TNF-α, 654-fold increase (pIC-LNP) vs. 197-fold increase (pIC-AL); and IL-1β, 7-fold increase (pIC-LNP) vs. 6-fold change increase (pIC-AL).

[0209] The extreme elevation of pro-inflammatory cytokine plasma levels observed with pIC-LNP suggests a "cytokine storm," which may be the underlying cause of mortality and liver injury.

[0210] Intravenous administration of anionic liposomes containing mRNA does not induce an immune inflammatory response, whereas LNPs containing the same mRNA do. Previous experiments have shown that current state-of-the-art nanoparticles (LNPs) for nucleic acid delivery exacerbate the immunotoxicity of the nucleic acid adjuvant poly(I:C), while encapsulation in anionic liposomes enhances efficacy without increasing immunotoxicity. To confirm the non-inflammatory properties of novel nucleic acid-lipid nanoparticles, we tested whether administration of non-immunostimulatory mRNA encapsulated in anionic liposomes [EPC:EPG:cholesterol (3:1:2)] lacks the induction of an immune response. This is particularly interesting in medical applications where inflammatory immune responses must be avoided, such as for the treatment of autoimmune diseases, diseases involving chronic inflammation, or genetic disorders. Immunogenic mRNA was not successfully encapsulated in anionic liposomes and LNPs using the same manufacturing process and conditions. Particle size distribution and RNA encapsulation efficiency were similar for both types of nanoparticles (Table N2). [Table 2]

[0211] A single dose of 10 μg of mRNA encapsulated in anionic liposomes or LNPs was intravenously administered to healthy mice. 24 hours later, immune responses in the splenic compartment were analyzed. Administration of mRNA-LNPs induced activation of splenic dendritic cells type 1 and type 2 (increased expression of the CD80 marker) and splenic T cells (increased expression of the CD25 marker), whereas administration of mRNA-AL did not induce activation of splenic dendritic cells type 1 and type 2 or T cells (Figures 25A-C). In addition, analysis of pro-inflammatory cytokine levels in the bloodstream 3 hours after injection of mRNA nanoparticles (Figures 23D-I) showed dramatic and significant increases in mRNA-LNPs compared to reference levels in the PBS group, unlike mRNA-AL: a 669-fold increase in IL-6 (mRNA-LNPs) vs. a 4-fold increase (mRNA-AL); a 579-fold increase in INFα (mRNA-LNPs) vs. no increase (mRNA-AL); a 60-fold increase in INF-β (mRNA-LNPs) vs. no increase (mRNA-AL); a 15-fold increase in INF-γ (mRNA-LNPs) vs. no increase (mRNA-AL); an 8-fold increase in TNFα (mRNA-LNPs) vs. a 2-fold increase (mRNA-AL); and a 2-fold increase in IL-1β (mRNA-LNPs) vs. no increase (mRNA-AL). These findings confirm the non-inflammatory properties of anionic liposomes containing nucleic acids.

[0212] conclusion In conclusion, it is possible to encapsulate multiple nucleic acids, such as dsRNA poly(I:C) and ssRNA (mRNA), into anionic liposomes using solvent mixing or microfluidics techniques. Extensive physicochemical characterization studies have shown that RNA is, to some extent, localized within the hydrophobic regions of the lipid bilayer. This may explain the increased stability of RNA within anionic liposomes. Encapsulation of poly(I:C) into anionic liposomes enhances immunostimulatory effects without significant toxicity compared to the current state-of-the-art delivery system, RNA-LNP. Encapsulation of poly(I:C) into anionic liposomes increases the localization of tumor-infiltrating lymphocytes to tumor tissue, making it a potent antineoplastic agent suitable for the treatment of malignant tumors both as monotherapy and in combination with ICIs. Additionally, the present invention facilitates its use as an adjuvant system in combination with antigens to improve vaccine efficacy. Furthermore, encapsulation of non-immunogenic mRNA in anionic liposomes does not elicit immune system activation, whereas mRNA within LNPs does. This unique feature allows the use of RNA-containing anionic liposomes to treat diseases with underlying inflammatory signatures.

Claims

1. 1. A composition comprising anionic nucleic acid-lipid particles, the anionic nucleic acid-lipid particles comprising: - one or more nucleic acids; one or more lipid bilayers comprising at least one anionic lipid and / or at least one neutral lipid, Including, at least a portion of the one or more nucleic acids is encapsulated by the nucleic acid-lipid particle, and less than 10% by weight of the one or more nucleic acids is exposed outside the nucleic acid-lipid particle; composition.

2. - at least 20% by weight of said one or more nucleic acids is encapsulated by said nucleic acid-lipid particles; - at least 20% by weight of said one or more nucleic acids are contained within the hydrophobic regions of said one or more lipid bilayers, preferably in the form of nucleic acid-lipid complexes; and / or - less than 5% by weight of the nucleic acid is exposed on the exterior of the nucleic acid-lipid particle; The composition of claim 1.

3. 3. The composition of claim 1, wherein the particle comprises one or more of a cationic lipid, a cationic polymer, and a multivalent cation in an amount of up to 20 mol % relative to the total lipid molar amount of the particle.

4. The composition of any one of claims 1 to 3, wherein the particles comprise at least 5 mol% of at least one sterol relative to the total lipid molar amount of the particles.

