Compositions and methods for delivery of active agents, including nucleic acids

Abstract

Disclosed herein are lipid nanoparticles for the delivery of active agents, including nucleic acids, and methods for making and using them.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 401,113, filed August 25, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Therapeutic nucleic acids include, for example, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, and immunostimulatory nucleic acids. These nucleic acids act through various mechanisms. In the case of siRNA or miRNA, these nucleic acids can downregulate the intracellular levels of specific proteins through a process called RNA interference (RNAi). After introduction of siRNA or miRNA into the cytoplasm, these double-stranded RNA constructs can bind to a protein called RISC. The sense strand of the siRNA or miRNA leaves the RISC complex and provides a template within RISC, which can recognize and bind to mRNAs with sequences complementary to those of the bound siRNA or miRNA. After binding to the complementary mRNA, the RISC complex cleaves the mRNA and releases the cleaved strand. RNAi can downregulate specific proteins by targeting the specific destruction of the corresponding mRNA that codes for protein synthesis.

[0003] Because siRNA and miRNA constructs can be synthesized with any nucleotide sequence directed against target protein, the therapeutic application of RNAi is very broad.To date, siRNA constructs have shown the ability to specifically down-regulate target protein in both in vitro and in vivo models.In addition, siRNA constructs are currently being evaluated in clinical studies.

[0004] Despite recent advances, there remains a need in the art for improved lipid therapeutic nucleic acid compositions suitable for general therapeutic use. These compositions, for example, encapsulate nucleic acids with high efficiency, have a high drug:lipid ratio, protect the encapsulated nucleic acid from degradation and clearance in serum, are suitable for systemic delivery, and provide intracellular delivery of the encapsulated nucleic acid. In addition, these lipid-nucleic acid particles should be well tolerated and provide an adequate therapeutic index so that patient treatment with an effective dose of nucleic acid is not associated with significant toxicity and / or risk to the patient. Summary of the Invention

[0005] Lipid nanoparticles (LNPs) have been shown to be effective delivery vehicles for mRNA, as demonstrated by the success of the mRNA-based COVID-19 vaccines developed by BioNTech and Moderna. However, intramuscular delivery of LNPs using current formulations results in systemic gene expression, which is associated with rare but significant adverse effects, such as myocarditis.

[0006] LNP compositions called QTsomes combine quaternary amine lipids and tertiary amine lipids in a lipid blend that forms LNPs. When previously investigated, QTsomes demonstrated excellent activity in anti-miR and antisense oligo delivery. However, these LNP compositions were not optimized for intramuscular mRNA delivery.

[0007] Key formulation parameters were examined herein to identify improved QTsome compositions. These LNP compositions can exhibit superior intramuscular mRNA delivery efficiency. In some embodiments, these LNP compositions can also exhibit superior intramuscular mRNA delivery efficiency while reducing concomitant systemic gene expression.

[0008] Through investigation, certain parameters for QTsome design were revealed. For example, the presence of cationic lipids (e.g., quaternary amine-containing lipids, e.g., DOTAP) in LNPs generally reduces systemic gene expression. Increasing the percentage of cationic lipids (e.g., DOTAP) in LNPs generally significantly reduces systemic gene expression. At relatively high mol% cationic lipids (e.g., 5-8% DOTAP), muscle gene expression is reduced along with systemic expression. At relatively low mol% cationic lipids (e.g., 1.5-3% DOTAP), muscle gene expression is not significantly reduced, but systemic gene expression is reduced. This suggests that relatively low mol% cationic lipids (e.g., 0.5 mol%-3.5 mol% or 1.5 mol%-3.0 mol% cationic lipids) are unexpectedly beneficial for achieving high intramuscular mRNA vaccine delivery without inducing side effects associated with systemic delivery, such as myocarditis. This is a major advantage with high potential impact for developing next-generation mRNA vaccines.

[0009] Also, in the case of helper lipids, DOPE has been found to be better in terms of activity than DSPC or DOPC. Thus, in some embodiments, the LNPs described herein can include DOPE as a helper lipid.

[0010] It has also been discovered that preparation of LNPs at elevated temperatures results in a dramatic loss of gene expression. Thus, in some embodiments, the LNP compositions described herein can be prepared at temperatures below 65°C (e.g., below 60°C, below 55°C, below 50°C, below 45°C, below 40°C, below 35°C, or below 30°C, below 25°C, or below 20°C).

[0011] It has also been discovered that a two-step mixing process in which the ethanol content of the lipid side is approximately 25% at the time of mixing results in superior LNP delivery efficiency relative to the single-step 1:3 volumetric mixing that is standard practice in the LNP field. Thus, in some embodiments, the LNP compositions described herein can be prepared using a two-step mixing process in which the ethanol content of the lipid side is approximately 25% at the time of mixing.

[0012] In addition, the ionizable lipid A066 has been found to improve mRNA delivery. Thus, in some embodiments, the LNPs described herein can contain A066. In some embodiments, A066 can be used in place of other ionizable lipids, such as SM-102.

[0013] It has also been discovered that the addition of cationic lipids (e.g., quaternary amine-containing lipids, such as DOTAP) alters the apparent pKa of ionizable lipids as determined by TNS pKa assays. As a result, the addition of cationic lipids (e.g., quaternary amine-containing lipids, such as DOTAP) can be used to fine-tune the apparent pKa value of LNPs, optimizing for local or systemic mRNA delivery and / or targeting delivery to certain tissues in the body.

[0014] Additionally, compositions comprising 1.5-3 mol% of a cationic lipid (e.g., a quaternary amine-containing lipid, e.g., DOTAP), 1.5-2 mol% of a PEGylated lipid (e.g., mPEG-DMG), an ionizable lipid (e.g., A066, DODMA, MC-3, and / or SM-102), and a helper lipid (e.g., DOPE, DSPC, and / or DOPC) have been discovered to be particularly suitable for nucleic acid delivery, including mRNA vaccine delivery, for COVID-19, other viruses, or cancer.

[0015] Thus, provided herein is a pharmaceutical composition comprising lipid particles encapsulating an active agent. The lipid particles can comprise one or more cationic lipids, one or more ionizable lipids, one or more neutral lipids, and one or more PEGylated lipids.

[0016] In some embodiments, the one or more cationic lipids and the one or more ionizable lipids are present in the lipid particle in an amount effective to provide an apparent pKa, as determined by a TNS pKa assay, of 6 to 10, e.g., 6 to 8. The apparent pKa may be determined by a TNS pKa assay, which is calculated using the following formula: Apparent pKa = pKa0 + k × Q / T was defined by pKa represents the pKa of the ionizable lipid; k represents an empirical constant determined by the TNS pKa assay; Q represents the mole % of cationic lipid; T represents the mole % of ionizable lipids.

[0017] In some embodiments, the one or more cationic lipids and the one or more ionizable lipids are present in the lipid particle according to the following formula: 6 <pKa0+k×Q / T<10 was present in an amount that satisfied pKa represents the pKa of the ionizable lipid; k represents an empirical constant determined by the TNS pKa assay; Q represents the mole % of cationic lipid; T represents the mole % of ionizable lipids.

[0018] In some embodiments, the one or more cationic lipids and the one or more ionizable lipids are present in the lipid particle according to the following formula: 6 <pKa0+k×Q / T<8 was present in an amount that satisfied pKa represents the pKa of the ionizable lipid; k represents an empirical constant determined by the TNS pKa assay; Q represents the mole % of cationic lipid; T represents the mole % of ionizable lipids.

[0019] In some embodiments, Q / T can be greater than 0 to 1 (e.g., greater than 0 to 0.25, greater than 0 to 0.5, greater than 0 to 0.75, 0.25 to 0.5, 0.25 to 0.75, 0.25 to 1, 0.5 to 0.75, 0.5 to 1, or 0.75 to 1).

[0020] In some embodiments, one or more cationic lipids are present in the lipid particle in an amount greater than 0 mol% to 10 mol%, based on the total components forming the lipid particle. In certain embodiments (e.g., for local delivery), one or more cationic lipids are present in the lipid particle in an amount of 0.5 mol% to 5 mol%, based on the total components forming the lipid particle. In certain embodiments (e.g., for systemic delivery), one or more cationic lipids are present in the lipid particle in an amount of 4 mol% to 8 mol%, based on the total components forming the lipid particle.

[0021] In some embodiments, the one or more ionizable lipids are present in the lipid particle in an amount of 20 mol % to 65 mol % based on the total components forming the lipid particle.

[0022] In some embodiments, the one or more neutral lipids are present within the lipid particle in an amount of 35 mol % to 80 mol % based on the total components forming the lipid particle.

[0023] In some embodiments, the one or more PEGylated lipids are present within the lipid particle in an amount of greater than 0 mol % to 5 mol % based on the total components forming the lipid particle.

[0024] Also provided is a pharmaceutical composition comprising lipid particles encapsulating an active agent, wherein the lipid particles comprise greater than 0 mol% to 10 mol% (e.g., 0.5 mol% to 10 mol%) of one or more cationic lipids, 20 mol% to 65 mol% of one or more ionizable lipids, 35 mol% to 80 mol% of one or more neutral lipids, and greater than 0 mol% to 5 mol% of one or more PEGylated lipids.

[0025] In certain embodiments (e.g., for local delivery), the one or more cationic lipids are present in the lipid particle in an amount of 0.5 mol% to 3.5 mol% based on the total components forming the lipid particle. In certain embodiments (e.g., for systemic delivery), the one or more cationic lipids are present in the lipid particle in an amount of 4 mol% to 8 mol% based on the total components forming the lipid particle.

[0026] In some embodiments, the one or more ionizable lipids are present in the lipid particle in an amount of 30 mol % to 50 mol % based on the total components forming the lipid particle.

[0027] In some embodiments, the one or more neutral lipids are present within the lipid particle in an amount of 30 mol % to 50 mol % of the total components forming the lipid particle.

[0028] In some embodiments, the one or more PEGylated lipids are present within the lipid particle in an amount of 0.5 mol % to 3 mol % of the total components forming the lipid particle.

[0029] In some cases, one or more ionizable lipids comprise a lipid head group containing a tertiary amine. In some examples, one or more ionizable lipids comprise N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), DLin-MC3-DMA, DLin-KC2-DMA, 1-(2,3-bis(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)propyl)pyrrolidine (A066), or any combination thereof.

[0030] In some examples, the one or more neutral lipids comprise dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), cholesterol, or any combination thereof.

[0031] In some examples, the one or more PEGylated lipids include PEG-ditetradecylacetamide, PEG-myristoyl diglyceride, PEG-diacylglycerol, PEG dialkyloxypropyl, PEG-phospholipid, PEG-ceramide, or any combination thereof.

[0032] In some cases, one or more cationic lipids comprise a lipid head group containing a quaternary amine. In some examples, one or more cationic lipids are selected from the group consisting of DOTMA ([1-(2,3-dioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE (dioleylphosphatidylethanolamine), DOSPA (2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamine). ammonium trifluoroacetate), DORIE (N-[1-(2,3-dioleyloxypropyl)]-N,N-dimethyl-N-hydroxyethylammonium bromide), DODAB, DOIC, DMEPC, DOGS: dioctadecylamidoglycylspermine, DIMRI: dimyristoxypropyldimethylhydroxyethylammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-alpha.-trimethylammonioacetyl)diethanolamine chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, lipids described in U.S. Pat. No. 5,049,386, N-[1-(2,3-dioleyloxypropyl)]-N,N-dimethyl-N-hydroxybenzoates disclosed in WO 91 / 16024 and WO 97 / 019675, and the like; DLinDMA disclosed in WO 2005 / 121348; and DLin-K-DMA disclosed in WO 2009 / 086558; and (3R,4R)-3,4-bis((Z)-hexadec-9-enyloxy)-1-methylpyrrolidine and N-methyl-N,N-bis(2-((Z)-octadec-6-enyloxy)ethyl)amine disclosed in WO 2011 / 13636, or any combination thereof.

[0033] The lipid particles can have an average diameter of less than 1 micron, e.g., 50 nm to 750 nm, 50 nm to 250 nm, 50 nm to 200 nm, 50 nm to 150 nm, or 50 nm to 100 nm. The lipid particles can have a polydispersity index (PDI) of less than 0.4.

[0034] The active agent encapsulated within the lipid particle can include any suitable active agent, such as a small molecule therapeutic agent, a diagnostic agent, a peptide, a protein, an antibody, or a nucleic acid, hi certain embodiments, the active agent can include a nucleic acid, such as an siRNA, an mRNA, or any combination thereof.

[0035] The compositions described herein can be used to deliver one or more active agents to cells (e.g., in vivo, ex vivo, or in vitro). Accordingly, provided herein are methods of delivering an active agent to a cell (e.g., in vivo, ex vivo, or in vitro), comprising contacting the cell with a composition described herein. Also provided are methods for delivering an active agent to a cell in vivo, comprising administering to a mammalian subject (e.g., a human) a composition described herein. In some embodiments, administration can include systemic administration (e.g., intravenous injection or infusion).

[0036] Also provided are methods for systemically administering an active agent to a subject in need thereof. These methods can include intravenously injecting a pharmaceutical composition comprising lipid particles encapsulating the active agent, the lipid particles comprising 0.5 mol% to 8 mol% of one or more cationic lipids, 20 mol% to 65 mol% of one or more ionizable lipids, 35 mol% to 80 mol% of one or more neutral lipids, and greater than 0 mol% to 5 mol% of one or more PEGylated lipids. In some cases, the active agent can include an anticancer drug.

[0037] Also provided are methods for administering an active agent to the liver of a subject. These methods can include intravenously injecting a pharmaceutical composition comprising lipid particles encapsulating the active agent, the lipid particles comprising 0.5 mol% to 3 mol% of one or more cationic lipids, 20 mol% to 65 mol% of one or more ionizable lipids, 35 mol% to 80 mol% of one or more neutral lipids, and greater than 0 mol% to 5 mol% of one or more PEGylated lipids. In some cases, the active agent can include an anti-cancer agent, for example, an active agent for treating liver cancer.

[0038] Also provided are methods for administering an active agent to a solid tumor in a subject. These methods can include intravenously injecting a pharmaceutical composition comprising lipid particles encapsulating the active agent, the lipid particles comprising 3 mol% to 6 mol% of one or more cationic lipids, 20 mol% to 65 mol% of one or more ionizable lipids, 35 mol% to 80 mol% of one or more neutral lipids, and greater than 0 mol% to 5 mol% of one or more PEGylated lipids. In some cases, the active agent can comprise an anticancer drug. These methods can allow the lipid particles to selectively target tumor angiogenesis.

[0039] Also provided are methods for administering an active agent to the lungs of a subject. These methods can include intravenously injecting a pharmaceutical composition comprising lipid particles encapsulating the active agent, the lipid particles comprising 6 mol% to 10 mol% of one or more cationic lipids, 20 mol% to 65 mol% of one or more ionizable lipids, 35 mol% to 80 mol% of one or more neutral lipids, and greater than 0 mol% to 5 mol% of one or more PEGylated lipids. In some cases, the active agent can include an anti-cancer agent, e.g., an active agent for the treatment of lung cancer.

[0040] Also provided are methods for locally administering an active agent to a subject in need thereof. These methods can include intramuscularly injecting a pharmaceutical composition comprising lipid particles encapsulating an active agent, the lipid particles comprising greater than 0 mol% to 5 mol% of one or more cationic lipids, 20 mol% to 65 mol% of one or more ionizable lipids, 35 mol% to 80 mol% of one or more neutral lipids, and greater than 0 mol% to 5 mol% of one or more PEGylated lipids. In some cases, the active agent can comprise a nucleic acid (e.g., siRNA, mRNA, or any combination thereof). In some cases, the active agent can comprise a vaccine. [Brief explanation of the drawings]

[0041] [Figure 1A]1 is a plot summarizing efforts to optimize cationic lipids for QTsome-based mRNA delivery. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 1B] 1 is a plot summarizing efforts to optimize cationic lipids for QTsome-based mRNA delivery. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 1C] 1 is a plot summarizing efforts to optimize cationic lipids for QTsome-based mRNA delivery. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 1D] 1 is a plot summarizing efforts to optimize ionizable lipids for QTsome-based mRNA delivery. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 1E] 1 is a plot summarizing efforts to optimize helper lipids for QTsome-based mRNA delivery. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 1F] 1 is a plot summarizing attempts to optimize formulation procedures for QTsome-based mRNA delivery. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 1G] 10 is a plot summarizing attempts to optimize temperature control for QTsome-based mRNA delivery. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 2A] 1 shows the results of particle characterization of QTPlus-AM21. Particle size is shown for QTsome Original or QTPlus encapsulating AM21. [Figure 2B] 1 shows particle characterization results for QTPlus-AM21. 2 shows cryo-EM images for QTPlus-AM21. [Figure 2C]1 shows particle characterization results of QTPlus-AM21. 1% agarose gel electrophoresis of QTPlus-AM21 with different nitrogen-to-phosphate (N / P) ratios is shown. [Figure 3A] Figure 1 shows miR-21 downstream gene regulation by 16mer and 20mer AM21 in free solution. [Figure 3B] 1 shows the regulation of downstream genes of miR-21 by 16mer and 20mer AM21 in QTPlus. [Figure 4A] The 16mer and 20mer AM21 are compared in the QTPlus-AM21 formulation. [Figure 4B] The 16mer and 20mer AM21 are compared in tumor growth inhibition in vitro. [Figure 4C] The 16mer and 20mer AM21 are compared in tumor growth inhibition in vitro. [Figure 5A] The ability of AM21 (FIG. 5B) and QTsome Original or QTPlus-AM21 (FIG. 5C) to regulate miR-21 downstream genes (FIG. 5A) is shown. [Figure 5B] The ability of AM21 (FIG. 5B) and QTsome Original or QTPlus-AM21 (FIG. 5C) to regulate miR-21 downstream genes (FIG. 5A) is shown. [Figure 5C] The ability of AM21 (FIG. 5B) and QTsome Original or QTPlus-AM21 (FIG. 5C) to regulate miR-21 downstream genes (FIG. 5A) is shown. [Figure 6A] 1 demonstrates that QTPlus-AM21 exhibited superior tumor growth inhibition in vitro compared to free AM21. [Figure 6B] 1 demonstrates that QTPlus-AM21 exhibited superior tumor growth inhibition in vitro compared to free AM21. [Figure 6C] 10 is a plot demonstrating that QTPlus-AM21 sensitized A549 to erlotinib chemotherapy. [Figure 7]1 is a plot showing the acute cytotoxicity of QTPlus-AM21. [Figure 8A] Figure 1 shows in vitro PTEN and EGFR expression in QTPlus-AM21 and erlotinib-treated A549 tumor models. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 8B] Figure 1 shows in vitro PTEN and EGFR expression in QTPlus-AM21 and erlotinib-treated A549 tumor models. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 8C] Figure 1 shows in vivo PTEN and EGFR expression in QTPlus-AM21 and erlotinib-treated A549 tumor models. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 8D] Figure 1 shows in vivo PTEN and EGFR expression in QTPlus-AM21 and erlotinib-treated A549 tumor models. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 9] 1 is a plot showing the in vivo antitumor activity of QTsome Original-AM21 in an A549 xenograft mouse model. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 10] 1 is a plot showing the in vivo antitumor activity of QTPlus-AM21 and erlotinib in an A549 xenograft mouse model. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 11A] Figure 11 shows that QTPlus-AM21 regulates PD1 / PD-L1 expression in THP-1 (Figure 11A) and RAW264.7 (Figure 11B) cell lines and induces macrophage polarization in THP-1 (Figure 11C) and RAW264.7 (Figure 11D) cell lines. [Figure 11B]Figure 11 shows that QTPlus-AM21 regulates PD1 / PD-L1 expression in THP-1 (Figure 11A) and RAW264.7 (Figure 11B) cell lines and induces macrophage polarization in THP-1 (Figure 11C) and RAW264.7 (Figure 11D) cell lines. [Figure 11C] Figure 11 shows that QTPlus-AM21 regulates PD1 / PD-L1 expression in THP-1 (Figure 11A) and RAW264.7 (Figure 11B) cell lines and induces macrophage polarization in THP-1 (Figure 11C) and RAW264.7 (Figure 11D) cell lines. [Figure 11D] Figure 11 shows that QTPlus-AM21 regulates PD1 / PD-L1 expression in THP-1 (Figure 11A) and RAW264.7 (Figure 11B) cell lines and induces macrophage polarization in THP-1 (Figure 11C) and RAW264.7 (Figure 11D) cell lines. [Figure 12] 1 shows that QTPlus-AM21 induces pro-inflammatory cytokines and chemokines in macrophages in vitro. [Figure 13A] QTPlus-AM21 inhibits tumor growth by modulating macrophages in vitro. QTPlus-AM21-treated RAW264.7 cells inhibit wound healing by MC38 colorectal cancer cells. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 13B] QTPlus-AM21 inhibits tumor growth by modulating macrophages in vitro. QTPlus-AM21 inhibits tumor growth by modulating RAW264.7 cells and enhancing apoptosis in MC38 cells. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 13C] These results demonstrate that QTPlus-AM21 inhibits tumor growth by modulating macrophages in vitro. QTPlus-AM21 also induced macrophage proliferation. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 13D]QTPlus-AM21 inhibits tumor growth by modulating macrophages in vitro. QTPlus-AM21 also induced population polarization. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 14] 1 is a plot showing the in vivo antitumor activity of QTPlus-AM21 in the MC38 syngeneic mouse model. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 15A] 1 shows that QTPlus-AM21 increased CD45+ tumor-infiltrating immune cells in the tumor microenvironment in vivo. [Figure 15B] 1 shows that QTPlus-AM21 increased CD45+ tumor-infiltrating immune cells in the tumor microenvironment in vivo. [Figure 15C] 1 shows that QTPlus-AM21 increased the CD86+M1 population in the tumor microenvironment in vivo. [Figure 15D] 1 shows that QTPlus-AM21 increased the CD86+M1 population in the tumor microenvironment in vivo. [Figure 16A] 1 shows that QTPlus-AM21 induced CXCL10, IFNa, and TNFα in spleen tissue. [Figure 16B] 1 shows that QTPlus and QTPlus regulated PD1 / PD-L1 expression in mouse tumors. [Figure 16C] 1 shows that QTPlus and QTPlus regulated PD1 / PD-L1 expression in spleen tissue. [Figure 17] 1 is a plot showing the in vivo antitumor activity of QTPlus-AM21 and atezolizumab in the MC38 syngeneic mouse model. One-way ANOVA: *p<0.05, **:p<0.01, ***p<0.001. [Figure 18] FIG. 1 is a schematic diagram of a method for preparing QTsomes. [Figure 19]10 is a plot showing firefly luciferase bioluminescence intensity measurements using a Bright-Glo luciferase kit in HEK293T cells after treatment with 100 ng of FFLuc mRNA using QTsome+ lipid nanoparticles. [Figure 20] 10 is a plot of firefly luciferase bioluminescence intensity measurements using a Bright-Glo luciferase kit in HEK293T cells after treatment with 100 ng of FFLuc mRNA using QTsome+ lipid nanoparticles. [Figure 21A] 1 shows the results of particle size analysis of firefly luciferase mRNA encapsulated QTsome+ lipid nanoparticles. [Figure 21B] 1 shows the results of particle size analysis of firefly luciferase mRNA encapsulated QTsome+ lipid nanoparticles. [Figure 22A] 1 shows the particle size of QTsome+ lipid nanoparticles encapsulating beta-galactosidase mRNA. [Figure 22B] 1 shows zeta potential analysis of QTsome+ lipid nanoparticles encapsulating beta-galactosidase mRNA. [Figure 22C] 1 shows a beta-galactosidase activity assay of QTsome+ lipid nanoparticles encapsulating beta-galactosidase mRNA. [Figure 23] 1 is a plot showing results obtained using firefly messenger RNA encapsulated QTsome+ lipid nanoparticles tested in A549 NSCLC cells. [Figure 24] Photographs showing encapsulation efficiency determination by agarose gel electrophoresis. [Figure 25] The apparent pKa of FFLuc mRNA-encapsulated QTsome+ lipid nanoparticles as determined by TNS assay is plotted. [Figure 26A] The in vivo and in vitro delivery results obtained using DSPC + 1.5% DOTAP and DOPE + 1.5% DOTAP are compared. [Figure 26B]The in vivo and in vitro delivery results obtained using DSPC + 1.5% DOTAP and DOPE + 1.5% DOTAP are compared. [Figure 27] 1 shows in vivo mRNA expression by IM injection as imaged by the IVIS system. [Figure 28] The results of the overall testing of FFLuc mRNA-encapsulated QTsome+ lipid nanoparticles via IM injection in vivo are summarized below. Bioluminescence was monitored by an IVIS system. [Figure 29] Modulation and downstream effects of the Akt signaling pathway are shown. [Figure 30A] Intensity-weighted DLS diameter measurements of empty QTsome+. [Figure 30B] Intensity-weighted DLS diameter measurements of encapsulated QTsome+. [Figure 31] Cryo-TEM micrograph of siAkt1 QTsome+. QTsome+ has a bilayered structure with smooth particle curvature. [Figure 32A] 1 shows the effect of siRNA-Akt on different targets in human liver cancer cell lines. Relative mRNA expression levels after transfection of 25 nM siRNA-scrambled and siRNA-Akt in Hep3B, huh7, SNU387, and HepG2 are shown. Si-Akt encapsulated by Lipofectamine 3000 was normalized to siRNA-scrambled encapsulated by Lipofectamine 3000, and siRNA-Akt encapsulated by QTsome+ was normalized to siRNA-scrambled encapsulated by Lipofectamine 3000. [Figure 32B]1 shows the effect of siRNA-Akt on different targets in human liver cancer cell lines. Relative mRNA expression levels after transfection of 25 nM siRNA-scrambled and siRNA-Akt in Hep3B, huh7, SNU387, and HepG2 are shown. Si-Akt encapsulated by Lipofectamine 3000 was normalized to siRNA-scrambled encapsulated by Lipofectamine 3000, and siRNA-Akt encapsulated by QTsome+ was normalized to siRNA-scrambled encapsulated by Lipofectamine 3000. [Figure 32C] Figure 1 shows how protein expression was demonstrated by Western blot analysis to assess the levels of different proteins in Hep3B and huh-7. β-actin served as an internal reference for both RT-PCR and Western blot assays. [Figure 32D] FIG. 1 shows how IL-6 and TNF-α release in human PBMCs was analyzed via ELISA. [Figure 32E] 1 shows how cell cycle was determined and analysis via flow cytometry. [Figure 33A] 1 is a plot showing the antitumor effect in male athymic BALB / C mice inoculated with the huh7 xenograft model. Doses were given via IV injection on days 1, 4, 7, and 10. Treatment began when tumor size was up to 100-150 mm3. Drug was given every 3 days via IV injection. Tumor size was efficiently suppressed by siRNA-Akt1 compared to the saline and siRNA control groups. Tumor size was calculated using the equation:

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[0042] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0043] "Aqueous solution" refers to a composition that comprises, in whole or in part, water.

[0044] "Organic lipid solution" refers to a composition that comprises, in whole or in part, an organic solvent with lipids. In some embodiments, the organic lipid solution can comprise an alkanol, most preferably ethanol. In certain embodiments, the compositions described herein can be free of organic solvents, such as ethanol.

[0045] "Lipid" refers to a group of organic compounds that are esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents, such as fats, oils, waxes, phospholipids, glycolipids, and steroids.

[0046] "Amphipathic lipids" include lipids whose hydrophilic character is derived from the presence of polar or charged groups, such as carbohydrate, phosphato, carboxy, sulfato, amino, sulfhydryl, nitro, hydroxy, and other such groups, and whose hydrophobic character can be imparted by the inclusion of polar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, as well as such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Examples include phospholipids, aminolipids, and sphingolipids. Phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Amphipathic lipids can also lack phosphorus, such as sphingolipids, glycosphingolipid families, diacylglycerols, and b-acyloxyacids.

[0047] An "anionic lipid" is any lipid that has a negative charge at physiological pH, and includes phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups linked to neutral lipids.

[0048] "Cationic lipids" carry a net positive charge at a selective pH, e.g., physiological pH, and include N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"), N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTMA"), N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (" Examples of cationic lipids include 3-(N-(N',N'-dimethylaminoethane)-carbamoyldicholesterol ("DC-Chol"), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"). Additionally, several commercially available preparations of cationic lipids are available that can be used in the present invention. These include, for example, LIPOFECTIN®, LIPOFECT AMINE®, and TRANSFECTAM®.

[0049] Pharmaceutical Compositions Provided herein is a pharmaceutical composition comprising lipid particles encapsulating an active agent. The lipid particles can comprise one or more cationic lipids, one or more ionizable lipids, one or more neutral lipids, and one or more PEGylated lipids.

[0050] In some embodiments, the one or more cationic lipids and the one or more ionizable lipids are present in the lipid particle in an amount effective to provide an apparent pKa, as determined by a TNS pKa assay, of 6 to 10, e.g., 6 to 8. The apparent pKa may be determined by a TNS pKa assay, which is calculated using the following formula: Apparent pKa = pKa0 + k × Q / T was defined by pKa represents the pKa of the ionizable lipid; k represents an empirical constant determined by the TNS pKa assay; Q represents the mole % of cationic lipid; T represents the mole % of ionizable lipids.

[0051] In some embodiments, the one or more cationic lipids and the one or more ionizable lipids are present in the lipid particle according to the following formula: 6 <pKa0+k×Q / T<10 was present in an amount that satisfied pKa represents the pKa of the ionizable lipid; k represents an empirical constant determined by the TNS pKa assay; Q represents the mole % of cationic lipid; T represents the mole % of ionizable lipids.

[0052] In some embodiments, the one or more cationic lipids and the one or more ionizable lipids are present in the lipid particle according to the following formula: 6 <pKa0+k×Q / T<8 was present in an amount that satisfied pKa represents the pKa of the ionizable lipid; k represents an empirical constant determined by the TNS pKa assay; Q represents the mole % of cationic lipid; T represents the mole % of ionizable lipids.

[0053] In some embodiments, Q / T can be greater than 0 to 1 (e.g., greater than 0 to 0.25, greater than 0 to 0.5, greater than 0 to 0.75, 0.25 to 0.5, 0.25 to 0.75, 0.25 to 1, 0.5 to 0.75, 0.5 to 1, or 0.75 to 1).

[0054] In some embodiments, one or more cationic lipids are present in the lipid particle in an amount greater than 0 mol% to 10 mol%, based on the total components forming the lipid particle. In certain embodiments (e.g., for local delivery), one or more cationic lipids are present in the lipid particle in an amount of 0.5 mol% to 5 mol%, based on the total components forming the lipid particle. In certain embodiments (e.g., for systemic delivery), one or more cationic lipids are present in the lipid particle in an amount of 4 mol% to 8 mol%, based on the total components forming the lipid particle.

[0055] In some embodiments, the one or more ionizable lipids are present in the lipid particle in an amount of 20 mol % to 65 mol % based on the total components forming the lipid particle.

[0056] In some embodiments, the one or more neutral lipids are present within the lipid particle in an amount of 35 mol % to 80 mol % based on the total components forming the lipid particle.

[0057] In some embodiments, the one or more PEGylated lipids are present within the lipid particle in an amount of greater than 0 mol % to 5 mol % based on the total components forming the lipid particle.

[0058] Also provided is a pharmaceutical composition comprising lipid particles encapsulating an active agent, wherein the lipid particles comprise greater than 0 mol% to 10 mol% (e.g., 0.5 mol% to 10 mol%) of one or more cationic lipids, 20 mol% to 65 mol% of one or more ionizable lipids, 35 mol% to 80 mol% of one or more neutral lipids, and greater than 0 mol% to 5 mol% of one or more PEGylated lipids.

[0059] In certain embodiments (e.g., for local delivery), the one or more cationic lipids are present in the lipid particle in an amount of 0.5 mol% to 3.5 mol% based on the total components forming the lipid particle. In certain embodiments (e.g., for systemic delivery), the one or more cationic lipids are present in the lipid particle in an amount of 4 mol% to 8 mol% based on the total components forming the lipid particle.

[0060] In some embodiments, the one or more ionizable lipids are present in the lipid particle in an amount of 30 mol % to 50 mol % based on the total components forming the lipid particle.

[0061] In some embodiments, the one or more neutral lipids are present within the lipid particle in an amount of 30 mol % to 50 mol % of the total components forming the lipid particle.

[0062] In some embodiments, the one or more PEGylated lipids are present within the lipid particle in an amount of 0.5 mol % to 3 mol % of the total components forming the lipid particle.

[0063] The lipid particles can have an average diameter of less than 1 micron, e.g., 50 nm to 750 nm, 50 nm to 250 nm, 50 nm to 200 nm, 50 nm to 150 nm, or 50 nm to 100 nm. The lipid particles can have a polydispersity index (PDI) of less than 0.4.

[0064] The components of these compositions are described in more detail below.

[0065] Ionizable lipids As mentioned above, the compositions described herein can comprise one or more ionizable lipids. " Ionizable lipids " are lipids that have a pH-dependent charge. One or more ionizable lipids in the compositions described herein can comprise ionizable cationic lipids that have a positive or neutral charge depending on pH.

[0066] Generally, in lipid-based formulations for nucleic acid delivery, either cationic lipids or ionizable lipids can be used to enable electrostatic interactions with negatively charged cargoes. Cationic lipids are defined as lipids that carry a permanent positive charge(s), typically derived from a quaternary amine. Examples of cationic lipids include DOTAP, DOTMA, DDAB, and DODAC. In contrast, ionizable lipids contain chemical moieties, such as tertiary amine(s), that are positively charged at acidic pH but uncharged at neutral to basic pH. Ionizable lipids can have pKa values ​​in a biologically relevant range. However, the pKa value of such lipids is highly dependent on the method used to measure it, resulting in a difference of up to 3 units in value for the same lipid. This is demonstrated in a recent paper by Carrasco et al., Communications Biology, Vol. 4, Paper No. 956 (2021).

[0067] Examples of ionizable lipids are DODMA (N,N-dimethyl-2,3-dioleyloxypropylamine), DODAP, DLinDMA (1,2-dilinoleyloxy-3-dimethylaminopropane), DLinMC3DMA (dilinoleylmethyl-4-dimethylaminobutyrate), DLinKC2DMA (2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), ALC-0315 ([(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)-1,1-dioxolane), and methyl ... and the like. Examples of suitable hydroxybenzoates include 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate), SM-102 (9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate), Merck-32 (see, e.g., WO2012 / 018754), Acuitas-5 (see, e.g., WO2015 / 199952), KL-10 (see, e.g., U.S. Patent Application Publication No. 2012 / 0295832), and C12-200 (see, e.g., Love, KT et al., PNAS, 107:1864 (2009)). Ionizable lipids also include those disclosed in U.S. Pat. Nos. 8,158,601, 9,593,077, 9,365,610, 9,567,296, 9,580,711, and 9,670,152, WO 2012 / 018754, WO 2015 / 199952, WO 2019 / 191780, and U.S. Patent Application Publication Nos. 2012 / 0295832, 2017 / 0190661, and 2017 / 0114010, each of which is incorporated herein by reference in its entirety.

[0068] In some embodiments, one or more ionizable lipids can comprise a lipid head group containing a tertiary amine. In certain embodiments, one or more ionizable lipids can comprise N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), MC-3, KC-2, or any combination thereof.

[0069] In some embodiments, the one or more ionizable lipids constitute at least 20 mol% (e.g., at least 25 mol%, at least 30 mol%, at least 35 mol%, at least 40 mol%, at least 45 mol%, at least 50 mol%, at least 55 mol%, or at least 60 mol%) of the total components forming the lipid particle. In some embodiments, the one or more ionizable lipids constitute 65 mol% or less (e.g., 60 mol% or less, 55 mol% or less, 50 mol% or less, 45 mol% or less, 40 mol% or less, 35 mol% or less, 30 mol% or less, or 25 mol%) of the total components forming the lipid particle.

[0070] The one or more ionizable lipids are present in the lipid particle in an amount ranging from any of the minimum values ​​described above to any of the maximum values ​​described above. For example, in some embodiments, the one or more ionizable lipids are present in the lipid particle in an amount ranging from 20 mol % to 65 mol % (e.g., 30 mol % to 50 mol %) of the total components forming the lipid particle.

[0071] neutral lipid As noted above, the compositions described herein can include one or more neutral lipids.

[0072] Examples of neutral lipids include phospholipids, such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (DPPG ... Examples of suitable phospholipids include diacylphosphatidylethanolamine (POPE), palmitoyloleyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0073] Additional examples of neutral lipids include sterols, such as cholesterol and its derivatives.Non-limiting examples of cholesterol derivatives include polar analogs, such as 5a-cholestanol, 5a-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs, such as 5a-cholestan, cholestenone, 5a-cholestanone, 5a-cholestanone, and cholesteryl decanoate; and mixtures thereof.In a preferred embodiment, the cholesterol derivative is polar analog, such as cholesteryl-(4'-hydroxy)-butyl ether. Other examples of neutral lipids include non-phosphorous acid-containing lipids, such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkylaryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and sphingomyelin.

[0074] In some embodiments, the one or more neutral lipids can include dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), cholesterol, or any combination thereof.

[0075] In some embodiments, the one or more neutral lipids constitute at least 35 mol% (e.g., at least 40 mol%, at least 45 mol%, at least 50 mol%, at least 55 mol%, at least 60 mol%, at least 65 mol%, at least 70 mol%, or at least 75 mol%) of the total components forming the lipid particle. In some embodiments, the one or more neutral lipids constitute 80 mol% or less (e.g., 75 mol% or less, 70 mol% or less, 65 mol% or less, 60 mol% or less, 55 mol% or less, 50 mol% or less, 45 mol% or less, or 40 mol%) of the total components forming the lipid particle.

[0076] The one or more neutral lipids are present in the lipid particle in an amount ranging from any of the above minimum values ​​to any of the above maximum values. For example, in some embodiments, the one or more neutral lipids are present in the lipid particle in an amount of 35 mol % to 80 mol % (30 mol % to 50 mol %) of the total components forming the lipid particle.

[0077] PEGylated lipids As noted above, the compositions described herein can include one or more PEGylated lipids, which are useful in that they can reduce or prevent aggregation of lipid particles.

[0078] PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified according to their molecular weight and include the following: monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol succinate (MePEG-S), monomethoxypolyethyleneglycol succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethyleneglycol amine (MePEG-NEh), monomethoxypolyethyleneglycol tresylate (MePEG-TRES), monomethoxypolyethyleneglycol imidazolyl carbonyl (MePEG-IM), and compounds containing terminal hydroxyl groups instead of terminal methoxy groups (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH).

[0079] Examples of PEG-lipids include, but are not limited to, PEG conjugated to dialkyloxypropyl (PEG-DAA), PEG conjugated to diacylglycerol (PEG-DAG), PEG conjugated to phospholipids, such as phosphatidylethanolamine (PEG-PE), PEG conjugated to glycerides to form glycols, such as 1,2-dimyristoyl-sn-glycerol, methoxy-PEG glycol (PEG-DMG), PEG conjugated to ceramide, PEG conjugated to cholesterol, or derivatives thereof and mixtures thereof. In some examples, one or more PEGylated lipids can include, for example, PEG-ditetradecylacetamide, PEG-myristoyl diglyceride, PEG-diacylglycerol, PEG-dialkyloxypropyl, PEG-phospholipid, PEG-ceramide, or any combination thereof.

[0080] The PEG moiety of the PEG-lipid conjugates described herein can comprise an average molecular weight ranging from 550 daltons to 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of 750 daltons to 5,000 daltons (e.g., 1,000 daltons to 5,000 daltons, 1,500 daltons to 3,000 daltons, 750 daltons to 3,000 daltons, 750 daltons to 2,000 daltons). In some embodiments, the PEG moiety has an average molecular weight of 2,000 daltons or 750 daltons.

[0081] In certain cases, PEG can be optionally substituted with alkyl, alkoxy, acyl, or aryl groups.PEG can be directly conjugated to lipid, or can be linked to lipid via a linker moiety.Any linker moiety suitable for linking PEG to lipid can be used, and linker moieties include, for example, non-ester-containing linker moieties and ester-containing linker moieties.In one embodiment, linker moiety is a non-ester-containing linker moiety. Suitable non-ester-containing linker moieties include, but are not limited to, amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-0-), succinyl (-(0)CCH2CH2C(0)-), succinamidyl (-NHC(0)CH2CH2C(0)NH-), ether, disulfide, and combinations thereof (e.g., linkers containing both carbamate and amide linker moieties). In some embodiments, a carbamate linker may be used to attach PEG to a lipid. In other embodiments, an ester-containing linker moiety may be used to attach PEG to a lipid. Suitable ester-containing linker moieties include, for example, carbonate (-OC(O)O-), succinoyl, phosphate (-O-(O)POH-O-), sulfonate, and combinations thereof.

[0082] The term "diacylglycerol" or "DAG" refers to a diacylglycerol consisting of two fatty acyl chains, R 1 and R 2 A compound having R 1 and R 2 and (iii) independently include compounds having 2 to 30 carbons attached by ester bonds to the first and second positions of glycerol. The acyl group may be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C12), myristoyl (C14), palmitoyl (C16), stearoyl (C1s), and icosoyl (C20). In a preferred embodiment, R 1 and R 2 are the same, i.e., R 1 and R 2 are both myristoyl (i.e., dimyristoyl), and R 1 and R 2 are both stearoyl (i.e., distearoyl).

[0083] The term "dialkyloxyalkyl" or "DAA" includes compounds having two alkyl chains, R and R', where both R and R' independently have 2 to 30 carbons. The alkyl groups can be saturated or have varying degrees of unsaturation.

[0084] Examples of PEG-DAA conjugates include PEG-didecyloxypropyl (C10), PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dipalmitoyloxypropyl (C16), and PEG-distearyloxypropyl (C18). In some of these embodiments, the PEG can have an average molecular weight of 750 or 2,000 daltons. In certain embodiments, the terminal hydroxyl group of the PEG can be substituted with a methyl group.

[0085] In addition to the above, other hydrophilic polymers can be used instead of PEG. Examples of suitable polymers that can be used instead of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized cellulose, such as hydroxymethylcellulose or hydroxyethylcellulose.

[0086] In some embodiments, one or more PEGylated lipids constitute more than 0 mol% (e.g., at least 0.5 mol%, at least 1 mol%, at least 1.5 mol%, at least 2 mol%, at least 2.5 mol%, at least 3 mol%, at least 3.5 mol%, at least 4 mol%, or at least 4.5 mol%) of the total components forming the lipid particle. In some embodiments, one or more PEGylated lipids constitute 5 mol% or less (e.g., 4.5 mol% or less, 4 mol% or less, 3.5 mol% or less, 3 mol% or less, 2.5 mol% or less, 2 mol% or less, 1.5 mol% or less, 1 mol% or less, or 0.5 mol% or less) of the total components forming the lipid particle.

[0087] The one or more PEGylated lipids are present in the lipid particle in an amount ranging from any of the above minimum values ​​to any of the above maximum values, for example, in some embodiments, the one or more PEGylated lipids are present in the lipid particle in an amount of greater than 0 mol % to 5 mol % of the total components forming the lipid particle.

[0088] cationic lipids As mentioned above, the lipid blends described herein can include one or more cationic lipids (e.g., lipids carrying a quaternary ammonium moiety). Examples of cationic lipids include, for example, DOTMA: [1-(2,3-thioleyloxy)propyl]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: dioleylphosphatidylethanolamine, DOSPA (2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trimethylammonium chloride), and the like. fluoroacetate), DORIE (N-[1-(2,3-dioleyloxypropyl)]-N,N-dimethyl-N-hydroxyethylammonium bromide), DODAB, DOIC, DMEPC, DOGS: dioctadecylamidoglycylspermine, DIMRI: dimyristoxypropyldimethylhydroxyethylammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-alpha.N-[1-(2,3-dioleyloxypropyl)]-N,N-dimethylammonium chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, lipids described in U.S. Pat. No. 5,049,386, N-[1-(2,3-dioleyloxypropyl)]-N,N-dimethylammonium chloride disclosed in WO 91 / 16024 and WO 97 / 019675, Examples of suitable amines include diethyl-N-hydroxyethylammonium bromide (DORIE), 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), DLinDMA disclosed in WO 2005 / 121348, DLin-K-DMA disclosed in WO 2009 / 086558, and (3R,4R)-3,4-bis((Z)-hexadec-9-enyloxy)-1-methylpyrrolidine and N-methyl-N,N-bis(2-((Z)-octadec-6-enyloxy)ethyl)amine disclosed in WO 2011 / 13636.

[0089] In some embodiments, the one or more lipids comprise more than 0 mol% (e.g., at least 0.5 mol%, at least 1 mol%, at least 1.5 mol%, at least 2 mol%, at least 2.5 mol%, at least 3 mol%, at least 3.5 mol%, at least 4 mol%, at least 4.5 mol%, at least 5 mol%, at least 5.5 mol%, at least 6 mol%, at least 6.5 mol%, at least 7 mol%, at least 7.5 mol%, at least 8 mol%, at least 8.5 mol%, at least 9 mol%, or at least 9.5 mol%) of the total components forming the lipid particle. In some embodiments, the one or more PEGylated lipids constitute 10 mol% or less (e.g., 9.5 mol% or less, 9 mol% or less, 8.5 mol% or less, 8 mol% or less, 7.5 mol% or less, 7 mol% or less, 6.5 mol% or less, 6 mol% or less, 5.5 mol% or less, 5 mol% or less, 4.5 mol% or less, 4 mol% or less, 3.5 mol% or less, 3 mol% or less, 2.5 mol% or less, 2 mol% or less, 1.5 mol% or less, 1 mol% or less, or 0.5 mol% or less) of the total components forming the lipid particle.

[0090] The one or more PEGylated lipids are present in the lipid particle in an amount ranging from any of the above minimum values ​​to any of the above maximum values. For example, in some embodiments, the one or more PEGylated lipids are present in the lipid particle in an amount greater than 0 mol% to 10 mol% (e.g., 0.5 mol% to 10 mol%, 0.5 mol% to 8 mol%, 0.5 mol% to 3.5 mol%, 4 mol% to 8 mol%, 0.5 mol% to 3 mol%, 3 mol% to 6 mol%, or 6 mol% to 10 mol%) of the total components forming the lipid particle.

[0091] activator As used herein, "active agent" refers to a therapeutic, diagnostic, or prophylactic agent. As discussed herein, therapeutic agents can be released from the disclosed compounds, compositions, and systems in a biologically active form.

[0092] As used herein, the term "therapeutic agent" is further understood to refer to one or more therapeutic agents, active ingredients, or substances that can be used to treat a medical condition. A therapeutic agent includes any synthetic or naturally occurring biologically active compound or composition that, when administered to an organism (human or non-human animal), induces a desired pharmacological, immunogenic, and / or physiological effect through local and / or systemic action. Thus, the term encompasses compounds or chemicals traditionally thought of as drugs, vaccines, and biopharmaceuticals, including molecules such as proteins, peptides, hormones, nucleic acids, genetic constructs, and the like. Examples of therapeutic agents are described in well-known references, such as the Merck Index (14th ed.), Physicians' Desk Reference (64th ed.), and The Pharmacological Basis of Therapeutics (12th ed.), and include, but are not limited to, medicines; vitamins and minerals, such as essential amino acids, calcium, iron, potassium, zinc, vitamin B12, and the like; substances used to treat, prevent, diagnose, cure, or mitigate a disease or disorder; substances that affect the structure or function of the body; or prodrugs that become biologically active or more active after being placed in a physiological environment. For example, the term "therapeutic agent" includes compounds or compositions for use in all of the major therapeutic areas, including adjuvants; antimicrobial agents (including antibiotics, antivirals, antiparasitics, and antifungals), anti-inflammatory agents (including steroids and nonsteroidal anti-inflammatory agents), anticoagulants, eye drops, gastrointestinal agents, antiplatelet agents, and antiseptics, steroidal drugs, antitumor agents, anticancer agents, antigens, antibodies (e.g., cetuximab, anti-CD24 antibodies, panitumumab), and the like. , and bevacizumab), birth control agents, progestational agents, anticholinergic agents, nutritional agents, analgesics, and analgesic combinations, such as acetaminophen and acetylsalicylic acid; anesthetics, such as lidocaine and xylocaine, anorexics, such as dexadrine and phendimetrazine tartrate; antiepileptics, local and general anesthetics, hypnotics, sedatives, antipsychotics, neuroleptics, antidepressants, such as isocarboxazid and amoxapine;Anti-anxiety drugs, antagonists, neuron blocking agents, anticholinergic and cholinergic agents, antimuscarinic and muscarinic agents, anti-Parkinson's agents, anti-Alzheimer's agents, anti-adrenergic agents, anti-arrhythmic agents, antihypertensive agents, hormones such as insulin, progestins, estrogens, corticoids, glucocorticoids, androgens, etc.; and nutrients, anti-arthritic drugs such as methylprednisolone, ibuprofen, etc.; anti-asthmatic drugs such as terbutaline sulfate, theophylline. , ephedrine, etc.; anticonvulsants, such as phenyloin sodium, diazepam, etc.; antiallergic drugs, antihistamines, such as diphenhydramine HCl, chlorpheniramine maleate, etc.; antiemetics, antineoplastic drugs, antipruritics, antipyretics; antispasmodics, such as belladonna alkaloids, dicyclomine hydrochloride, etc.; cardiovascular drugs, such as prazosin HCl, nitroglycerin, propranolol HCl, hydralazine HCl, pancrelipase, succinic acid dehydrogenase genases and the like; vasoactive agents, cardiovascular preparations (including calcium channel blockers, beta blockers, beta agonists, and antiarrhythmics), antihypertensives, diuretics such as furosemide, spironolactone, and the like; vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostic agents; bone growth stimulants and bone resorption inhibitors; muscle relaxants; psychostimulants; sedatives; tranquilizers such as thorazine, diazepam, chlorpromazine HCl, reserpine, chlordiazepoxide HCl, etc.; antiulcer drugs, such as rantidine HCl, cimetidine HCl, etc.; antiasthmatics, antidiarrheals, antiobesity agents, antithrombotic agents, antitussives, antiuricemic agents, antianginal agents, appetite suppressants, expectorants, hyperglycemic agents, hypoglycemic agents, thyroid and antithyroid agents, tissue growth agents, uterine relaxants, immunomodulators, including cytokines, interleukins, interferons, colony stimulating factors, tumor necrosis factors, etc.; immunosuppressants, such as rapamycin, tacrolimus, etc.; immunological agents;Antigens, factors, growth factors, amino acids, peptides, and proteins, and fragments thereof (whether naturally occurring, chemically synthesized, or recombinantly produced), such as LHRH, somatostatin, calcitonin, growth hormone, glucagon-like peptides, growth hormone-releasing factor, angiotensin, FSH, EGF, bone morphogenetic proteins (BMPs), erythropoietin (EPO), interferons, interleukins, collagen, fibrinogen, insulin, Factor VIII, Factor IX, Enbrel®, Rituxam®, Herceptin®, alpha glucosidase, Ceraz Drugs include, but are not limited to, cerebrospinal fluid (Ceredose®), vasopressin, ACTH, human serum albumin, gamma globulin, structural proteins, blood product proteins, complex proteins, antigens or antigenic polypeptides, enzymes, antibodies, monoclonal antibodies, etc.; and nucleic acid molecules (polymeric forms of two or more nucleotides, polynucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA), including both double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, etc.), small molecules (e.g., doxorubicin), and other biologically active macromolecules, such as proteins and enzymes. Drugs can be biologically active agents used in medical applications, including veterinary medicine, and in agriculture, such as plants, and other areas. In certain embodiments of the present disclosure, the drug to be delivered can be a mixture of active agents.

[0093] Representative examples of antibiotics include amikacin, amoxicillin, ampicillin, atovaquone, azithromycin, aztreonam, bacitracin, carbenicillin, cefadroxil, cefazolin, cefdinir, cefditoren, cefepime, cefiderocol, cefoperazone, cefotetan, cefoxitin, cefotaxime, cefpodoxime, cefprozil, ceftaroline, ceftazidime, ceftibuten, and cefti Zoxime, ceftriaxone, chloramphenicol, colistimethate, cefuroxime, cephalexin, cephradine, cilastatin, cinoxacin, ciprofloxacin, clarithromycin, clindamycin, dalbavancin, dalfopristin, daptomycin, demeclocycline, dicloxacillin, doripenem, doxycycline, eravacycline, ertapenem, erythromycin, fidaxoma Isin, fosfomycin, gatifloxacin, gemifloxacin, gentamicin, imipenem, lefamulin, lincomycin, linezolid, lomefloxacin, loracarbef, meropenem, metronidazole, minocycline, moxifloxacin, nafcillin, nalidixic acid, neomycin, norfloxacin, ofloxacin, omadacycline, oritavancin, oxacillin, oxytetracycline, paclitaxel These include bromomycin, penicillin, pentamidine, piperacillin, plazomycin, quinupristin, rifaximin, sarecycline, secnidazole, sparfloxacin, spectinomycin, sulfamethoxazole, sulfisoxazole, tedizolid, telavancin, telithromycin, ticarcillin, tigecycline, tobramycin, trimethoprim, trovafloxacin, and vancomycin.

[0094] Representative examples of antiviral agents include abacavir, acyclovir, adefovir, amantadine, amprenavir, atazanavir, balavir, baloxavir marboxil, boceprevir, cidofovir, cobicistat, daclatasvir, darunavir, delavirdine, didanosine, docasanol, dolutegravir, doravirine, ecoliever, edoxudine, efavirenz, and elvitegravir. , emtricitabine, enfuvirtide, entecavir, etravirine, famciclovir, fomivirsen, fosamprenavir, forscarnet, fosnonet, famciclovir, favipravir, fomivirsen, foscavir, ganciclovir, ibacitabine, idoxuridine, indinavir, inosine, inosine pranobex, type I interferon, type II interferon, type III interferon lamivudine, letermovir, lopinavir, loviride, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nitazoxanide, oseltamivir, peginterferon alfa-2a, peginterferon alfa-2b, penciclovir, peramivir, pleconaril, podophyllotoxin, pyramidine, raltegravir, remdesevir, ribavirin, rilpivirine, rimantadine, lintatrix These include, but are not limited to, modavir, molnupiravir, ritonavir, saquinavir, simeprevir, sofosbuvir, stavudine, talabivirine, telaprevir, telbivudine, tenofovir alafenamide, tenofovir disoproxil, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, umifenovir, valacyclovir, valganciclovir, vidarabine, zalcitabine, zanamivir, and zidovudine.

[0095] Representative examples of anticoagulants include, but are not limited to, heparin, warfarin, rivaroxaban, dabigatran, apixaban, edoxaban, enoxaparin, and fondaparinux.

[0096] Representative examples of antiplatelet agents include, but are not limited to, clopidogrel, ticagrelor, prasugrel, dipyridamole, dipyridamole / aspirin, ticlopidine, and eptifibatide.

[0097] Representative examples of antifungal agents include, but are not limited to, voriconazole, itraconazole, posaconazole, fluconazole, ketoconazole, clotrimazole, isavuconazonium, miconazole, caspofungin, anidulafungin, micafungin, griseofulvin, terbinafine, flucytosine, terbinafine, nystatin, and amphotericin b.

[0098] Representative examples of steroidal anti-inflammatory drugs include, but are not limited to, hydrocortisone, dexamethasone, prednisolone, prednisone, triamcinolone, methylprednisolone, budesonide, betamethasone, cortisone, and deflazacort. Representative examples of non-steroidal anti-inflammatory drugs include ibuprofen, naproxen, ketoprofen, tolmetin, etodolac, fenoprofen, flurbiprofen, diclofenac, piroxicam, indomethacin, sulindax, meloxicam, nabumetone, oxaprozin, mefenamic acid, and diflunisal.

[0099] Other examples of active agents include chloroquine, hydrochloroquine, pyridoxal phosphate, vitamin D, and vitamin C.

[0100] Representative examples of anti-cytokine or immunomodulatory agents include, but are not limited to, tocilizumab, sarilumab, bevacizumab, fingolimod, imiquimod, and eculizumab.

[0101] Immunotherapeutic agents may include, but are not limited to, anti-CD40 antibodies, anti-PDL1 antibodies (e.g., atezolizumab, durvalumab, or avelumab), anti-PD1 antibodies, anti-CTLA4 antibodies, programmed death protein 1 (PD-1) inhibitors, or programmed death protein ligand 1 or 2 inhibitors (e.g., nivolumab (BMS), pembrolizumab (Merck), pidilizumab (CureTech / Teva), AMP-244 (Amplimmune / GSK), BMS-936559 (BMS), and MEDI4736 (Roche / Genentech)), or combinations thereof.

[0102] Representative examples of contraceptives include, but are not limited to, progestins, estrogens, or any combination thereof. For example, suitable progestins include, but are not limited to, natural and synthetic compounds with progestational activity, such as progesterone, chlormadinone acetate, norethindrone, cyproterone acetate, norethindrone acetate, desogestrel, levonorgestrel, drospirenone, trimegestone, norgestrel, norgestimate, norelgestromin, etonogestrel, gestodene, and other natural and / or synthetic gestagens. For example, suitable estrogens include, but are not limited to, natural and synthetic compounds having estrogenic activity, such as estradiol (17β-estradiol), 17α-estradiol, estriol, estrone, and esters thereof, including acetate, sulfate, valerate, or benzoate esters of these compounds, such as estradiol 17β-cypionate, estradiol 17-propionate, estradiol 3-benzoate, and piperazine estrone sulfate; ethinyl estradiol; conjugated estrogens (natural and synthetic); mestranol; agonist antiestrogens; and selective estrogen receptor modulators. Other examples of contraceptives include gonodotropin-releasing hormone (GnRh) or its analogs, such as deslorelin, avorelin, leuprolide, triptorelin, nafarelin, goserelin, buserelin, and fertirelin.

[0103] The term "steroid" refers to compounds belonging to or related to the following exemplary families of compounds: corticosteroids, mineralosteroids, and sex steroids (e.g., including potentially androgenic or estrogenic, or antiandrogenic and antiestrogenic molecules). Included among these are, for example, prednisone, prednisolone, methyl-prednisolone, triamcinolone, fluocinolone, aldosterone, spironolactone, danazol (also known as OPTINA), and others. In some embodiments, the therapeutic agent may comprise a steroid.

[0104] Exemplary cancer drugs or anti-cancer agents can include, but are not limited to, antimetabolic and anti-mitotic anti-cancer agents, and combinations thereof. A variety of such agents, including single antimetabolic and anti-mitotic anti-cancer agents or combinations of such agents, can be used in the methods and compositions described herein.

[0105] Anti-cancer antimetabolites are typically structurally similar to natural metabolites involved in the normal metabolic processes of cancer cells, such as the synthesis of nucleic acids and proteins. However, antimetabolites are sufficiently different from natural metabolites that they interfere with the metabolic processes of cancer cells. In cells, antimetabolites are mistaken for similar metabolites and are processed by cells in a manner similar to normal compounds. The presence of "decoy" metabolites prevents cells from performing essential functions, preventing cells from growing and surviving. For example, antimetabolites can exert cytotoxic activity by substituting these incorrect nucleotides for cellular DNA, thereby preventing cell division, or by inhibiting important cellular enzymes that prevent DNA replication.

[0106] Thus, in one embodiment, the anti-cancer antimetabolite is a nucleotide or nucleotide analog. In certain embodiments, for example, the antimetabolite can include a purine (e.g., guanine or adenosine) or analog thereof, or a pyrimidine (cytidine or thymidine) or analog thereof, with or without an attached sugar moiety.

[0107] Suitable antimetabolite anticancer agents for use in the present disclosure can be generally classified according to the metabolic processes they affect and can include, but are not limited to, analogs and derivatives of folic acid, pyrimidines, purines, and cytidine. Thus, in one aspect, the antimetabolite(s) is selected from the group consisting of cytidine analogs, folic acid analogs, purine analogs, pyrimidine analogs, and combinations thereof.

[0108] In one particular embodiment, for example, the antimetabolite is a cytidine analog. According to this embodiment, for example, the cytidine analog can be selected from the group consisting of cytarabine (cytosine arabinoside), azacytidine (5-azacytidine), and salts, analogs, and derivatives thereof.

[0109] In another specific embodiment, for example, the antimetabolite is a folic acid analog. Folic acid analogs or antifolates generally function by inhibiting dihydrofolate reductase (DHFR), an enzyme involved in the formation of nucleotides; when this enzyme is blocked, nucleotides are not formed, disrupting DNA replication and cell division. In certain embodiments, for example, the folic acid analog can be selected from the group consisting of denopterin, methotrexate (amethopterin), pemetrexed, pteropterin, raltitrexed, trimetrexate, and salts, analogs, and derivatives thereof.

[0110] In another specific embodiment, for example, the antimetabolite is a purine analog. Purine-based antimetabolites function by inhibiting DNA synthesis, for example, by interfering with the production of purine-containing nucleotides, adenine and guanine, thereby stopping DNA synthesis and thereby stopping cell division. Purine analogs can also be incorporated into DNA molecules themselves during DNA synthesis, which can interfere with cell division. According to certain embodiments, for example, the purine analog may be selected from the group consisting of acyclovir, allopurinol, 2-aminoadenosine, arabinosyladenine (ara-A), azacitidine, azathiprine, 8-aza-adenosine, 8-fluoro-adenosine, 8-methoxy-adenosine, 8-oxo-adenosine, cladribine, deoxycoformycin, fludarabine, gancylovir, 8-aza-guanosine, 8-fluoro-guanosine, 8-methoxy-guanosine, 8-oxo-guanosine, guanosine diphosphate, guanosine diphosphate-beta-L-2-aminofucose, guanosine diphosphate-D-arabinose, guanosine diphosphate-2-fluorofucose, guanosine diphosphate fucose, mercaptopurine (6-MP), pentostatin, thiamiprine, thioguanine (6-TG), and salts, analogs, and derivatives thereof.

[0111] In yet another particular embodiment, for example, the antimetabolite is a pyrimidine analogue.Similar to the purine analogues discussed above, pyrimidine-based antimetabolites block the synthesis of pyrimidine-containing nucleotides (cytosine and thymine in DNA, cytosine and uracil in RNA).By acting as a "decoy", pyrimidine-based compounds can prevent the production of nucleotides and / or can be incorporated into growing DNA chains, causing their termination. According to certain embodiments, for example, the pyrimidine analog is ancitabine, azacitidine, 6-azauridine, bromouracil (e.g., 5-bromouracil), capecitabine, carmofur, chlorouracil (e.g., 5-chlorouracil), cytarabine (cytosine arabinoside), cytosine, dideoxyuridine, 3'-azido-3'-deoxythymidine, 3'-dideoxycytidin-2'-ene, 3'-deoxy-3'-deoxythymidin-2'-ene, dihydrouracil, doxifluridine, enocitabine, floxuridine, 5-furan, The pyrimidine analog may be selected from the group consisting of fluorocytosine, 2-fluorodeoxycytidine, 3-fluoro-3'-deoxythymidine, fluorouracil (e.g., 5-fluorouracil (also known as 5-FU)), gemcitabine, 5-methylcytosine, 5-propynylcytosine, 5-propynylthymine, 5-propynyluracil, thymine, uracil, uridine, and salts, analogs, and derivatives thereof. In one embodiment, the pyrimidine analog is other than 5-fluorouracil. In another embodiment, the pyrimidine analog is gemcitabine or a salt thereof.

[0112] In certain embodiments, the antimetabolite is selected from the group consisting of 5-fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and salts, analogs, derivatives, and combinations thereof. In other embodiments, the antimetabolite is selected from the group consisting of capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and salts, analogs, derivatives, and combinations thereof. In a particular embodiment, the antimetabolite is other than 5-fluorouracil. In a particularly preferred embodiment, the antimetabolite is gemcitabine, or a salt thereof (e.g., gemcitabine HCl (Gemzar®)).

[0113] Other antimetabolite anticancer drugs include, among others, acantifolic acid, aminothiadiazole, brequinar sodium, Ciba-Geigy CGP-30694, cyclopentylcytosine, cytarabine phosphate stearate, cytarabine conjugate, Lilly DATHF, Merrel Dow DDFC, desaguanine, dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC, Wellcome EHNA, Merck & Co. EX-015, fazarabine, fludarabine phosphate, N-(2'-furanidyl)-5-fluorouracil, Daiichi Seiyaku FO-152, 5-FU-fibrinogen, isopropylpyrrolidine, Lilly LY-188011; Lilly LY-264618, metobenzaprim, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA, pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, Takeda TAC-788, tiazofurin, Erbamont TIF, tyrosine kinase inhibitors, Taiho UFT, and ulishitin.

[0114] In one embodiment, the antimitotic anticancer drug is a microtubule inhibitor or a microtubule stabilizer. Generally, microtubule stabilizers, such as taxanes and epothilones, bind to the inner surface of beta-microtubule strands and enhance microtubule assembly by promoting the nucleation and elongation phases of the polymerization reaction and by reducing the concentration of critical tubulin subunits required for microtubule assembly. Unlike microtubule inhibitors, such as vinca alkaloids, which prevent microtubule assembly, microtubule stabilizers, such as taxanes, reduce the lag time and dramatically shift the dynamic equilibrium between tubulin dimers and microtubule polymers to polymerization. Thus, in one embodiment, the microtubule stabilizer is a taxane or epothilone. In another embodiment, the microtubule inhibitor is a vinca alkaloid.