5. The particles are at least 0.5 mol%, preferably at least 5 mol%, more preferably at least 15 mol% of at least one anionic lipid relative to the total lipid molar amount of said particle; and / or at least 1 mol%, preferably at least 5 mol%, more preferably at least 15 mol% of at least one neutral lipid relative to the total lipid molar amount of said particle, The composition according to any one of claims 1 to 4, comprising:

6. the nucleic acid-lipid particle is - have a core-shell structure and / or a core encapsulated by said one or more lipid bilayers, the core comprises at least 50% by volume of an aqueous solvent relative to the total core volume; - does not contain cationic lipids and / or comprises at most 1 mol% of cationic lipids relative to the total lipid molar amount of said particle; - does not contain cationic polymer and / or contains at most 1 mol% of cationic polymer relative to the total lipid molar amount of said particle; and / or - free of polyvalent cations and / or containing up to 1 mol% of polyvalent cations relative to the total lipid molar amount of the particle; The composition according to any one of claims 1 to 5.

7. the nucleic acid-lipid particle is - have an average hydrodynamic diameter of 50 to 300 nm as determined by dynamic light scattering (DLS); - having a polydispersity index (PDI) of 0.01 to 0.5 as determined by dynamic light scattering (DLS); and / or - have a zeta potential between -100 and -10 mV as determined by electrophoretic light scattering (ELS), The composition according to any one of claims 1 to 6.

8. said one or more nucleic acids are DNA, RNA, or DNA or RNA analogues, and / or said one or more nucleic acids are an immunological adjuvant, and / or a TOll-like receptor agonist (TLR), preferably a TLR3, TLR7 / 8 or TLR9 agonist, more preferably a TLR3 agonist, most preferably poly(I:C), poly(C:G) or poly(A:U); wherein the one or more lipids are neutral lipids, anionic lipids, and sterols, more preferably the one or more lipids are phosphatidylcholine, phosphatidylglycerol, and sterols, most preferably EPC, EPG, and cholesterol; The composition according to any one of claims 1 to 7.

9. the particle comprises 0.5 to 40 mol% of at least one anionic lipid relative to the total lipid molar amount of the particle, and / or the composition is an immunostimulatory composition; The composition according to any one of claims 1 to 8.

10. 10. The composition of any one of claims 1 to 9, wherein the particles comprise 0.5 to 80 mol% of at least one anionic lipid relative to the total lipid molar amount of the particle, and / or the composition is an immunosilent composition, and / or the particles are non-immunogenic and do not result in dendritic cell activation, T cell activation, and / or IFN-alpha secretion.

11. The composition according to any one of claims 1 to 10, wherein the composition is used in combination with one or more selected from the group consisting of an antigen, an immunological adjuvant and / or a vaccine.

12. The composition of any one of claims 1 to 11, wherein the composition is used in combination with an anti-neoplastic agent.

13. The antineoplastic agent is - cell-based anti-neoplastic agents; lymphocyte-based anti-neoplastic agents, preferably selected from B cells, αβT cells, γδT cells, NK cells, NKT cells, autologous tumor infiltrating lymphocytes (TIL), autologous NK cells, CAR-T cells, CAR-B cells, CAR-NK cells, CAR-NKT cells; - a myeloid-based antineoplastic agent, preferably selected from a dendritic cell-based, macrophage-based, or neutrophil-based antineoplastic agent; - an antibody or an immune checkpoint inhibitor, wherein said immune checkpoint inhibitor is preferably an antibody, more preferably an a-CTLA4 antibody, an anti-PD1 antibody and / or an anti-PD-L1 antibody; - small molecule drugs, preferably selected from alkylating agents, antibiotics, antimetabolites, hormone antagonists, photosensitizers, protein kinase inhibitors, poly(ADP-ribose) polymerase inhibitors, taxanes and / or topoisomerase inhibitors; - Radiation therapy; cytokines, preferably chosen from IL-2, IL-12, IL-15 and IL-21; growth factors, preferably selected from the CSF family and Flt3L; and / or - steroidal or non-steroidal anti-inflammatory drugs, 13. The composition of claim 12, wherein:

14. A composition according to any one of claims 1 to 13 for use in preventing and / or treating a disease.

15. 15. The composition for use according to claim 14, wherein the disease is one or more of cancer, infectious disease, chronic inflammation, genetic disease and autoimmune disease.

16. 16. A method for preparing a composition comprising the nucleic acid-lipid particles of any one of claims 1 to 15, said method comprising: a) providing one or more organic solutions comprising at least one anionic lipid and / or at least one neutral lipid; b) providing one or more aqueous solutions containing one or more nucleic acids; c) combining said one or more organic solutions with said one or more aqueous solutions, thereby producing said composition comprising the nucleic acid-lipid particles of any one of claims 1-15; A method comprising:

17. 17. The method of claim 16, wherein the combining in step c) comprises laminar mixing of the one or more organic solutions with the one or more aqueous solutions.

18. the flow ratio between said one or more aqueous solutions and said one or more organic solutions is between 24:1 and 1:10; - the at least one anionic lipid and / or the at least one neutral lipid is provided in the organic solution at a concentration of 0.1 to 50 mM; - the one or more organic solutions are ethanol solutions containing at least 80% (v / v), preferably at least 90% (v / v), more preferably at least 97.5% (v / v), most preferably at least 99% (v / v) ethanol; and / or - said one or more nucleic acids are provided in said aqueous solution at a concentration of between 10 and 5000 μg / ml, The method according to any one of claims 16 to 17.

19. wherein the one or more organic solutions in step a) are - at least 20 mol% of at least one anionic lipid and / or at least one neutral lipid relative to the total lipid molar amount in said one or more organic solutions; - at most 10 mol% of at least one cationic lipid relative to the total lipid molar amount in said organic solution; and / or - at least 5 mol % of at least one sterol relative to the total lipid molar amount in said organic solution; The method according to any one of claims 16 to 18, comprising:

20. - the organic solution in step a) is free of cationic lipids and / or comprises at most 1 mol% of at least one cationic lipid relative to the total lipid molar amount in the organic solution, and / or - the method does not involve the use of cationic lipids and / or cationic polymers, The method according to any one of claims 16 to 19.

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