[0115] In some embodiments, the anticancer agent may include a taxane, or a derivative or analog thereof. Taxanes may be naturally occurring compounds or related forms, or may be chemically synthesized compounds or derivatives thereof with antitumor properties. Taxanes are a family of terpenes, including, but not limited to, paclitaxel (Taxol®) and docetaxel (Taxotere®), which are derived primarily from the Pacific yew tree, Taxus brevifolia, and have activity against certain tumors, particularly breast and ovarian tumors. In one aspect, the taxane is docetaxel or paclitaxel. Paclitaxel is a preferred taxane and is considered an antimitotic agent, promoting the assembly of microtubules from tubulin dimers and stabilizing microtubules by preventing depolymerization. This stability results in the inhibition of the normal dynamic reorganization of the microtubule network, which is essential for important interphase and mitotic cellular functions.

[0116] Also included are various known taxane derivatives, including both hydrophilic and hydrophobic derivatives.Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application No. 99 / 18113; the piperazino and other derivatives described in WO99 / 14209; the taxane derivatives described in WO99 / 09021, WO98 / 22451, and U.S. Patent No. 5,869,680; the 6-thio derivatives described in WO98 / 28288; the sulfenamide derivatives described in U.S. Patent No. 5,821,263; deoxygenated paclitaxel compounds, such as those described in U.S. Patent No. 5,440,056; and the taxol derivatives described in U.S. Patent No. 5,415,869. As noted above, taxanes further include prodrugs of paclitaxel, including, but not limited to, those described in WO 98 / 58927, WO 98 / 13059, and U.S. Patent No. 5,824,701. Taxanes can also be taxane conjugates, such as paclitaxel-PEG, paclitaxel-dextran, paclitaxel-xylose, docetaxel-PEG, docetaxel-dextran, docetaxel-xylose, and the like. Other derivatives are mentioned, inter alia, in "Synthesis and Anticancer Activity of Taxol Derivatives," D.G.I. Kingston et al., Studies in Organic Chemistry, Vol. 26, entitled "New Trends in Natural Products Chemistry" (1986), edited by Atta-ur-Rabman and P.W. Le Quesne (Elsevier, Amsterdam 1986). Each of these references is incorporated herein by reference in its entirety.

[0117] Various taxanes may be readily prepared using techniques known to those skilled in the art (see also WO 94 / 07882, WO 94 / 07881, WO 94 / 07880, WO 94 / 07876, WO 93 / 23555, WO 93 / 10076; U.S. Pat. Nos. 5,294,637, 5,283,253, 5,279,949, 5,274,137, 5,202,448, 5,200,534, 5,229,529; and EP 590,267), each of which is incorporated herein by reference in its entirety, or may be obtained from a variety of commercial sources, including, for example, Sigma-Aldrich Co., St. Louis, Mo.

[0118] Alternatively, the antimitotic anticancer drug can be a microtubule inhibitor, and in a preferred embodiment, the microtubule inhibitor is a vinca alkaloid. Generally, vinca alkaloids are spindle poisons. Vinca alkaloids act during mitosis, when chromosomes divide and begin to move along the spindle tube toward one of their poles before cell separation. Under the influence of these spindle poisons, the spindle is disorganized by the dispersion of chromosomes during mitosis, affecting cell proliferation. In certain embodiments, for example, the vinca alkaloid is selected from the group consisting of vinblastine, vincristine, vindesine, vinorelbine, and their salts, analogs, and derivatives.

[0119] The antimitotic anticancer agent can also be an epothilone. Generally, members of the epothilone class of compounds stabilize microtubule function according to a mechanism similar to that of taxanes. Epothilones can also cause cell cycle arrest at the G2-M transition phase, resulting in cytotoxicity and ultimately apoptosis. Suitable epithilones include epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, and epothilone F, as well as salts, analogs, and derivatives thereof. One particular epothilone analog is ixabepilone (Ixempra™), an epothilone B analog.

[0120] In certain embodiments, the antimitotic anticancer agent is selected from the group consisting of taxanes, epothilones, vinca alkaloids, and salts and combinations thereof. Thus, for example, in one embodiment, the antimitotic agent is a taxane. In this embodiment, the antimitotic agent is more preferably paclitaxel or docetaxel, and even more preferably paclitaxel. In another embodiment, the antimitotic agent is an epothilone (e.g., an epothilone B analog). In another embodiment, the antimitotic agent is a vinca alkaloid.

[0121] Examples of cancer drugs that can be used in the present disclosure include, but are not limited to, thalidomide; platinum coordination compounds, such as cisplatin (cis-DDP), oxaliplatin, and carboplatin; anthracenediones, such as mitoxantrone; substituted ureas, such as hydroxyurea; methylhydrazine derivatives, such as procarbazine (N-methylhydrazine, MIH); adrenocortical suppressants, such as mitotane (o,p'-DDD) and aminoglutethimide; RXR agonists, such as bexarotene; and tyrosine kinase inhibitors, such as sunitimibe and imatinib.

[0122] Examples of additional cancer drugs include alkylating agents, antimetabolites, natural products, hormones and antagonists, and miscellaneous drugs. Aliases are shown in parentheses. Examples of alkylating agents include nitrogen mustards, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan (sarcolysin), and chlorambucil; ethyleneimines and methylmelamines, such as hexamethylmelamine and thiotepa; alkyl sulfonates, such as busulfan; nitrosoureas, such as carmustine (BCNU), semustine (methyl-CCNU), lomustine (CCNU), and streptozotocin; DNA synthesis antagonists, such as estramustine phosphate; and triazines, such as dacarbazine (DTIC, dimethyl-triazenoimidazole carboxamide) and temozolomide. Examples of antimetabolites include folic acid analogs, such as methotrexate (amethopterin); pyrimidine analogs, such as fluorouracil (5-fluorouracil, 5-FU, SFU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside), and gemcitabine; purine analogs, such as mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG), and pentostatin (2'-deoxycoformycin, deoxycoformycin), cladribine, and fludarabine; and topoisomerase inhibitors, such as amsacrine.Examples of natural products include vinca alkaloids, such as vinblastine (VLB) and vincristine; taxanes, such as paclitaxel, protein-bound paclitaxel (Abraxane), and docetaxel (Taxotere); epipodophyllotoxins, such as etoposide and teniposide; camptothecins, such as topotecan and irinotecan; antibiotics, such as dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, histrelin, bleomycin, mitomycin (mitomycin C), idarubicin, epirubicin; enzymes, such as L-asparaginase; and biological response modifiers, such as interferon alpha and interleukin 2. Examples of hormones and antagonists include luteinizing-releasing hormone agonists, such as buserelin; corticosteroids, such as prednisone and related preparations; progestins, such as hydroxyprogesterone caproate, lunedroxyprogesterone acetate, and megestrol acetate; estrogens, such as diethylstilbestrol and ethinylestradiol and related preparations; estrogen antagonists, such as tamoxifen and anastrozole; androgens, such as testosterone propionate and fluoxymesterone and related preparations; androgen antagonists, such as flutamide and bicalutamide; and gonadotropin-releasing hormone analogs, such as leuprolide.These and other examples of cancer drugs have aliases and trade names, and their use methods, including dosage and administration regimens, known to those skilled in the art.

[0123] In some embodiments, the anti-cancer agent may comprise a chemotherapeutic agent. Suitable chemotherapeutic agents include, but are not limited to, alkylating agents, antibacterial agents, antimetabolites, hormonal agents, plant-derived agents and their synthetic derivatives, anti-angiogenic agents, differentiation inducers, cell growth arrest inducers, apoptosis inducers, cytotoxic agents, agents that affect cellular bioenergetics, i.e., agents that affect cellular ATP levels and molecules / activities that regulate these levels, biological agents such as monoclonal antibodies, kinase inhibitors, and inhibitors of growth factors and their receptors, gene therapy agents, cell therapy, e.g., stem cells, or any combination thereof.

[0124] According to these embodiments, the chemotherapeutic agent is selected from the group consisting of cyclophosphamide, chlorambucil, melphalan, mechlorethamine, ifosfamide, busulfan, lomustine, streptozocin, temozolomide, dacarbazine, cisplatin, carboplatin, oxaliplatin, procarbazine, uramustine, methotrexate, pemetrexed, fludarabine, cytarabine, fluorouracil, floxuridine, gemcitabine, capecitabine, vinblastine, vincristine, vinorelbine, etoposide, paclitaxel, docetaxel, doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone, bleomycin, mitomycin, hydroxyurea, topotecan, irinotecan, amsacrine, teniposide, erlotinib hydrochloride, and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0125] Antitumor agents include abiraterone acetate, Abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), Adrucil (fluorouracil), afatinib dimaleate, Afinitor (everolimus), Akynzeo (netupitant and palonosetron hydrochloride), and Aldara (imiki). Modo), aldesleukin, alemtuzumab, Alimta (pemetrexed disodium), Aloxi (palonosetron hydrochloride), Ambochlorin (chlorambucil), Aminolevulinic acid, Anastrozole, Aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Arranon (nelarabine), Arsenic trioxide, Arzerra (ofatumumab), asparaginase Erwinia chrysanthemumii, Ava bevacizumab, axitinib, azacitidine, BEACOPP, Becenum (carmustine), Beleodaq (belinostat), belinstat, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, Bexxar (tositumomab and iodine-131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, busulfan, Busulfex (busulfan) rufan), cabazitaxel, cabozantinib-S-malate, CAF, Camppath (alemtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, carboplatin, carboplatin-TAXOL, carfilzomib, Carmubris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CeeNU (lomustine), ceritinib, Cerubidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, chlorambucil,Chlorambucil-prednisone, CHOP, cisplatin, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Clolar (clofarabine), CMF, Cometriq (cabozantinib-S-malate), COPP, COPP-ABV, Cosmegen (dactinomycin), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine, liposomal, Cytosar-U (cytarabine), Cytox an (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, dasatinib, daunorubicin hydrochloride, decitabine, degarelix, denileukin diftitox, denosumab, DepoCyt (liposomal cytarabine), DepoFoam (liposomal cytarabine), dexrazoxane hydrochloride, dinutuximab, docetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome) dm), DTIC-Dome (dacarbazine), Efudex (fluorouracil), Elitek (rasburicase), Ellence (epirubicin hydrochloride), Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinaze (asparaginase erwinia chrysanthemum), Etopophos (phosphorazine) etoposide acid), etoposide, etoposide phosphate, Evacet (doxorubicin hydrochloride liposomal), everolimus, Evista (raloxifene hydrochloride), exemestane, Fareston (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil), fluorouracil, Folex (methotrexate), Folex PFS (methotrexate), FOLFIRI,FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRINOX, FOLFOX, Folotyn (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV nonavalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant HPV nonavalent vaccine, recombinant HPV quadrivalent vaccine, recombinant Hycamtin (topotecan hydrochloride), Hyper-CVAD, Ibrance (palbociclib), ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, Ifex (ifosfamide), ifosfamide, Ifosfamidum (ifosfamide), imatinib mesylate, Imbruvica (ibrutinib), imiquimod, Inlyta (axitinib), interferon alfa-2b, recombinant Intron A (recombinant interferon alfa-2b), iodine-131 tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, Istodax (romidepsin), ixabepilone, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate), Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kyprolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium,Leukeran (chlorambucil), leuprolide acetate, Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (liposomal doxorubicin hydrochloride), liposomal cytarabine, lomustine, Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lupron Depot-3 Month (leuprolide acetate), Lupron Depot-4 Month Month (leuprolide acetate), Lynparza (olaparib), Marqibo (vincristine sulfate liposome), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, Megace (megestrol acetate), megestrol acetate, Mekinist (trametinib), mercaptopurine, mesna, Mesnex (mesna), Methazolastone (temozolomide), methotrexate, methotrexate Rexate LPF (methotrexate), Mexate (methotrexate), Mexate-AQ (methotrexate), mitomycin C, mitoxantrone hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (plelixafor), Mustargen (mechlorethamine hydrochloride), Mutamycin (mitomycin C), Myleran (busulfan), Mylosar (azacytidine), M ylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine tartrate), nelarabine, Neosar (cyclophosphamide), netupitant and palonosetron hydrochloride, Neupogen (filgrastim), Nexavar (sorafenib tosylate), nilotinib, nivolumab, Nolvadex (tamoxifen citrate) Nplate (romiplostim), obinutuzumab, Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, omacetaxine mepesuccinate, Oncaspar (pegaspargase), ondansetron hydrochloride, Ontak (denileukin diftitox), Opdivo (nivolumab), OPPA, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD,Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, Pegaspargase, Peginterferon alfa-2b, PEG-Intron (Peginterferon alfa-2b), Pembrolizumab, Pemet Rexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor, pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag olamine), Provenge (sipuleucel-T), Purinethol (mercaptopurine), Purixan (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonavalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, Recombinant interferon alfa-2b, regorafenib, R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), Rituxan (rituximab), rituximab, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), ruxolitinib phosphate, Sclerosol intrapleural aerosol (talc), siltuximab, sipuleucel-T, Somatuline Depot (lanreotide acetate), sonidegib, sorafenib tosylate, Sprycel (dasatinib), STANFORD V, sterile talc powder (talc), Steritalc (talc), Stivarga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Sylatron (peginterferon alfa-2b), Sylvant (siltuximab), Synovir (thalidomide), Synribo (omacetaxine mepesuccinate), TAC,Tafinlar (dabrafenib), talc, tamoxifen citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel, ), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), thiotepa, Toposar (etoposide), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab and iodine-131 tositumomab, Totect (dexrazoxane hydrochloride), TPF, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), Trisenox (trioxide) Arsenic), Tykerb (lapatinib ditosylate), Unituxin (dinutuximab), vandetanib, VAMP, Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), vemurafenib, VePesid (etoposide), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposome, vinorelbine tartrate, VIP, vismodegib, Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab) ), Zaltrap (Ziv-aflibercept), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Zykadia (ceritinib), and Zytiga (abiraterone acetate).

[0126] Growth factors useful as therapeutic agents include transforming growth factor-alpha ("TGF-α"), transforming growth factor ("TGF-β"), platelet-derived growth factor ("PDGF"), fibroblast growth factor ("FGF"), including acidic isoforms 1 and 2, basic form 2 of FGF, and FGFs including FGF4, 8, 9, and 10, nerve growth factor ("NGF"), including NGF 2.5s, NGF 7.0s, and beta-NGF, as well as neurotrophins, brain-derived neurotrophic factor, cartilage-derived factor, bone growth factor (BGF), basic fibroblast growth factor (BGF), and fibroblast growth factor (FGF). Growth factors include, but are not limited to, insulin-like growth factors (IGFs), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (G-CSF), insulin-like growth factors (IGF) I and II, hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocyte growth factor (KGF), transforming growth factors (TGFs) including TGF alpha, beta, beta 1, beta 2, beta 3, skeletal growth factors, bone matrix-derived growth factors, and bone-derived growth factors, and mixtures thereof.

[0127] Immunoglobulins useful in the present disclosure include, but are not limited to, IgG, IgA, IgM, IgD, IgE, and mixtures thereof. Some preferred growth factors include VEGF (vascular endothelial growth factor), NGF (nerve growth factor), PDGF-AA, PDGF-BB, PDGF-AB, FGFb, FGFa, and BGF.

[0128] Other molecules useful as anti-cancer agents include, but are not limited to, growth hormone, leptin, leukemia inhibitory factor (LIF), tumor necrosis factor alpha and beta, endostatin, thrombospondin, bone morphogenetic protein-1, bone morphogenetic proteins 2 and 7, osteonectin, somatomedin-like peptide, and osteocalcin.

[0129] Tumor antigens can be based on specific mutations (neoepitopes) and those expressed by cancer germline genes (antigens common to tumors found in multiple patients, referred to herein as "traditional cancer antigens" or "common cancer antigens"). In some embodiments, traditional antigens are those known to be commonly found in cancers or tumors or to be found in specific types of cancers or tumors. In some embodiments, traditional cancer antigens are non-mutated tumor antigens. In some embodiments, traditional cancer antigens are mutated tumor antigens.

[0130] Diagnostic agents include gases; metals; commercially available imaging agents used in positron emission tomography (PET), computed tomography (CAT), single-photon emission computed tomography, X-ray, fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents.Examples of suitable materials for use as contrast agents in MRI include gadolinium chelate, and iron, magnesium, manganese, copper, and chromium.Examples of materials useful for CAT and X-ray imaging include iodine-based materials.

[0131] In some embodiments, the active agent can comprise a vaccine or a component thereof. Vaccines can include isolated proteins or peptides, inactivated organisms and viruses, killed organisms and viruses, genetically modified organisms or viruses, cell extracts, and RNA encoding at least one antigenic polypeptide or immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response to the antigenic polypeptide). The active agent can be combined with interleukins, interferons, cytokines, and adjuvants, such as cholera toxin, alum, Freund's adjuvant, and the like. The prophylactic agent may contain infectious agents, such as antigens of bacterial organisms, such as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema antigens such as L. pallidum, Leptospirosis interrogans, Borrelia burgdorferi, and Camphylobacter jejuni;Antigens of viruses, such as human metapneumovirus (hMPV), human parainfluenza virus (hPIV) types 1, 2, and 3 (hPIV1, hPIV2, and hPIV3, respectively), respiratory syncytial virus (RSV), measles virus (MeV), coronaviruses (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1), poxviruses (e.g., antigens of viruses such as smallpox, monkeypox, African swine virus, influenza A and B, HIV, varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E; antigens of fungal, protozoan, and parasitic organisms, such as Cryptococcus The antigens may include antigens from bacteria such as Bacillus subtilis, Bacillus anthracis, Bacillus subtilis, Bacillus niger ...

[0132] In certain embodiments, the active agent is a polynucleotide. Polynucleotides or oligonucleotides that can be introduced by the methods herein include all types of DNA, cDNA, and RNA sequences. For example, the polynucleotide can be double-stranded DNA, single-stranded DNA, complexed DNA, encapsulated DNA, naked RNA, encapsulated RNA, messenger RNA (mRNA), tRNA, small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), antisense RNA (asRNA), self-amplifying mRNA (saRNA), guide RNA (gRNA), cRNA, and combinations thereof. The polynucleotide can also be a DNA construct, such as an expression vector, encoding a desired gene product (e.g., a gene product homologous or heterologous to the target into which the gene product is introduced).

[0133] Nucleic acids (e.g., polynucleotides or oligonucleotides) encoding peptides can be used to produce antigenic peptides in vitro. NAs can be, for example, DNA, cDNA, PNA, CNA, RNA, single-stranded and / or double-stranded, or polynucleotides in natural or stabilized forms, such as polynucleotides with phosphorothioate backbones, or combinations thereof, and may contain introns or may not contain introns as long as they encode the peptide. In one embodiment, in vitro translation is used to produce the peptide. There are many exemplary systems that can be used by those skilled in the art.

[0134] In some embodiments, the active agent can comprise an expression vector capable of expressing mRNA or a polypeptide. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, DNA is inserted into an expression vector such as a plasmid in the appropriate orientation and correct reading frame for expression, and if necessary, the DNA can be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria), although such controls are generally available in the expression vector. The vector is then introduced into the host bacteria using standard techniques and cloned (see, for example, Sambrook et al. (1989) MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratory, NY).

[0135] The term "nucleic acid encoding a polypeptide" encompasses NAs that contain only the coding sequence for the polypeptide, as well as NAs that contain additional coding and / or non-coding sequences. NAs can be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or non-coding (antisense) strand.

[0136] A NA can include a coding sequence for a peptide, either an antibody or an antigen, fused in the same reading frame to a polynucleotide that aids in the expression and / or secretion of the polypeptide from a host cell (e.g., a leader sequence that functions as a secretory sequence to control transport of the polypeptide from the cell). Polypeptides with leader sequences can be preproteins, with the leader sequence being cleaved by the host cell to form the mature form of the polypeptide.

[0137] NA sequences encoding a polypeptide of interest can be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide, and codons preferred in the host cell in which the recombinant polypeptide of interest will be produced can be selected. Standard methods can be applied to synthesize isolated polynucleotide sequences encoding isolated polypeptides of interest. Oligomers containing NA sequences encoding specific isolated polypeptides can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0138] Once assembled (e.g., by synthesis, site-directed mutagenesis, or otherwise), the polynucleotide sequence encoding the particular isolated polypeptide of interest is inserted into an expression vector and, optionally, operably linked to expression control sequences suitable for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, to obtain high levels of expression of a transfected gene in a host, the gene can be operably linked to transcriptional and translational expression control sequences that function in the selected expression host.

[0139] Recombinant expression vectors can be used to amplify and express DNA encoding antibodies or antigenic peptides. Recombinant expression vectors are replicable DNA constructs containing synthetic or cDNA-derived DNA fragments operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcription unit generally comprises an assembly of one or more genetic elements having a regulatory role in gene expression, such as a transcriptional promoter or enhancer, a structural or coding sequence to be transcribed into mRNA and translated into protein, and appropriate transcriptional and translational initiation and termination sequences, as described in detail herein. Such regulatory elements may also include an operator sequence to control transcription. Generally, operably linked means contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by the host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it may contain an N-terminal methionine residue, which can optionally be subsequently cleaved from the expressed recombinant protein to provide the final product.

[0140] "Ribonucleic acid" or "RNA" refers to a polymer containing at least two ribonucleotides. A "ribonucleotide" contains the sugar ribose, a base, and a phosphate group. Nucleotides are linked together through the phosphate group. "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0141] The RNA may be in the form of oligonucleotide RNA, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA, siRNA (small interfering RNA), self-replicating RNA, ribozymes, chimeric sequences, or derivatives of these groups.

[0142] The RNA contains one or more nucleotides with modified nucleobases (in addition to any 5' cap structure), including m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-0-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine); Am (2'-0-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine)). (cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonylcarbamoyladenosine); ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A (N6-hydroxynorvalylcarbamoyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalylcarbamoyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); m1 I (1-methylinosine); m'1m (1,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-fungylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4-acetyl2TOmethylcytidine); k2C (lysidine); m1G (1-methylguanosine); m2G (N2-methylguanosine); m7G ( 7-methylguanosine; Gm (2'-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (incompletely modified hydroxywybutosine);imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine); m5Um (5,2'-0-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um (2-thio-2'-0-methyluridine) );acp3U (3-(3-amino-3-carboxypropyl)uridine);ho5U (5-hydroxyuridine);mo5U (5-methoxyuridine);cmo5U (uridine 5-oxyacetic acid);mcmo5U (uridine 5-oxyacetic acid methyl ester);chm5U (5-(carboxyhydroxymethyl)uridine));mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester);mcm5U (5-methoxycarbonylmethyluridine);mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine);m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2'-0-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethyl 5-aminomethyl-2-L-O-methyluridine; cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2'-O-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosine); rn62Am (N6,N6,O-2-trimethyladenosine);m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-O-methylcytidine); m1Gm (1,2'-O-dimethylguanosine); m'Am (1,2-O-dimethyladenosine) irinomethyluridine; tm5s2U (S-taurinomethyl-2-thiouridine) ));imG-14 (4-demethylguanosine);imG2 (isoguanosine);or ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, their 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)- Alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanin 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, or abasic nucleotide;

[0143] The RNA may optionally include one or more UNA molecules, such as those disclosed in U.S. Patent Nos. 8,314,227, 9,051,570, 9,303,260, 9,297,009, and 9,340,789, and U.S. Patent Publication No. 2016 / 0168567, which are incorporated herein in their entireties.

[0144] The RNA or self-replicating RNA can contain one or more modified pyrimidine nucleobases, such as pseudouridine and / or 5-methylcytosine residues. The RNA can include a 5' cap containing 7'-methylguanosine, and the first one, two, or three 5' ribonucleotides can be methylated at the 2' position of the ribose. The RNA can contain a 5' trinucleotide cap structure as described by Tanis, et al., U.S. Application No. 15 / 788,742, filed October 19, 2017, which is incorporated herein by reference in its entirety.

[0145] Natural RNA has a phosphate backbone; the RNA described herein may contain other types of backbones and bases, including peptide nucleic acids, phosphothioate, phosphoramidate, phosphorothioate, and / or methylphosphonate linkages.

[0146] "Antisense" is a polynucleotide that interferes with the function of DNA and / or RNA. This can result in the inhibition of expression.

[0147] "Gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide or precursor. The polypeptide may be encoded by a full-length coding sequence or by any portion of the coding sequence, so long as the desired activity or functional property (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the full-length polypeptide or a fragment thereof is retained.

[0148] In some embodiments, RNA (e.g., mRNA) can be used to induce a balanced immune response to an infectious agent. In some embodiments, RNA (e.g., mRNA) can be used to induce a balanced immune response to an infectious agent. In some embodiments, RNA (e.g., mRNA) can be used to induce a balanced immune response to an infectious agent, such as a metapneumovirus, e.g., human metapneumovirus (hMPV), a parainfluenza virus, e.g., human parainfluenza virus (hPIV) types 1, 2, and 3 (hPIV1, hPIV2, and hPIV3, respectively), respiratory syncytial virus (RSV), measles virus (MeV), a coronavirus (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NL64, HCoV-NL65, HCoV-NL66, HCoV-NL67, HCoV-NL68, HCoV-NL69, HCoV-NL70, HCoV-NL71, HCoV-NL72, HCoV-NL73, HCoV-NL74, HCoV-NL75, HCoV-NL76, HCoV-NL77, HCoV-NL78, HCoV-NL79 ...9, HCoV-NL71, HCoV-NL72, HCoV-NL73, HCoV-NL74, HCoV-NL75, HCoV-NL76, HCoV-NL77, HCoV-NL78, V-NH, HCoV-HKU1), poxvirus (e.g., smallpox, monkeypox), African swine virus, influenza A and B, HIV, varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E viruses, and the like.

[0149] In some embodiments, RNA (e.g., mRNA) can be used to induce a balanced immune response against respiratory viruses. As used herein, the term "respiratory virus" refers to a virus that causes respiratory disease. For example, negative-sense single-stranded RNA viruses of the Paramyxoviridae family, such as human metapneumovirus (hMPV), human parainfluenza virus (hPIV) types 1, 2, and 3 (hPIV1, hPIV2, and hPIV3, respectively), RSV, and measles virus (MeV). Another example of a respiratory virus is coronavirus. Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome and a helical symmetry nucleocapsid. Coronaviruses are a species of virus belonging to the subfamily Coronavirinae in the family Coronaviridae in the order Nidovirales.

[0150] Representative examples of betacoronaviruses include, but are not limited to, envecoviruses (e.g., betacoronavirus 1, human coronavirus OC43, Chinese Rattus coronavirus HKU24, human coronavirus HKU1, and murine coronavirus), hibecoviruses (e.g., bat Hp-betacoronavirus Zhejiang 2013), merbecoviruses (e.g., hedgehog coronavirus 1, Middle East respiratory syndrome-associated coronavirus (MERS-CoV), Pipistrellus bat coronavirus HKU5, and Tylonycteris bat coronavirus HKU4), nobecoviruses (e.g., Rousettus bat coronavirus GCCDC1 and Rousettus bat coronavirus HKU9), and sarbecoviruses (e.g., severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)).

[0151] Representative examples of gammacoronaviruses include, but are not limited to, Segakoviruses (eg, beluga coronavirus SQ1) and Igakoviruses (eg, avian coronavirus (IBV)).

[0152] Representative examples of deltacoronaviruses include, but are not limited to, anddecoviruses (e.g., Wigeon coronavirus HKU20), burdecoviruses (e.g., Brown-eared ...

[0153] In some embodiments, the coronavirus is a human coronavirus. Representative examples of human coronaviruses include, but are not limited to, human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome-related coronavirus (MERS-CoV).

[0154] In some embodiments, the antigenic polypeptides include at least one (e.g., at least two, three, four, or five) human metapneumovirus (hMPV) antigenic polypeptide, human parainfluenza virus (hPIV) types 1, 2, and 3 (hPIV1, hPIV2, and hPIV3, respectively) antigenic polypeptide, respiratory syncytial virus (RSV) antigenic polypeptide, measles virus (MeV) antigenic polypeptide, varicella-zoster antigenic polypeptide, influenza virus antigenic polypeptide, herpes simplex virus 1 (HSV1) antigenic polypeptide, herpes simplex virus 2 (HSV2) antigenic polypeptide, poxvirus (e.g., smallpox, monkeypox) antigenic polypeptide, African swine virus antigenic polypeptide, cytomegalovirus antigenic polypeptide, Epstein-Barr virus antigenic polypeptide, rotavirus antigenic polypeptide, An RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an antigen polypeptide, a rhinovirus antigen polypeptide, an adenovirus antigen polypeptide, a papillomavirus antigen polypeptide, a poliovirus antigen polypeptide, a mumps antigen polypeptide, a rabies antigen polypeptide, a rubella antigen polypeptide, a coxsackievirus antigen polypeptide, an equine encephalitis antigen polypeptide, a Japanese encephalitis antigen polypeptide, a yellow fever antigen polypeptide, a Rift Valley fever antigen polypeptide, a hepatitis A, B, C, D, and E virus antigen polypeptide, or a coronavirus (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) antigen polypeptide.As used herein, the term "antigenic polypeptide" refers to an immunogenic fragment of an antigenic polypeptide (e.g., human metapneumovirus (hMPV), human parainfluenza virus (hPIV) types 1, 2, and 3 (hPIV1, hPIV2, and hPIV3, respectively), respiratory syncytial virus (RSV), measles virus (MeV), varicella-zoster, influenza virus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), poxvirus (e.g., smallpox, monkeypox), African swine virus, cytomegalovirus, Epstein-Barr virus, The present invention encompasses immunogenic fragments that induce (or are capable of inducing) an immune response to rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E virus, or coronavirus (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1), or a combination of any of them.

[0155] How to use These compositions can be prepared as described herein or elsewhere and can be administered by a variety of routes, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical (including transdermal, epidermal, ocular, and mucosal, including intranasal, vaginal, and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, or intranasal), oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, or injection or infusion, or intracranial (e.g., intrathecal or intraventricular administration). Parenteral administration can be in the form of a single bolus dose or can be, for example, by a continuous perfusion pump. In some embodiments, the compounds provided herein or pharmaceutically acceptable salts thereof are suitable for parenteral administration. In some embodiments, the compounds provided herein are suitable for intravenous administration. In some embodiments, the compounds provided herein are suitable for oral administration. In some embodiments, the compounds provided herein are suitable for topical administration.

[0156] Pharmaceutical compositions and formulations for topical administration may include, but are not limited to, transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. In some embodiments, the pharmaceutical compositions provided herein are suitable for parenteral administration. In some embodiments, the pharmaceutical compositions provided herein are suitable for intravenous administration. In some embodiments, the pharmaceutical compositions provided herein are suitable for oral administration. In some embodiments, the pharmaceutical compositions provided herein are suitable for topical administration.

[0157] The compositions described herein can be used to deliver one or more active agents to cells (e.g., in vivo, ex vivo, or in vitro). Accordingly, provided herein are methods of delivering an active agent to a cell (e.g., in vivo, ex vivo, or in vitro), comprising contacting the cell with a composition described herein. Also provided are methods for delivering an active agent to a cell in vivo, comprising administering to a mammalian subject (e.g., a human) a composition described herein. In some embodiments, administration can include systemic administration (e.g., intravenous injection or infusion).

[0158] Also provided are methods for systemically administering an active agent to a subject in need thereof. These methods can include intravenously injecting a pharmaceutical composition comprising lipid particles encapsulating the active agent, the lipid particles comprising 0.5 mol% to 8 mol% of one or more cationic lipids, 20 mol% to 65 mol% of one or more ionizable lipids, 35 mol% to 80 mol% of one or more neutral lipids, and greater than 0 mol% to 5 mol% of one or more PEGylated lipids. In some cases, the active agent can include an anticancer drug.

[0159] Also provided are methods for administering an active agent to the liver of a subject. These methods can include intravenously injecting a pharmaceutical composition comprising lipid particles encapsulating the active agent, the lipid particles comprising 0.5 mol% to 3 mol% of one or more cationic lipids, 20 mol% to 65 mol% of one or more ionizable lipids, 35 mol% to 80 mol% of one or more neutral lipids, and greater than 0 mol% to 5 mol% of one or more PEGylated lipids. In some cases, the active agent can include an anti-cancer agent, for example, an active agent for treating liver cancer.

[0160] Also provided are methods for administering an active agent to a solid tumor in a subject. These methods can include intravenously injecting a pharmaceutical composition comprising lipid particles encapsulating the active agent, the lipid particles comprising 3 mol% to 6 mol% of one or more cationic lipids, 20 mol% to 65 mol% of one or more ionizable lipids, 35 mol% to 80 mol% of one or more neutral lipids, and greater than 0 mol% to 5 mol% of one or more PEGylated lipids. In some cases, the active agent can comprise an anticancer drug. These methods can allow the lipid particles to selectively target tumor angiogenesis.

[0161] Also provided are methods for administering an active agent to the lungs of a subject. These methods can include intravenously injecting a pharmaceutical composition comprising lipid particles encapsulating the active agent, the lipid particles comprising 6 mol% to 10 mol% of one or more cationic lipids, 20 mol% to 65 mol% of one or more ionizable lipids, 35 mol% to 80 mol% of one or more neutral lipids, and greater than 0 mol% to 5 mol% of one or more PEGylated lipids. In some cases, the active agent can include an anti-cancer agent, e.g., an active agent for the treatment of lung cancer.

[0162] Also provided are methods for locally administering an active agent to a subject in need thereof. These methods can include intramuscularly injecting a pharmaceutical composition comprising lipid particles encapsulating an active agent, the lipid particles comprising greater than 0 mol% to 5 mol% of one or more cationic lipids, 20 mol% to 65 mol% of one or more ionizable lipids, 35 mol% to 80 mol% of one or more neutral lipids, and greater than 0 mol% to 5 mol% of one or more PEGylated lipids. In some cases, the active agent can comprise a nucleic acid (e.g., siRNA, mRNA, or any combination thereof). In some cases, the active agent can comprise a vaccine.

[0163] Reference will now be made in detail to the present exemplary embodiments, examples of which are illustrated in the accompanying drawings. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice disclosed herein. The embodiments are further described in the following examples. These examples do not limit the scope of the claims, but merely serve to clarify certain particular embodiments. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit being indicated by the following claims. [Example]

[0164] The present invention will be described in more detail using specific examples. The following examples are provided for illustrative purposes and are not intended to limit the present invention in any way. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to produce essentially the same results.

[0165] Example 1: pH-Sensitive Nanoemulsion (PSNE) for Delivery of Nucleic Acid Cargo overview The ability of lipid nanoparticles (LNPs) to deliver genetic materials has shown great potential in therapeutics for treating a wide variety of diseases. However, due to the poor accessibility of LNPs encapsulating genetic payloads to the tumor microenvironment, only a few such nanomedicines have been developed and are under clinical investigation. QTsome is an LNP platform that facilitates drug delivery using a combination of quaternary amine-based cationic lipids and tertiary amine-based ionizable lipids. Here, we specifically evaluated and optimized QTsome components for delivering nucleic acid cargo. As an example, we also developed an antisense oligonucleotide against miR-21 in cancer. QTsomes encapsulating AM21 demonstrated superior antitumor activity in multiple tumor models and demonstrated synergistic effects with the tyrosine kinase inhibitor erlotinib and the anti-PDL1 therapy atezolizumab.

[0166] IntroductionMicroRNAs (miRNAs) are small non-coding RNAs that can regulate gene expression through mRNA degradation or partial translational repression. miRNA-based gene regulation can be achieved in the miRNA-induced silencing complex (miRISC) complex, which contains a miRNA guide strand that binds to the 3'-untranslated region (3'-UTR) of target messenger RNA (mRNA) via the miRNA's seeding region, the first 2-7 nucleotides from the 5' end. This classical interaction typically results in mRNA deadenylation, translational repression, and mRNA degradation. However, in most cases, the interaction between miRNAs and their target mRNAs is not completely complementary. This has led to the idea that a single miRNA could potentially target multiple downstream mRNAs or multiple sites on the same mRNA, suggesting that a broader range of biological processes may be involved in miRNA-based gene regulation. Due to the robust regulatory effects of miRNAs on biological processes and gene expression, research has also shown that miRNAs can not only be found intracellularly but also migrate to extracellular compartments and systemic fluids, making them effective biomarkers for multiple diseases. The first miRNA used as a biomarker was miRNA-21 (miR-21), and researchers found high expression of miR-15, miR-21, and miR-210 in the serum of patients with diffuse large B-cell lymphoma (DLBCL) in 2008. Specifically, miR-21 showed the greatest differential expression in clinical samples. miR-21 is highly associated with disease progression and patient survival. Subsequently, miR-21 overexpression has also been demonstrated to be a promising biomarker for the diagnosis and poor prognosis of many solid tumor types, including non-small cell lung cancer (NSCLC) and colorectal cancer.Recently, researchers have found that miR-21 can function not only as a biomarker but also as an oncogenic miRNA, regulating epigenetic levels of cell apoptosis, DNA repair, cell proliferation, tumor metastasis, and drug resistance by downregulating tumor suppressor genes such as AKT1, DDAH1, PTEN, and PDCD4. In addition, studies have also shown that miR-21 is associated with a tumor-promoting immune response with a higher M2 macrophage population, suggesting that miR-21 may induce drug resistance not only to chemotherapy but also to immunotherapy. However, despite the continued discovery of miRNAs as biomarkers in cancer, efficient therapeutic agents targeting tumor-promoting miRNAs have yet to be fully developed.

[0167] Antisense oligonucleotides (ASOs) are single-stranded deoxyribonucleotides complementary to their targets. The mechanism of antisense targeting is to induce gene downregulation by recruiting RNAse H endonuclease activity, which can cleave the heteroduplex formed by the ASO and the target gene. ASOs can target not only mRNA but also oligonucleotides, such as miRNAs or small interfering RNAs (siRNAs), making ASOs a promising approach for therapeutically inhibiting miRNAs. However, unmodified anti-miRNA antisense oligonucleotides (ASOs) are susceptible to nucleases that are easily removed from systemic fluids. To overcome this obstacle, many types of chemical modifications of ASOs have been introduced. Modifications of the phosphate backbone, nucleobases, and ribose sugar moieties have been widely used to improve the pharmacokinetics, pharmacodynamics, and biodistribution of drugs. In addition, chemical conjugation of ASOs, including small molecules, peptides, aptamers, and antibodies, has been developed to improve tissue-specific biodistribution and therapeutic efficacy. However, the free drugs of ASOs contain a high density of negative charges, which makes them less accessible to the tumor microenvironment (TME) via cellular uptake. Therefore, an efficient delivery platform is needed for therapeutic ASOs.

[0168] QTsome is a lipid nanoparticle (LNP) platform that facilitates drug delivery using a combination of quaternary amine-based cationic lipids and tertiary amine-based ionizable lipids. The design using a cocktail of cationic and ionizable lipids in QTsome can achieve improved pH-dependent drug loading and release profiles. Preliminary studies have shown that the conventional QTsome design was able to deliver oligonucleotides. However, the conventional QTsome (QTsome Original) contains a high amount of PEG lipids and outdated functional lipids, which hinder cellular uptake and release of nucleic acid cargo into the cytoplasm, making gene delivery by QTsome Original less efficient than other emerging LNP platforms from Moderna, Pfizer / BioNTech, Alnylam Pharmaceuticals, and others. In this example, QTsome Original was optimized into QTsome Plus (QTPlus) by reducing the amount of cationic lipids and PEG lipids, increasing the amount of ionizable lipids, and screening for candidates for ionizable lipids and helper lipids. Finally, QTPlus demonstrated significantly enhanced gene delivery, including that of oligonucleotides and mRNA, compared with the LNP standard with QTsome Original and Moderna formulations. QTPlus was further used to deliver a 16-mer anti-miR-21 ASO (AM21), and QTPlus demonstrated significant miR-21 inhibition through downstream gene regulation. Additionally, QTPlus-encapsulated AM21 (QTPlus-AM21) demonstrated significant antitumor activity in A549 NSCLC and MC38 colorectal tumor models. QTPlus-AM21 significantly induced chemotherapy sensitization in NSCLC in the presence of the tyrosine kinase inhibitor (TKI) erlotinib and demonstrated enhanced antitumor immunity in combination with the anti-PDL1 therapy atezolizumab, suggesting that QTPlus-AM21 may also be a potent antitumor candidate for combination therapy with chemotherapy and immunotherapy.

[0169] Materials and Methods Materials. 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) was purchased from MedChemExpress (Monmouth Junction, NJ, United States). 1-(2,3-bis(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)propyl)pyrrolidine (A-066) was purchased from Hangzhou Dragonpharm Co., Ltd. (Hangzhou, China). Cholesterol was purchased from Avanti Polar Lipids, Inc. (Birmingham, AL, United States). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) were purchased from NOF America (Cambridge, MA, United States). CleanCap® Firefly Luciferase (FLuc) mRNA was purchased from TriLink Biotechnologies (San Diego, CA, United States). AM21 was purchased from Integrated DNA Technologies, Inc. (Coralville, IA, United States). (20-mer AM21 sequence: 5'-+C*+A*+A*+C*A*+T*C*+A*G*T*+C*+T*G*A*+T*A*+A*G*+C*+T-3'. 16-mer AM21 sequence: 5'-A*+T*+C*A*G*+T*+C*+T*G*A*+T*A*A*G*+C*+T-3'. Scrambled 16-mer oligonucleotide sequence: 5'-+C*A*C*G*+T*+C*+T*A*+T*A*+C*G*+C*+C*+C*A*-3'. "+" represents locked nucleic acid, LNA, base. "*" represents phosphorothioated backbone.) Erlotinib was purchased from Cayman Chemical (Ann Arbor, MI, United States). Atezolizumab was kindly obtained by Arthur G. James Cancer Hospital from The Ohio State University (Columbus, OH, United States).

[0170] Formulation and Characterization of QTPlus-mRNA and QPlus-AM21. Specifically, for QTPlus-mRNA, DOTAP, A-066, DOPE, cholesterol, and DMG-PEG2000 were prepared in a molar ratio of 1.5 / 50 / 12 / 35 / 1.5 in ethanol. FLuc mRNA was prepared in DEPC-treated water. The lipid mixture in ethanol was directly injected into the FLuc mRNA phase, where QTPlus self-assembly and mRNA encapsulation occurred simultaneously. This QTPlus formulation protocol was referred to as one-step formulation.

[0171] Empty QTsome Original and QTPlus were prepared by manually and rapidly injecting the lipid mixture into acetate buffer. DOTAP, A-066, DOPE, cholesterol, and DMG-PEG2000 were prepared in a molar ratio of 1.5 / 50 / 12 / 35 / 1.5 in ethanol. FLuc mRNA, AM21, and scrambled oligonucleotide solutions were prepared in DEPC-treated water. The oligonucleotide solution was mixed with the empty QTsome Original or QTPlus phase in equal volumes to reach a final lipid-to-oligo ratio of 10 / 1 (w / w). This formulation protocol was referred to as two-step formulation. The final lipid concentration of QTsome was 10 mg / ml, and the final oligonucleotide concentration was 1 mg / ml. The particle size of QTsome-encapsulated oligonucleotides was measured by dynamic light scattering (DLS) using a NICOMP NANO ZLS Z3000 (Entegris, Billerica, MA, United States). Gel electrophoresis was performed using 1% agarose gels loaded with 1 μg of oligonucleotide per well. Ethidium bromide fluorescence imaging was performed after 20 minutes of gel electrophoresis at 100 volts. Cryo-EM images of QTPlus-AM21 were obtained from the Center for Electron Microscopy and Analysis at The Ohio State University (Columbus, OH, United States).

[0172] Cell Culture. The RAW264.7 murine macrophage cell line and the MC38 murine colorectal cancer cell line were kindly provided by Dr. Peixuan Guo and Dr. Christopher Coss, respectively, at The Ohio State University College of Pharmacy. The THP-1 human monocyte cell line was kindly provided by Dr. Joshua Englert at The Ohio State University Wexner Medical Center. The KB and A549 cell lines were purchased from Millipore Sigma (Burlington, MA, United States). RAW264.7 and MC38 were grown in DMEM supplemented with 10% FBS and 1x antibiotic-antimycotic. THP-1, KB, and A549 were grown in RPMI supplemented with 10% FBS. Cells were maintained at 37°C and grown in a humidified atmosphere containing 5% CO2.

[0173] In vitro bioluminescence assay. HEK293, A549, and KB were seeded in 96-well plates at 3,000 cells / well 24 hours before treatment. Cells were treated with 0.1 μg of FLuc mRNA in QTsome Original or QTPlus. Cells were allowed to uptake QTsome Original or QTPlus, which encapsulates mRNA, overnight. mRNA expression was determined using the Bright-Glo™ Luciferase Assay System (Promega, Madison, WI).

[0174] In vitro gene regulation. KB, A549, THP-1, MC38, and RAW264.7 cells were cultured in 6-well plates at 3 × 10 5Cells were seeded at 1000 cells / well 24 hours before treatment. Cells were treated with scrambled oligonucleotides or AM21 in free solution, Lipofectamine, QTsome Original, and QTPlus. Total RNA was extracted using TRI Reagent (Zymo Research) according to the manufacturer's protocol. cDNA was prepared using a High-Capacity cDNA Reverse Transcription Kit (Invitrogen, Waltham, MA, USA), and real-time qPCR (RT-qPCR) was performed on a QuantStudio7 Flex Real-Time PCR System using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad Laboratories, Hercules, CA). All RT-qPCR primers were purchased from Sigma-Aldrich. Beta-actin (Actb) was selected as a housekeeping gene control. Relative amounts of RNA levels were calculated and compared according to the 2-ΔΔCt method.

[0175] Colony formation assay. A549 cells were seeded in 24-well plates at 100 cells / well 24 hours prior to treatment. Cells were treated with the 16-mer and 20-mer AM21 at concentrations of 1 μM and 10 μM in free solution and QTPlus. Cells were allowed to form colonies for up to one week, followed by fixation with methanol and colony detection with crystal violet.

[0176] MTS assay. A549 cells were seeded in 96-well plates at 3,000 cells / well 25 hours prior to treatment. Cells were treated with QTPlus AM21 (concentrations ranging from 0 nM to 6.4 μM), erlotinib (0 μM to 200 μM), and a combination of QTPlus AM21 and erlotinib at a fixed concentration ratio (6.4 / 200 QTPlus AM21 / erlotinib). After 72 hours of treatment, cell viability was assessed using CellTiter96® AQueous One Solution (Promega, Madison, WI) according to the manufacturer's protocol. Synergistic effects of QTPlus AM21 and erlotinib were determined using CompuSyn software (The ComboSyn, Inc.).

[0177] Macrophage-tumor cell co-culture study wound healing assay. MC38 and RAW264.7 cells were cultured at 6 x 10 per well in a 6-well plate. 6 A total of 100 cells were seeded at a fixed macrophage to tumor cell ratio of 3 / 1. Cells were treated with 400 μM AM21 in free solution or QTPlus with or without 1 μg / ml lipopolysaccharide (LPS) stimulation. Cells were incubated at 37°C for 24 hours and collected for flow cytometry analysis.

[0178] For wound healing studies, a scratch wound healing model was performed to examine the migration ability of MC38 cells in the presence of macrophages after treatment. A scratch wound across the well was created immediately before treatment using a 10 μl pipette tip. Cells were washed with PBS and incubated with complete medium containing free solution and 400 μM AM21 in QTPlus, with or without 1 μg / ml LPS stimulation. Cells were grown at 37°C for 24 hours. The distance between the edges of the wound was measured using a Nikon Eclipse Ti-S microscope (Nikon, Tokyo, Japan).

[0179] Enzyme-linked immunosorbent assay (ELISA). Human PTEN matched antibody pair kit was purchased from Abcam (Cambridge, UK). Human EGFR matched ELISA antibody pair set was purchased from Sino Biological (Beijing, China). A549 cells were cultured at 8 × 10 in a 60 mm culture dish. 5 Cells were seeded at 1000 cells / plate 24 hours prior to treatment. Cells were treated with 10 μg of QTPlus-AM21 and 20 μM of erlotinib, individually or in combination. After overnight treatment, cells were harvested and homogenized in Pierce RIPA buffer (Thermo Fisher Scientific). Total protein was extracted after incubation on ice for 30 minutes and centrifuged at 14,000 × g for 30 minutes at 4°C. Protein concentrations were quantified and integrated using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). PTEN and EGFR concentrations were measured according to the manufacturer's protocol.

[0180] In vivo antitumor efficacy study. To determine the antitumor efficacy of QTsome Original encapsulated AM21, A549 NSCLC xenograft mouse models were cultured in nude mice at 1 × 10 per mouse. 7 Tumors were generated by subcutaneous seeding of 100 cells onto the right flank. 3 Treatment began when the AKT-dependent vasoconstriction (vasoconstriction) reached 100%. Mice (n=5) were treated intravenously with saline, 4 mg / kg QTsome Original-encapsulated ASO against AKT, and 3.5 mg / kg QTsome Original-encapsulated AM21. The study was conducted by Bioduro-Sandia (San Diego, CA, United States).

[0181] To evaluate the antitumor efficacy of QTPlus-AM21 and the combination therapy of QTPlus-AM21 with erlotinib, A549 xenograft mouse models were established in nude mice at 2.5 × 10 per mouse. 6Tumors were generated by subcutaneous seeding of 100 cells onto the right flank. 3 Treatment was initiated when the IL-16 receptor agonist (IL-16) ...

[0182] To evaluate the combination therapy of QTPlus-AM21 with anti-PDL1 therapy, the MC38 mouse colorectal syngeneic model was administered to C57BL / 6 mice (obtained from Charles River Laboratories) at 1 × 10 per mouse. 6 Tumors were generated by subcutaneous seeding of 100 cells onto the right flank. 3 Treatment was initiated when the sigma-inducing factor (SIF) reached 1.5. Mice (n=5) were treated intraperitoneally with saline, 3 mg / kg QTPlus-encapsulated scrambled oligonucleotide, 3 mg / kg QTPlus-AM21, 10 mg / kg atezolizumab, and the QTPlus-AM21 / atezolizumab combination (3 mg / kg QTPlus-AM21 and 10 mg / kg atezolizumab).

[0183] All mice were dosed every three days for five doses. Tumor growth and body weight were monitored, and tumor volume was calculated according to the following formula:

number

[0184] All animal studies were reviewed and approved by The Ohio State University Institutional Laboratory Animal Care and Use Committee (IACUC). All mice were euthanized one day after the last dose, when immune activation and protein expression in the TME peak. Terminal tumor growth inhibition (%TGI) was determined by the following formula:

number

[0185] Flow cytometry. Alexa Fluor 700 anti-mouse CD45 (30-F11), APC / cyanine 7 anti-mouse CD3e (145-2C11), FITC anti-mouse CD4 (RM4-5), PE / cyanine 7 anti-mouse CD8a (53-6.7), PE anti-mouse FOXP3 (MF-14), and BV-650 anti-mouse NK1.1 (PK136) were used to detect lymphocyte populations in mouse tumors. FITC anti-mouse F4 / 80 (BM8), PE anti-mouse CD206 (C068C2), BV-605 anti-mouse CD86 (PO3), APC-Cy7 anti-mouse CD11b (M1 / 70), and BV-650 anti-mouse Gr-1 (RB6-8C5) were used to detect myeloid cell populations in mouse tumors. All fluorophore-conjugated antibodies and True-Nuclear transcription factor buffer sets for FOXP3 staining were purchased from BioLegend (San Diego, CA, USA). Single-cell suspensions of tumor tissue in FACS staining buffer were stained according to the manufacturer's protocol. Stained cells were analyzed at the Flow Cytometry Shared Resources (FCSR) at The Ohio State University Comprehensive Cancer Center using an LSR II flow cytometer.

[0186] Statistical analysis. All studies were performed in triplicate. Data are presented as mean ± standard deviation unless otherwise indicated. Statistical analysis was performed using Microsoft Excel. One-way ANOVA was used to determine the variation in means between two or more treatment groups. Student's t-test was used as a post-hoc analysis to determine statistically significant differences between any two groups. A p-value of 0.05 was selected as the cutoff for statistical significance.

[0187] result Optimization of QTsome Original into the QTPlus formulation. The mRNA delivery efficiency of QTPlus with different components was compared in vitro with QTsome Original, Lipofectamine, and the LNP standard from Moderna (Moderna STD) (Figures 1A–1G). Regarding the cationic lipid, a higher amount of cationic lipid showed higher mRNA delivery efficiency (Figures 1A, 1B, and 1C). We also evaluated the mRNA delivery efficiency of QTPlus with different types of commercially available ionizable lipids. In this analysis, A-066 was most suitable for mRNA delivery in the QTsome platform (Figure 1D), and DOPE was most suitable as a helper lipid (Figure 1E). QTPlus was also formulated based on two different protocols (one-step and two-step). Surprisingly, in contrast to the standard one-step LNP / mRNA formulation protocol, QTPlus-mRNA developed by the two-step formulation showed superior mRNA delivery to HEK293 cells (Figure 1F). Finally, in vitro mRNA expression was impaired as formulation temperature increased (Figure 1G). Therefore, QTPlus-mRNA was developed at room temperature instead of pre-incubating at 60°C before formulation.

[0188] QTPlus delivers both mRNA and oligonucleotides. The particle sizes of empty QTPlus and QTPlus-AM21 were larger than those of empty QTsome Original and QTsome Original-AM21, ranging from 80 to 90 nm (empty QTPlus) and 110 to 120 nm (QTPlus-AM21) (Figure 2A). Furthermore, the particle size of QTPlus-AM21 increased slightly to 140 nm after titration of the final product into a pH-neutral solution (Figure 2A). QTPlus-AM21 exhibited a compact spherical structure with a single lipid layer surrounding the exterior (Figure 2B), and was capable of encapsulating any amount of oligonucleotide at an N / P ratio of 3 to 10 (Figure 2C).

[0189] 16mer vs. 20mer AM21. AM21 oligonucleotides of different lengths were designed to evaluate sequence-length-dependent gene regulation and antitumor effects (Figures 3A-3B and 4A-4C). The 16mer and 20mer AM21 exhibited distinct miR-21 downstream gene regulation profiles when A549 cells were treated in free solution and QTPlus formulation (Figures 3A-3B). However, the 16mer AM21 was more effective at reducing particle aggregation when the final QTPlus-AM21 product was titrated into a pH-neutral solution (Figure 4A). In addition, the 16mer AM21 was more effective at inhibiting A549 cell growth in vitro when treated in free solution or QTPlus formulation (Figures 4B and 4C).

[0190] Gene regulation by QTPlus-AM21 in tumor cells in vitro. In KB and A549 cells, AM21 consistently induced Akt1 downregulation, Ddah1 upregulation, and Pdl1 upregulation (Figures 5B and 5C). However, the role of AM21 in regulating Bcl2, Pten, and Pdcd4 expression remains controversial when AM21 is transfected into cells in free solution and with QTsome Original or QTPlus (Figures 5B and 5C). Based on these profiles, QTPlus-AM21 demonstrated the highest level of gene regulation in A549 cells compared with KB and Hep3b cells in vitro (Table 1). Again, QTPlus also demonstrated higher gene delivery efficiency than QTsome Original (Figure 5C). [Table 1]

[0191] Synergistic Effect between QTPlus-AM21 and Erlotinib. QTPlus-AM21 did not show acute cytotoxicity in A549 cells when treated at concentrations ranging up to 6.4 μM (Figure 7). However, when AM21 was subjected to long-term treatment in A549 cells and colony formation was evaluated, QTPlus-AM21 showed a much higher inhibitory effect on colony formation compared to free AM21 (Figures 6A and 6B). Interestingly, when QTPlus-AM21 was treated with erlotinib at a fixed concentration ratio, QTPlus-AM21 was able to sensitize A549 cells to erlotinib cytotoxicity (Figure 6C). The combination index between QTPlus-AM21 and erlotinib was 0.60, and the dose reduction index (DRI) for QTPlus-AM21 and erlotinib was 6.26 and 2.26, respectively. Based on the ELISA results, the combination treatment of QTPlus-AM21 and erlotinib also enhanced PTEN expression (FIG. 8A) and inhibited EGFR expression (FIG. 8B) pharmacologically in vitro based on ELISA.

[0192] In vivo antitumor activity of QTsome / QTPlus-AM21 against NSCLC. QTsome Original was initially used for in vivo AM21 delivery. A QTsome Original-encapsulated ASO against AKT (QTsome Original-anti-AKT ASO) was also designed as a control group to compare the antitumor activity between the inhibition of miR-21 and one of its downstream oncogenes. Although the antitumor efficacy of QTsome Original-AM21 was limited, it was still higher than that of QTsome Original-anti-AKT ASO (Figure 9). When QTPlus was applied to deliver AM21 in vivo, the antitumor response significantly increased (TGI% from 37.9% ± 30.3 for QTsome Original-AM21 to 81.0% ± 4.9 for QTPlus-AM21). Although not statistically significant, the combination of QTPlus-AM21 and erlotinib demonstrated greater in vivo antitumor activity (84.4% ± 7.2% TGI%) (Figure 10). In addition, synergistic PTEN upregulation and EGFR downregulation were observed in vivo with the combination of QTPlus-AM21 and erlotinib (Figures 8C and 8D). There was no significant difference in body weight between mice treated with QTPlus-AM21 and erlotinib individually or in combination, with mild systemic toxicity.

[0193] Immune modulation by QTPlus-AM21. Pd1 upregulation was observed in naive human and mouse macrophages without LPS stimulation (Figures 11A and 1B). In addition, QTPlus-AM21 was able to enhance Cd86 upregulation in both human and mouse macrophage cell lines when they were polarized to the M1 population by LPS stimulation (Figures 11C and 11D). Furthermore, activated M1 macrophages treated with QTPlus-AM21 enhanced Cxcl10, Il-12p40, and Tnfa expression in vitro (Figure 12). When mouse macrophages RAW264.7 were cocultured with mouse colorectal cancer cells MC38, treatment with QTPlus-AM21 was able to increase macrophage proliferation and polarization to the M1 population (Figures 13C and 13D). This macrophage polarization by QTPlus-AM21 was also associated with increased apoptosis in MC38 cancer cell populations (Figure 13B), which ultimately reduced MC38 growth and wound healing efficacy in vitro (Figure 13A).

[0194] Preliminary animal studies showed that QTPlus-AM21 also exhibited antitumor activity in the MC38 syngeneic mouse model (Figure 14). Mice treated with QTPlus-AM21 showed a final TGI% of 68.33% ± 12.4. However, the antitumor activity of QTPlus-AM21 was not significantly improved when the dose was increased from 3 mg / kg to 6 mg / kg (Figure 14). However, treatment with QTPlus-AM21 showed increased CD45+ tumor-infiltrating immune cells (Figures 15A and 15B) and F4 / 80+CD86+M1 populations (Figures 15C and 15D). Significant upregulation of Cxcl10, Ifna, and Tnfa was observed in spleens from mice treated with QTPlus-AM21 (Figure 16A). IL-12 expression was not affected in vivo by QTPlus-AM21 compared to in vitro results (Figure 12). Pdl1 / Pd1 upregulation was observed in tumor (Fig. 16B) and spleen (Fig. 16C) tissues from mice treated with QTPlus-AM21.

[0195] Combination therapy of QTPlus-AM21 and atezolizumab. In the MC38 syngeneic mouse model, both QTPlus-AM21 monotherapy and the QTPlus-AM21 / atezolizumab combination demonstrated significant antitumor responses. Although not statistically significant, QTPlus-AM21 combination therapy demonstrated synergistic antitumor activity with atezolizumab in the MC38 syngeneic mouse model. There was no significant difference in body weight between mice treated with QTPlus-AM21 and atezolizumab individually or in combination, with mild systemic toxicity.

[0196] Consideration QTPlus as an Efficient Nonviral Gene Delivery Platform. LNPs have been developed as a promising platform for delivering various therapeutic agents. Liposomes were initially developed to encapsulate small molecule chemotherapy as anticancer therapeutics. The benefits of using LNPs to deliver small molecules are enhanced therapeutic efficacy by increasing the half-life of the active compound in systemic fluids and targeting tumors via the EPR effect. To maintain these advantages, components of conventional LNPs for small molecule delivery typically contain a certain amount of PEG lipid to prevent degradation of LNP / drug complexes in systemic fluids and reduce particle size due to the EPR effect. However, because gene delivery requires rapid drug release from the LNP into the cytoplasm to achieve therapeutic gene regulation or expression, these designs for conventional LNPs may not be capable of delivering gene payloads. Therefore, when applied for gene delivery, LNPs are expected to prevent degradation in systemic fluids but rapidly release their cargo by endosomal escape upon internalization by cells, suggesting that lower amounts of PEG lipids and higher amounts of functional lipids should be considered when designing LNPs for gene delivery.

[0197] By reducing the amount of cationic lipid and PEG lipid, QTPlus demonstrated a significant increase in in vitro mRNA delivery compared to QTsome Original (Figures 1A-1C). Phosphatidylethanolamine (PE) lipids, which have unsaturated fatty acid chains, are inverted hexagonal (H) lipids, which can greatly facilitate endosomal escape. II ) phase. Indeed, QTPlus with DOPE as a helper lipid showed the highest in vitro mRNA expression (Figure 1E). Gene delivery efficiency by ionizable lipids can vary based on the specific design of different LNPs. Here, in the QTPlus platform, A-066 showed the highest mRNA delivery efficiency compared to other commercially available ionizable lipids (Figure 1D). Regarding the formulation procedure, mixing empty QTPlus with RNA solution may generate a more sealed particle structure, preventing contact between the encapsulated RNA and nucleases in systemic fluids. This may be the reason why the two-step QTPlus-mRNA formulation showed higher mRNA expression than the two-step formulation (Figure 1F). Finally, the QTPlus-mRNA product can be developed at room temperature to prevent RNA hydrolysis after heating (Figure 1G).

[0198] Regarding oligonucleotide delivery, QTPlus was also able to deliver AM21. Although QTPlus-AM21 exhibited a larger size than QTsome Original-AM21 (Figure 2A), particle sizes within 200 nm are considered favorable for cellular uptake. QTPlus also demonstrated a high encapsulation rate for AM21 at N / P ratios of 3 to 10, as demonstrated by gel electrophoresis (Figure 2C), suggesting that the N / P ratios possible with QTPlus may be more tolerable than current LNP-based vaccines. Overall, QTPlus with its optimized composition demonstrated significant increases in both oligonucleotide and mRNA delivery in vitro.

[0199] AM-21 as a potent antitumor agent for NSCLC. While 16-mer and 20-mer AM21 exhibit distinct miR-21 downstream gene regulation profiles (Figures 3A-B), 16-mer AM21 was superior to 20-mer AM21 in inhibiting tumor cell growth in vitro (Figures 4B-C). This is because mature miR-21 is often complexed with Ago2 in the cytoplasm, and the binding domain between miR-21 and Ago2 is located at the first and 17-21 nucleotides from the 5' end. Therefore, 16-mer ASOs targeting miR-21 bypass the Ago2 binding domain and achieve fully complementary binding with miR-21, whereas 20-mer ASOs do not. The distinct gene regulation profiles of AM21 in multiple cancer cell lines suggest that NSCLC is most sensitive to dysregulated miR-21 biological processes compared to other tumor types (Table 1). Therefore, NSCLC was selected as the primary indication for QTPlus-AM21.

[0200] Furthermore, QTPlus significantly enhanced AM21 delivery compared with AM21 transfection in free solution (Figures 6A and 6B), and tumor growth inhibition was entirely dependent on miR-21 inhibition, rather than on acute cytotoxicity by QTPlus (Figure 7). Pharmacologically, miR-21 modulation also shares several signaling pathways that can enhance or restore therapeutic mechanisms with chemotherapy, such as tyrosine kinase inhibitors (TKIs) (30, 31). Studies have shown that treatment with TKIs can restore PTEN expression in lung cancer cells. However, EGFR-mutant lung cancer develops TKI drug resistance by inhibiting PTEN expression. Here, treatment with QTPlus-AM21 could promote PTEN expression and inhibit EGFR levels in the A549 TME (Figures 8A-8D), which ultimately led to promising antitumor efficacy with QTPlus-AM21 monotherapy and QTPlus-AM21 / erlotinib combination therapy (Figure 10), suggesting that QTPlus-AM21 is a promising anticancer agent as monotherapy or in combination with TKI-based first-line chemotherapy for NSCLC.

[0201] AM-21 resulted in high-temperature tumors that are beneficial for anti-PDL1 therapy. Researchers have demonstrated that miR-21 deficiency results in macrophage polarization toward the M1 population and other anti-tumor immune responses in transgenic miR-21-depleted mice, but there is little evidence that antisense oligonucleotide therapy can also effectively induce a bidirectional therapeutic effect targeting both tumors and the immune system. Here, QTPlus-AM21 induced upregulation of CD86 in both human and mouse macrophages (Figures 11C and 11D), which was further demonstrated by flow cytometry results to induce macrophage proliferation and M1 polarization (Figures 13C and 13D). Furthermore, M1 macrophages stimulated by QTPlus-AM21 could inhibit MC38 cancer cell growth and wound healing (Figure 12), and simultaneously, QTPlus-AM21 could induce apoptosis in the MC38 cancer cell population (Figures 13A and 13B). This effect may be explained by cytokine- or chemokine-dependent cytotoxicity, in which secreted CXCL10, IL-12, and TNFα from QTPlus-AM21-stimulated macrophages could inhibit MC38 cancer cell growth in vitro. While 3 mg / kg QTPlus-AM21 demonstrated significant antitumor activity in the MC38 syngeneic mouse model, 6 mg / kg QTPlus-AM21 did not demonstrate a significantly higher response (Figure 14). This may be due to the upregulated PD1 / PD-L1 expression by QTPlus-AM21 and the enhanced immune escape of tumors, as demonstrated in vitro and in vivo (Figures 11A and 11B, 16B and 16C). However, QTPlus-AM21 treatment of syngeneic tumor-bearing mice successfully increased CD45+ tumor-infiltrating immune cells and F4 / 80+CD86+M1 populations in the TME (Figures 15A-15C), suggesting that QTPlus-AM21 can result in "hot" tumors that are beneficial for additional immunotherapy.Finally, both QTPlus-AM21 monotherapy and QTPlus-AM21 / atezolizumab combination therapy demonstrated significant antitumor responses in the MC38 syngeneic mouse model, suggesting that QTPlus-AM21 is also a potent anticancer agent for colorectal cancer as monotherapy or in combination with PD1 / PD-L1 immune checkpoint blockade.

[0202] conclusion Increasing numbers of approved nucleic acid therapeutics have demonstrated the potential to treat diseases through in vivo gene regulation. However, their clinical translation depends on delivery technologies that can improve stability, drug release via endosomal escape, and gene regulation profiles. LNPs provide a lipid compartment for nucleic acid cargo, which can isolate it from serum nuclease activity and facilitate cellular uptake. In this study, the composition of QTsome was optimized for gene delivery, including oligonucleotides and mRNA. The optimized QTPlus demonstrated significant gene delivery efficiency compared with QTsome Original. AM21 was developed for cancer and encapsulated within QTPlus to evaluate its antitumor efficacy in vitro and in vivo. Pharmacologically, AM21 demonstrated significant miR-21 downstream gene regulation and could polarize macrophages toward the M1 population, which benefits the antitumor immune response. QTPlus-AM21 demonstrated synergistic antitumor activity in vivo, either as monotherapy or in combination with erlotinib and atezolizumab, highlighting its great potential as a monotherapy for NSCLC and colorectal cancer, as well as an adjuvant therapy with TKI-based first-line chemotherapy and immune checkpoint blockade.

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[0204] Example 2. Novel composition of lipid nanoparticles for intramuscular delivery of mRNA with reduced systemic gene expression. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is known as the pathogen that causes coronavirus disease 2019 (COVID-19). Coronaviruses are single-stranded RNA viruses coated with a lipid membrane containing envelope (E), membrane (M), and spike (S) proteins. Over the past two years, the COVID-19 pandemic has had a significant impact on the entire world. By November 2021, there had been nearly 250 million coronavirus cases worldwide, including approximately 5 million deaths. The high morbidity and mortality of SARS-CoV-2 spurred the need for an effective vaccine. In 2020, two mRNA vaccine candidates, mRNA-1273 and BNT162b2, were developed by Moderna and Pfizer / BioNTech, respectively, for the prevention of COVID-19. Messenger RNA (mRNA) is a type of single-stranded nucleic acid molecule transcribed from DNA. mRNA plays an essential role in transmitting genetic messages from the nucleus to ribosomes, where proteins are synthesized. As a vaccine, mRNA has the advantage of being able to encode any protein without the need for nuclear delivery. The leading mRNA vaccine candidate encodes the S protein to activate antibody responses against the trimeric S protein of SARS-CoV-2. While mRNA therapeutics are a promising treatment strategy for many diseases, drawbacks limit their development. First, mRNA is easily degraded by RNases in plasma and has a short circulation time without protection. Furthermore, the negative charge of mRNA prevents it from passing through negatively charged cell membranes and further targeting the cytosol. In some situations, mRNA may also cause the activation of unwanted immune responses. Lipid nanoparticles (LNPs) enable efficient delivery of mRNA. LNPs containing ionizable lipids with positively charged head groups can efficiently and sufficiently encapsulate nucleic acids through a self-assembly process. The mRNA is then trapped within the internal aqueous phase and protected from RNases and macrophages by the LNPs.Generally, LNPs prepared for mRNA delivery consist of helper lipids, ionizable lipids, cholesterol, and polyethylene glycol (PEG) to form stable formulations with relatively long circulation times. For example, mRNA-1273 was encapsulated with distearoylphosphatidylcholine (DSPC), cholesterol, methoxypoly(ethylene glycol) dimyristoylglycerol (PEG-DMG), and SM-102, a high-performance pH-responsive ionizable lipid, patented by Moderna.

[0205] Although mRNA vaccines from Pfizer / BioNTech and Moderna have been revolutionary in terms of their impact on public health during the COVID-19 pandemic, there have been incidences of severe adverse reactions. Current SARS-CoV-2 mRNA vaccines have been reported to cause severe myopericarditis. Although extremely rare, these adverse reactions are a common reason cited for vaccine hesitancy. A possible cause of adverse reactions is systemic gene expression, which could be reduced if mRNA delivery could be restricted to muscle, i.e., the local administration site. A series of novel lipid nanoparticle compositions for IM-injected mRNA vaccines, called QTsomes, are described in this example. QTsomes are LNPs incorporating a combination of quaternary (cationic) and tertiary amine (ionizable) lipids. They can also contain other lipids. In some instances, QTsomes can be formed from a helper lipid, cholesterol, PEG-DMG, an ionizable lipid, and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a cationic lipid that permanently carries a positive charge. By incorporating the cationic lipid into LNPs, QTsomes are expected to achieve efficient mRNA delivery via IM injection and reduced systemic gene expression because the majority of the mRNA is restricted to muscle.

[0206] Materials and Methods DSPC, DOPC, DOPE, DOTAP, and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL). DODMA and DMG-PEG2000 were purchased from NOF America (White Plains, NY). SM-102 was synthesized by DC Chemical (Shanghai, China). A066 was synthesized by Dragon Pharma (Shanghai, China). Firefly luciferase messenger RNA (FFLuc mRNA) was purchased from TriLink Biotechnologies (San Diego, CA). Any chemicals or buffers described elsewhere were purchased from Fisher Scientific (Hampton, NH).

[0207] Helper lipids (including DSPC, DOPC, and DOPE), DOTAP, ionizable lipids (including SM-102, A066, and DODMA), cholesterol, and DMG-mPEG2000 were mixed in a lipid-ethanol mixture at molar ratios ranging from 12:3 to 8:45 to 40:3 and 8.5:1.5. The lipid-ethanol mixture was diluted to a final lipid concentration of 8 mg / mL. Messenger RNA was diluted to a concentration of 0.133 mg / mL in 25 mM citrate buffer, pH 2.75. mRNA-lipid QTsome+ nanoparticles were formulated by injecting 250 μL of the lipid-ethanol solution into 750 μL of the mRNA / sodium citrate solution at room temperature using a 29G-1 / 2-inch insulin syringe under vortexing. The solution was thoroughly mixed under gentle vortexing for 15 minutes. Next, 100 μL of 0.2 M Tris-HCl solution (pH 8.0) was added to the lipid QTsome+ nanoparticle solution, and the pH was titrated to pH 7.4 using 2.0 M sodium hydroxide solution. The pH was confirmed by pH paper. To complete the product, 1000 μL of 20 mM Tris-HCl solution (pH 7.4) was added, and the final solution was dialyzed in PBS overnight at 4°C.

[0208] The particle size and zeta potential (ζ) of the nucleic acid-loaded pH-sensitive nanoemulsions were analyzed by dynamic light scattering using a NICOMP Z3000 Nano DLS / ZLS system (Entegris, Billerica, MA). The mRNA concentration was measured using the Quant-it™ RiboGreen RNA Assay Kit.

[0209] ICR CD-1 Swiss mice were purchased from Charles River Laboratory. Animals were housed in a temperature-controlled room with a 12-hour light / 12-hour dark cycle and fed regular chow. All animal studies were reviewed and approved by The Ohio State University Institutional Laboratory Animal Care and Use Committee. Both male and female mice were used for the experiments.

[0210] In vivo mRNA expression was quantified by measuring firefly luciferase bioluminescence. Mice treated with IM injection of 1.5 μg of FFLuc mRNA lipid QTsome+ nanoparticles per leg were intraperitoneally injected with luciferin 5 minutes before bioluminescence measurement. Bioluminescence images were captured by an in vivo imaging system (IVIS) under optimal exposure settings.

[0211] A 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS) fluorescence assay was designed to evaluate the apparent pKa of lipid QTsome+ nanoparticles containing different lipid combinations. TNS binds to positively charged lipids via electrostatic interactions and emits strong fluorescence upon excitation. A series of universal buffers (combinations of 10 mM Na citrate, 10 mM Na phosphate, 10 mM Na borate, and 150 mM NaCl) with pH ranging from 3 to 12 were prepared. QTsome+ was diluted in these buffers to a final ionizable lipid concentration of 75 μM (including SM-102, A066, DODMA, and DOTAP). TNS was then added to a final concentration of 6.0 μM. After thoroughly mixing TNS and QTsome+, the pH was measured using a pH meter. 200 μL of sample was transferred in triplicate into a black opaque plate and analyzed using a SpectraMax M5 plate reader with excitation at 325 nm (λ ex ) and emission at 435 nm (λ em ) to obtain TNS fluorescence. In the presence of amino lipids, TNS fluorescence reached a maximum when 100% of the amino lipids were ionized, and it was assumed that TNS had almost no fluorescence when the amino lipids were in the non-ionized state. The apparent pKa of QTsome+ lipid nanoparticles can be elucidated by the following equation with FL versus pH after fitting the data points with a three-parameter sigmoidal function:

number

[0212] Hypotheses and Key Findings Adding quaternary amines (permanently charged cationic lipids) limits liver penetration and reduces systemic toxicity, providing a compelling reason for selecting the QTsomes described herein as a delivery platform.

[0213] result To test the delivery efficiency of QTsome+ lipid nanoparticles, FFLuc mRNA was selected as the reporter gene of interest to be encapsulated within different QTsome+ formulations. Bioluminescence intensity was measured using a Bright-Glo luciferase kit, which was used to quantify expressed firefly luciferase after mRNA transfection. We treated 10,000 HEK293T cells overnight with 100 ng of FFLuc mRNA using different formulations and Lipofectamine 3000 as a positive control. Here, we showed that bioluminescence intensity significantly decreased when mRNA lipid nanoparticles were formulated at elevated temperatures. mRNA lipid nanoparticles had the highest bioluminescence intensity when formulated at room temperature. Therefore, the following samples were formulated at room temperature instead of 65°C or 37°C.

[0214] Next, we compared the delivery efficiency of different helper lipids, including DSPC, DOPC, and DOPE. Here, we demonstrated that DOPE performed better than DOPC and DSPC in delivering mRNA into cells in vitro using QTsome+ lipid nanoparticles. We also compared the delivery efficiency of QTsome+ lipid nanoparticles containing different DOTAP percentages. We fixed the overall charged / chargeable lipid composition at 48%. Here, the results demonstrated that bioluminescence intensity decreased as the DOTAP percentage increased. See Figures 19 and 20.

[0215] To further explore options for QTsome+ lipid nanoparticles, we further reduced the DOTAP percentage in the formulation and changed the ionizable lipid to the industry standard DLin-MC3-DMA and non-proprietary A066. Bioluminescence results suggested that substituting MC3 slightly increased delivery efficiency compared to SM-102. Furthermore, further reducing DOTAP to 1.5% or increasing the ionizable lipid to 50% facilitated the in vitro delivery process.

[0216] Compared to SM102, QTsomes containing A066 have larger particle sizes, which may be due to the different structure of A066, which has a short branched tail, which can more efficiently encapsulate nucleic acids with small molecular weights, such as siRNA and miRNA, but is not as good as SM102 when encapsulating larger nucleic acids, such as mRNA (see Figures 21A-B).

[0217] When β-Gal was encapsulated, the particle size of QTsomes containing SM102 was larger than that of the same QTsome formulation encapsulating mRNA-luc. This is due to the larger molecular weight of β-Gal than mRNA-luc. 100 μl of each sample was diluted with 3 ml of distilled water. Compared to the other formulations, the absolute value of the zeta potential of Qtsomes containing DOPC was smaller than that of the other formulations. This resulted in a larger particle size. Based on the results from the 100 ng / well β-Gal reporter assay in HEK cells, the QTM1-DOPE formulation had the best performance, and the Moderna formulation performed better than Qtsome Plus containing DOPC and DSPC, although not as well as QTM1-DOPE. See Figures 22A-22C.

[0218] To evaluate the effect of preparation temperature on transfection efficiency, several mRNA-luc LNPs were prepared at different temperatures. After treatment with 100 ng of mRNA-luc LNPs in A549 cell lines, each group was incubated at 37°C for 20 hours. The transfection efficiency increased significantly with decreasing temperature. LNPs prepared at room temperature had the most efficient transfection. See Figure 23.

[0219] According to the gel electrophoresis images (see Figure 24), we can conclude that FFLuc mRNA was highly encapsulated within QTsome+ lipid nanoparticles.

[0220] Based on the TNS assay results (Figure 25), the apparent pKa of QTsome+ (fitting the data from the TNS assay) was highly correlated with the % quaternary amine, consistent with our hypothesis that DOTAP is added as an agent to adjust the zeta potential and apparent pKa of specific ionizable lipids. DOTAP can be thought of as an ionizable lipid with an infinite pKa (which does not release protons but has sufficient positive charge to support membrane disruption).

[0221] Interestingly, the helper lipid plays an important role in regulating the apparent pKa. Under the same % of DOTAP, DSPC resulted in the lowest pKa of all, which was reflected in the in vivo delivery efficiency (higher FFLuc expression). DOPC and DOPE have similar pKa, and the in vivo delivery efficiency was mainly governed by the fusion ability of DOPE.

[0222] It is worth comparing DSPC + 1.5% DOTAP and DOPE + 1.5% DOTAP to see which one provides better delivery efficiency, see Figures 26A-B.

[0223] The in vivo injection results showed that there was no significant difference in FLuc expression between DSPC, DOPC, or DOPE. Further injection runs were performed to increase the N value for statistics. See Figure 27.

[0224] The in vivo injection results showed an inverse trend of FLuc expression versus DOTAP percentage. The Q+T percentage of all samples was fixed at 48%, and DOTAP percentage was the parameter of interest in this experiment. However, one-way ANOVA revealed no significant differences between groups (left panel). When we discarded the two low-expressing samples (marked in orange arrows), the trend was more obvious. One-way ANOVA significance was shown between 1.5 vs. 5.0, 1.5 vs. 8.0, and 3.0 vs. 8.0. See Figure 28.

[0225] By combining two IM injections, the in vivo results showed that there was no significant difference between different helper lipids. However, DOPE still performed better than PC-based helper lipids. We also showed that increasing the DOTAP percentage in the formulation reduced the in vivo delivery efficiency, which was consistent with the in vitro experimental results.

[0226] Consideration Lipid nanoparticles (LNPs) have been developed for nucleic acid delivery for decades because they can protect nucleic acids from degradation by nucleases and clearance from the circulation. mRNA LNPs containing ionizable lipids, such as mRNA-1273, can reduce the zeta potential of mRNA LNPs, thereby helping to reduce cytotoxicity. However, liver penetration of mRNA after IV or IM injection has been observed, which may further cause systemic toxicity. In this example, we discovered a strategy to limit liver penetration of mRNA and reduce systemic toxicity by adding an additional cationic lipid containing a quaternary amine, such as 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTMA), which permanently carries a positive charge. Based on this hypothesis, we propose this advanced nucleic acid delivery system, QTsome, as a preferred platform for mRNA vaccine formulation.

[0227] References 1.Wu,D.,Wu,T.,Liu,Q.&Yang,Z.The SARS-CoV-2 outbreak:What we know.Int.J.Infect.Dis.94,44-48(2020). 2.World Health Organization.WHO Coronavirus(COVID-19)Dashboard. 3.Jackson,L.A.et al.An mRNA Vaccine against SARS-CoV-2-Preliminary Report.N.Engl.J.Med.383,1920-1931(2020). 4.Walsh,E.E.et al.Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates.N.Engl.J.Med.383,2439-2450(2020). 5.Polack,F.P.et al.Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine.N.Engl.J.Med.383,2603-2615(2020). 6.Hoffmann,M.et al.SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.Cell 181,271-280.e8(2020). 7.Kaczmarek,J.C.,Kowalski,P.S.&Anderson,D.G.Advances in the delivery of RNA therapeutics:from concept to clinical reality.Genome Med.9,60(2017). 8.Klugar,M.et al.Side Effects of mRNA-Based and Viral Vector-Based COVID-19 Vaccines among German Healthcare Workers.Biology(Basel).10,752(2021). 9.Kadali,R.A.K.,Janagama,R.,Peruru,S.&Malayala,S.V.Side effects of BNT162b2 mRNA COVID-19 vaccine:A randomized,cross-sectional study with detailed self-reported symptoms from healthcare workers.Int.J.Infect.Dis.106,376-381(2021). 10.Ossato,A.et al.Comparison of medium-term adverse reactions induced by the first and second dose of mRNA BNT162b2(Comirnaty,Pfizer-BioNTech)vaccine:a post-marketing Italian study conducted between 1 January and 28 February 2021.Eur.J.Hosp.Pharm.ejhpharm-2021-002933(2021)doi:10.1136 / ejhpharm-2021-002933. 11.Li,C.et al.Intravenous Injection of Coronavirus Disease 2019(COVID-19)mRNA Vaccine Can Induce Acute Myopericarditis in Mouse Model.Clin.Infect.Dis.(2021)doi:10.1093 / cid / ciab707. 12.Wayment-Steele,H.K.et al.Theoretical basis for stabilizing messenger RNA through secondary structure design.Nucleic Acids Res.49,10604-10617(2021). 13.Zourabian, R., Votruba, J. & Chaloupka, J. Effect of temperature on translation of mRNA coding for an extracellular proteinase and cell proteins in Bacillus megaterium. Curr. Microbiol. 24, 337-342 (1992).

[0228] Example 3. Downregulation of Akt1 by siRNA encapsulated in lipid nanoparticles and combination therapy with lenvatinib for the treatment of hepatocellular carcinoma As the third most common cause of cancer deaths, hepatocellular carcinoma (HCC) resulted in approximately 800,000 deaths in 2020. HCC is the main type of liver cancer, accounting for approximately 90% of liver cancer cases. Orthotopic liver transplantation (OLT) is considered one of the optimal strategies for treating HCC. However, most patients with early-stage HCC cannot undergo OLT in time. With the development of diagnostic and therapeutic technologies, HCC patients are more likely to be cured early. Because aberrant gene expression leading to uncontrolled proliferation of hepatocytes subsequently causes HCC formation, systemic targeted therapy, such as sorafenib, has been applied to regulate oncogene-related pathways. To appropriately suppress the abnormal growth of cancer cells and achieve the expected therapeutic effect, it is beneficial to target two or more pathways.

[0229] In this study, small interfering RNA (siRNA) specifically targeting Akt1 was designed to silence Akt1 mRNA expression, thus downregulating protein translation and affecting multiple downstream pathways. Akt1 plays an essential role in cell migration, proliferation, and apoptosis. Akt1 is thought to regulate anti-apoptosis in many cell death paradigms. Downregulation of Akt1 gene expression suppresses downstream signaling and promotes cell death in cancer cells (Figure 29). The siRNA was encapsulated within an advanced drug delivery system consisting of both cationic and ionizable lipids, termed QTsome Plus. QTsome Plus was developed by Yung et al. in 2016. 6 This study is based on the previous QTsome version designed in [1]. Generally, nucleic acids, such as siRNA and miRNA, are delivered by lipid nanoparticles composed of ionizable lipids instead of cationic lipids to avoid cytotoxicity. However, ionizable lipids are neutral under physiological conditions, and the uncharged surface of lipid nanoparticles cannot be easily taken up by cells. In this study, to enhance the delivery efficiency of encapsulated siRNA, QTsome Plus was designed to consist of both quaternary ammonium and tertiary amine. Quaternary ammonium has a permanent positive charge, and its ratio is much lower than that of pH-sensitive tertiary amine. Therefore, the lipid nanoparticles do not aggregate and can fuse with negatively charged membranes under physiological pH conditions without causing severe cytotoxicity.

[0230] To enhance the antitumor effect in HCC models, we also used the recently FDA-approved drug lenvatinib in combination with siRNA-Akt1 QTsome plus. Lenvatinib acts as a kinase inhibitor against multiple kinases, including vascular endothelial growth factor receptors (VEGFR) 1-3 and fibroblast growth factor receptors (FGFR) 1-4. 7 Lenvatinib has demonstrated antitumor activity against multiple cancers, including melanoma, thyroid cancer, and hepatocellular carcinoma. 8、9The objective of this study was to evaluate the antitumor effects of a novel vehicle-encapsulated siRNA targeting Akt1 and the combination therapy of siRNA and lenvatinib in an HCC cell line-derived xenograft (CDX) model.

[0231] Materials and Methods Materials. Cholesterol and PEG-DMG were purchased from Avanti® Polar Lipids. Lipids were purchased from MedChemExpress. Primers for RT-qPCR were purchased from ThermoFisher®. SYBR Green SuperMix was purchased from BioRad. Applied Biosystem™ High-Capacity cDNA Reverse Transcription Kit was purchased from FisherSci. Primary and secondary antibodies for Western blot were purchased from Cell Signaling Technologies (CST), except for the antibody for human FRS2, which was purchased from R&D systems. Mini-protein TGX gel, Tris / glycine, was purchased from BIO-RAD. PI / RNase staining buffer, BD Annexin V-FITC, and RNase A were purchased from FisherSci. siRNA sequences were synthesized by Integrated DNA Technologies (IDT). Lenvatinib was purchased from Caymen Chemical. Peripheral blood mononuclear cells (PBMCs) were purchased from IQ Biosciences. Human ELISA kits for IL-6, TNF-α, and IFN-γ were purchased from ThermoFisher.

[0232] Preparation of siRNA-encapsulated lipid nanoparticle formulation. Lipid, cholesterol, and PEG-DMG stock solution are mixed together and injected into 25mM citrate RNase-free water. Empty lipid nanoparticles are sonicated and then sterile filtered through a 45nm membrane. siRNA is added to the empty lipid nanoparticles at a drug-lipid ratio of 1:12. Then, dialysis is performed to remove ethanol and adjust pH.

[0233] Cells and cell culture. Cell lines were purchased from the JCRB Cell Bank. Huh7 was cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). HepG2, Hep3B, and SNU387 were cultured in Eagle's minimum essential medium (EMEM) supplemented with FBS at 37°C in a 5% CO2 incubator. 100 U / ml penicillin and 100 μg / ml streptomycin were added to the culture medium as complete medium.

[0234] Xenograft animal model. Huh-7 cell + Matrigel suspension was injected subcutaneously (sc) into the flank of one hind leg of 6-8 week-old male athymic BALB / C mice. Tumor size was approximately 100 mm. 3 The mice were continuously monitored until the tumor size reached 3000 mm. The mice were then divided into several groups, and each group received treatment according to the study design. Tumor size and body weight were measured daily or every two days. The study was terminated when the tumor size reached 3000 mm. 3 The tumor size was determined when the tumor reached 100 μg / ml or when severe necrosis, ulceration, or bleeding occurred. In this case, the mice were humanely sacrificed. Tumor and tissue collection was performed for further studies, if required. MC38 were dissolved in 10 mL of PBS. 6 The cells were resuspended at a density of 10 / ml. 10 6 MC38 cells were inoculated. 3 Treatment was initiated when tumors grew to a range of 0.01 to 0.01 mm. Treatment was administered every 3 days. Tumor size and weight were monitored daily. Hydrogel was administered upon weight loss of more than 10%. Mice were sacrificed 5 days after the dose. Blood samples were collected for further study.

[0235] Dynamic Light Scattering (DLS). Particle size was tested with a Nicomp Nano Z3000 DLS / ZLS system (Entegris). Samples were diluted to 50-100 μg / ml in PBS buffer, and then particle size was tested by dynamic light scattering measurements. Intensity-weighted size distribution was reported by the instrument.

[0236] Cryo-transmission electron microscopy (cryo-EM). Cryo-EM images were acquired by the Center for Electron Microscopy and Analysis (CEMAS) at The Ohio State University. A suspension of lipid nanoparticles was placed on a copper grid coated with a carbon film. Excess liquid was then removed. The lipid nanoparticles were quickly frozen in liquid ethane. The grid with the sample was then transferred to the specimen chamber of a transmission electron microscope (TEM). The specimen chamber was cooled with liquid helium.

[0237] Real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Total RNA was extracted from in vitro tissues or cells using TRIzol Reagent (Thermo Fisher Scientific). cDNA was reverse transcribed from mRNA to cDNA using a High-Capacity cDNA Reverse Transcription Kit (Fisher Scientific). The cDNA was then amplified for 40 cycles with the required primers. Relative cDNA levels were analyzed using SYBR Green and Applied Biosystems QuantStudio7, and comparative C to analyze mRNA expression. T Method (ΔΔC T β-actin was used as an internal control.

[0238] Western blot analysis. Cells were grown in 100 mm Petri dishes for 48 hours and then lysed with lysis buffer (0.5 M EDTA and 1% Triton X-100) in phosphate-buffered saline (PBS) containing a protease inhibitor and phosphatase inhibitor cocktail. Proteins were measured and diluted to the same concentration, followed by the addition of Laemmli buffer with 10% β-ME and boiling for 5 minutes. Proteins were then loaded into each well of a 10% Mini-protein TGX gel and run in Tris / glycine buffer at 100 V for 80 minutes. Proteins were then transferred to a membrane in transfer buffer at 180 A for at least 1 hour. The membrane was air-dried and blocked with non-fat dry milk. The membrane was washed three times with TBST. The membrane is incubated overnight at 4°C with the primary antibody at 1:1000 in TBS buffer with 5% BSA, and then with the secondary antibody at 1:5000 in TBS with 5% BSA. The membrane is washed three times with TBST. Western blots are analyzed after imaging and ECL reagent is added.

[0239] In vitro enzyme-linked immunosorbent assay. Cytokine levels were analyzed using commercially available ELISA kits from ThermoFisher to detect human IL-6, TNF-α, and IFN-γ after treatment. ELISA was performed according to the protocol provided by the manufacturer. Plates were read at a wavelength of 450 nm in a microplate reader.

[0240] Flow cytometry analysis. Cells were seeded in monolayers, incubated overnight, and then harvested with 0.25% trypsin, 0.02% EDTA. Cells were resuspended and counted, then washed in PBS with 0.5% BSA and collected by centrifugation. For cell cycle distribution assays, cells were washed with cold PBS and then fixed with 75% ethanol for at least 12 hours. Dying and dead cells were stained with propidium iodide and excluded from analysis. Duplicates and dead cells were also excluded by gating on FSC and SSC.

[0241] In vivo antitumor study. In nude mice study, mice were treated with 3×10 siRNA against Akt1. 6 A suspension of huh-7 cells was inoculated via sc. Tumors grew to approximately 100 mm 3 When the tumors reached 100 μg / ml, the mice were divided into groups with similar average tumor sizes. siAkt1 was administered every 3 days. After treatment, the mice were monitored for 2 days and then sacrificed. In nude mouse studies, mice were treated with 2×10 siRNA in combination with lenvatinib. 6 A suspension of huh-7 cells was inoculated via sc. Tumors grew to approximately 100 mm 3 When the tumors reached 3000 mm3, the mice were divided into groups with similar average tumor sizes. After six doses, the animals were observed and tumors were measured at 3000 mm3. 3 Mice were sacrificed when they reached adulthood or when severe necrosis, hemorrhage, or ulceration occurred.

[0242] Statistical analysis. All in vitro studies were performed in triplicate. In all in vivo studies, each group had at least five mice. Student's t-test was used to determine the significance of differences between groups, and data analysis with a value of P<0.05 was considered to have statistical significance.

[0243] result Physical characteristics of QTsome+-encapsulated siRNA. Particle size was measured by dynamic light scattering (DLS). siRNA was encapsulated by QTsome+ at a drug-lipid ratio of 1:12. Ionizable lipid, cholesterol, and PEG-DMG were mixed in ethanol and rapidly injected into an acidic citrate buffer solution. After probe sonication and filtration through a 0.45 nm membrane, the intensity-weighted diameter of empty QTsome+ was 86.8 nm, and the polydispersity index (PDI) was 0.189. An aqueous siRNA solution was then injected into the empty QTsome+ solution under vortexing. After titration to pH 7.4, the intensity-weighted diameter was 140.1 nm, and the PDI was 0.234. This benefits distribution to tumor sites via the enhanced permeability and retention (EPR) effect (Figures 30A and 30B). A single peak was observed in each sample, and the PDI values ​​of the two samples were relatively comparable. This indicates that QTsome+ were nearly monodisperse during and after preparation. The intensity-weighted diameter of encapsulated QTsome+ was larger than that of empty ones, indicating that siRNA was encapsulated. The structure of siRNA-encapsulated QTsome+ was characterized via cryo-EM. According to the data provided by the software, 99% of the empty vesicle distribution was below 238.9 nm, and 99% of the siRNA-encapsulated vesicle distribution was below 322.1 nm.

[0244] As shown in Figure 31, QTsome+ exhibits a bilayer structure with smooth particle curvature. Several small vesicles exhibiting a bilayer structure were also present inside the lipid nanoparticles, indicating that the siRNA was surrounded by ionizable or cationic lipids due to interactions between the positive charges of the lipid head groups and the negative charges of the nucleic acid, forming a water / oil / water system. [Table 2]

[0245] siRNA-Akt1 down-regulates Akt1 gene and protein levels in HCC cell lines. The effect of siRNA suppressing Akt1 mRNA was initially evaluated in huh7, Hep3B, SNU387, and HepG2 cell cultures. siRNA was delivered via either Lipofectamine 3000 reagent or QTsome+ and transfected into cells (Figure 32A). After 24 hours of transfection with 25 nM dose of siRNA-scrambled or siRNA-Akt1, mRNA was extracted and reverse transcribed into cDNA. mRNA expression was analyzed by SYBR Green and Applied Biosystems QuantStudio7, and comparative C for analysis. T The expression of siRNA was quantified using the ELISA method. For each vehicle, siRNA-scrambled served as an internal control for siRNA-Akt1, reducing the vehicle's effect on gene expression. Significant downregulation of Akt1 mRNA was observed in huh7 and Hep3B cell lines treated with siRNA-Akt1, regardless of the vesicle used to deliver the siRNA. Regarding SNU387, Akt1 downregulation was significant in cells treated with QTsome+, but the transfection efficiency of siRNA encapsulated by Lipofectamine 3000 was not as good as that of QTsome+. Although slight downregulation of Akt1 was observed, neither Lipofectamine 3000 nor QTsome+ achieved good transfection efficiency in HepG2 cells. This indicates that HepG2 is not an optimal model for siRNA-Akt1 transfection. Different doses of siRNA were also tested in huh-7 and Hep3B to assess the relationship between Akt1 downregulation and dose (Figure 32B). Higher doses, from 0.3 nM to 30 nM, resulted in greater levels of downregulation of mRNA, while at 0.3 nM, almost no downregulation was observed.

[0246] Western blot analysis was then performed to evaluate the effect of siRNA-Akt1 on Akt1 protein expression. Cells were transfected with PBS, 25 nM siRNA-scrambled, and 25 nM siRNA-Akt1 encapsulated by QTsome+ for 48 hours. Figure 32C shows that Akt1 protein expression was suppressed in huh-7, Hep3B, and SNU387 cell lines after treatment with siRNA-Akt1 QTsome+ compared with the groups treated with PBS or siRNA-scrambled. This demonstrates that siRNA-Akt1 can suppress Akt1 protein expression in these cell lines, confirming that QTsome+ can deliver transmembrane siRNA to the cytosol and efficiently target mRNA.

[0247] The pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α are known to play essential roles as mediators of immunity and inflammation, and in promoting macrophage polarization and inflammatory activity of macrophages. See Figure 32D.

[0248] Cell cycle status was assessed by flow cytometry. Cells were transfected with 25 nM PBS, siRNA-scrambled, or siRNA-Akt1 for 48 hours and then fixed and stained. Cell separation into G0 / G1, S, and G2 / M phases was determined by linear fluorescence intensity after staining with propidium iodide (PI). As shown in Figure 32E, siRNA-Akt1 induced cell cycle arrest in S phase. The percentage of cells in G0 / G1 phase decreased, accompanied by a slight increase in the percentage of cells in S phase. In huh-7 cells, the percentage of cells in G0 / G1 phase decreased from 59.5% to 46.5% with siRNA-Akt1 treatment, while S phase increased from 11.1% to 17.3% compared to PBS. A similar trend was observed in Hep3B and SNU387 cells. This indicates that downregulation of Akt1 by siRNA is involved in S-phase accumulation in huh-7, Hep3B, and SNU387 cells.

[0249] In vivo treatment with si-Akt1 results in antitumor activity in huh7 xenografts without severe side effects in nude mice. To test the in vivo efficacy of si-Akt1, nude mice bearing huh7 xenografts were treated with saline, siRNA-scrambled QTsome+, or siRNA-Akt1 QTsome+ for 11 days. Nude male mice aged 8-10 weeks were treated with 3 × 10 6 huh7 cells were inoculated. The average tumor size was 100-150 mm. 3 Treatment was initiated when tumors reached a tumor size of 1000 μg / kg. Animal data showed that siRNA-Akt1 encapsulated by QTsome+ could significantly suppress tumor growth at both 2 mg / kg and 4 mg / kg doses, with tumor growth inhibition (TGI) rates of 25.90% and 40.07%, respectively. The antitumor effect of 4 mg / kg siRNA-Akt1 was better than that of 2 mg / kg, indicating a certain degree of positive correlation between dose and effect (Figure 33A). Although the mechanism of antitumor activity is unclear, this demonstrates that Akt1 is a good target. The hypothesis is that siRNA targets macrophages, induces macrophage polarization, and subsequently modifies the tumor microenvironment. The average tumor size of mice treated with siRNA-scrambled was not significantly different from that of those treated with saline, indicating that QTsome+ has minimal effect on tumor suppression. The mean body weights of all groups treated with siRNA QTsome+ decreased at a relatively similar level, indicating that QTsome+ has slight toxicity as a vehicle, but Akt1 downregulation has a negligible effect on weight loss. No severe side effects, such as dizziness, asthenia, dehydration, or ulcers, occurred (Figure 33B). [Table 3]

[0250] siRNA-Akt1 can suppress the growth of MC38 xenografts in C57BL6 mice. To evaluate the antitumor efficacy of siRNA-Akt1 on the mouse cell line MC38, an in vivo pharmacodynamic study was performed in C57BL6 mice. Cells from the mice were diluted in PBS at 10 6 The cells were resuspended at a density of 10 / ml. 10 6 MC38 cells were inoculated. 3 Treatment was initiated when tumors reached a range of 0.01 to 0.01 mmHg. Treatment was administered every 3 days. Tumor size and weight were monitored daily. Hydrogel was administered upon weight loss of more than 10%. Mice were sacrificed 2 days after the 4th dose. Blood samples were collected for further study.

[0251] Figure 34 shows that siRNA-Akt1 showed a significant difference compared to siRNA-scrambled at a dose of 4 mg / kg, indicating that downregulation of Akt1 can induce anti-tumor effects in vivo. This may be caused by the induction of the M1 subtype of macrophage, which suppresses the growth of exogenous tumor cells. Compared with the saline-treated group, siRNA-scrambled showed slight tumor inhibition, but the difference was not statistically significant, suggesting that the vehicle may have some effect on tumor suppression. The combination of siRNA-Akt1 with the anti-PDL1 monoclonal antibody atezolizumab showed a sufficient anti-tumor effect (72.4% TGI compared to saline), and the significant difference between the combination group and the atezolizumab-only group demonstrated that downregulation of Akt1 can promote the tumor-suppressing efficacy of anti-PDL1, indicating that simultaneous inhibition of both Akt1 and PDL1 may be a good option for cancer therapy.

[0252] These results are further described in the attached Appendix, which is incorporated herein by reference in its entirety.

[0253] The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of some aspects of the claims. Any compositions and methods that are functionally equivalent are intended to be within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to be within the scope of the appended claims. Furthermore, although only certain representative compounds, components, compositions, and method steps disclosed herein are specifically recited, other combinations of compounds, components, compositions, and method steps are also intended to be within the scope of the appended claims even if not specifically recited. Thus, although combinations of steps, elements, components, or components may not be explicitly recited herein, or may be less, other combinations of steps, elements, components, and components are included even if not explicitly recited.

[0254] As used herein, the term "comprising" and variations thereof are used synonymously with the term "including" and variations thereof and are open, non-limiting terms. Although the terms "comprising" and "including" are used herein to describe various embodiments, the terms "consisting essentially of" and "consisting of" may be used in place of "comprising" and "including" to provide more specific embodiments of the present invention and are disclosed. Except where stated, all numbers expressing geometry, dimensions, and the like used in the specification and claims are to be understood as at least and are not intended to limit the application of the doctrine of equivalents to the claims, and are to be interpreted in light of the number of significant digits and ordinary rounding approaches.

[0255] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications cited herein and the material for which they are cited are specifically incorporated by reference.

Claims

1. 1. A pharmaceutical composition comprising lipid particles encapsulating an active agent, said lipid particles comprising: one or more cationic lipids, one or more ionizable lipids, one or more neutral lipids, and one or more PEGylated lipids, A pharmaceutical composition wherein the one or more cationic lipids and the one or more ionizable lipids are present in the lipid particles in an amount effective to provide an apparent pKa of 6 to 10, e.g., 6 to 8, as determined by a TNS pKa assay.

2. 1. A pharmaceutical composition comprising lipid particles encapsulating an active agent, said lipid particles comprising: one or more cationic lipids, one or more ionizable lipids, one or more neutral lipids, and one or more PEGylated lipids, The one or more cationic lipids and the one or more ionizable lipids are present within the lipid particle in a composition having the following formula: 6<pKa ０ +k×Q / T<10 was present in an amount that satisfied pKa ０ represents the pKa of the ionizable lipid; k represents an empirical constant determined by TNS pKa assay; Q represents the mole percent of the cationic lipid; wherein T represents the mole percent of said ionizable lipid.

3. The one or more cationic lipids and the one or more ionizable lipids are present within the lipid particle in a composition having the following formula: 6<pKa ０ +k×Q / T<8 was present in an amount that satisfied pKa ０ represents the pKa of the ionizable lipid; k represents an empirical constant determined by TNS pKa assay; Q represents the mole percent of the cationic lipid; 3. The composition of claim 2, wherein T represents the mole percent of the ionizable lipid.

4. 4. The composition of claim 2 or 3, wherein Q / T is greater than 0 to 1.

5. 5. The composition of claim 1, wherein the one or more cationic lipids are present in the lipid particle in an amount of from greater than 0 mol% to 10 mol%, based on the total components forming the lipid particle.

6. 6. The composition of claim 5, wherein the one or more cationic lipids are present in the lipid particle in an amount of 0.5 mol % to 5 mol %, based on the total components forming the lipid particle.

7. 6. The composition of claim 5, wherein the one or more cationic lipids are present in the lipid particle in an amount of 4 mol % to 8 mol %, based on the total components forming the lipid particle.

8. 8. The composition of claim 1, wherein the one or more ionizable lipids are present in the lipid particle in an amount of 20 mol % to 65 mol %, based on the total components forming the lipid particle.

9. 9. The composition of claim 1, wherein the one or more neutral lipids are present in the lipid particle in an amount of 35 mol% to 80 mol%, based on the total components forming the lipid particle.

10. 10. The composition of any one of claims 1 to 9, wherein the one or more PEGylated lipids are present in the lipid particle in an amount of greater than 0 mol% to 5 mol%, based on the total components forming the lipid particle.

11. 1. A pharmaceutical composition comprising lipid particles encapsulating an active agent, said lipid particles comprising: greater than 0 mol% to 10 mol% of one or more cationic lipids; 20 mol % to 65 mol % of one or more ionizable lipids; 35 mol% to 80 mol% of one or more neutral lipids, and A pharmaceutical composition comprising greater than 0 mol% to 5 mol% of one or more PEGylated lipids.

12. The one or more cationic lipids and the one or more ionizable lipids are present within the lipid particle in a composition having the following formula: 6<pKa ０ +k×Q / T<10 was present in an amount that satisfied pKa ０ represents the pKa of the ionizable lipid; k represents an empirical constant determined by TNS pKa assay; Q represents the mole percent of the cationic lipid; 12. The composition of claim 11, wherein T represents the mole percent of the ionizable lipid.

13. The one or more cationic lipids and the one or more ionizable lipids are present within the lipid particle in a composition having the following formula: 6<pKa ０ +k×Q / T<8 was present in an amount that satisfied pKa ０ represents the pKa of the ionizable lipid; k represents an empirical constant determined by TNS pKa assay; Q represents the mole percent of the cationic lipid; 13. The composition of claim 12, wherein T represents the mole percent of the ionizable lipid.

14. 14. The composition of any one of claims 11 to 13, wherein the one or more cationic lipids are present in the lipid particle in an amount of 0.5 mol% to 10 mol%, based on the total components forming the lipid particle.

15. 15. The composition of any one of claims 11 to 14, wherein the one or more cationic lipids are present in the lipid particle in an amount of 0.5 mol% to 3.5 mol%, based on the total components forming the lipid particle.

16. 15. The composition of any one of claims 11 to 14, wherein the one or more cationic lipids are present in the lipid particle in an amount of 4 mol% to 8 mol%, based on the total components forming the lipid particle.

17. 17. The composition of any one of claims 1 to 16, wherein the one or more ionizable lipids are present in the lipid particle in an amount of 30 mol% to 50 mol%, based on the total components forming the lipid particle.

18. 18. The composition of any one of claims 1 to 17, wherein the one or more ionizable lipids comprise a lipid head group that includes a tertiary amine.

19. 19. The composition of any one of claims 1-18, wherein the one or more ionizable lipids comprise N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), DLin-MC3-DMA, DLin-KC2-DMA, 1-(2,3-bis(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)propyl)pyrrolidine (A066), or any combination thereof.

20. 20. The composition of any one of claims 1 to 19, wherein the one or more neutral lipids are present in the lipid particle in an amount of 30 mol% to 50 mol% of the total components forming the lipid particle.

21. 21. The composition of any one of claims 1 to 20, wherein the one or more neutral lipids comprise dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), cholesterol, or any combination thereof.

22. 22. The composition of any one of claims 1 to 21, wherein the one or more PEGylated lipids are present in the lipid particle in an amount of 0.5 mol% to 3 mol% of the total components forming the lipid particle.

23. 23. The composition of any one of claims 1-22, wherein the one or more PEGylated lipids comprise PEG-ditetradecylacetamide, PEG-myristoyl diglyceride, PEG-diacylglycerol, PEG dialkyloxypropyl, PEG-phospholipid, PEG-ceramide, or any combination thereof.

24. 24. The composition of any one of claims 1 to 23, wherein the one or more cationic lipids comprise a lipid head group comprising a quaternary amine.

25. The one or more cationic lipids may be DOTMA: [1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: dioleylphosphatidylethanolamine, DOSPA (2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate), DORIE (N-[1-(2,3-dioleyloxypropyl)]-N,N-dimethyl-N-hydroxyethylammonium bromide), DODAB, DOIC, DMEPC, DOGS: dioctadecylamidoglycylspermine, DIMRI: dimyristoxypropyldimethylhydroxyethylammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-.alpha. -trimethylammonioacetyl)diethanolamine chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, lipids described in U.S. Pat. No. 5,049,386, WO 97 / 04909 N-[1-(2,3-dioleyloxypropyl)]-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), and the like, disclosed in WO 91 / 16024 and WO 97 / 019675; DLinDMA, and the like, disclosed in WO 2005 / 121348; and DLin-K-DMA, and the like, disclosed in WO 2009 / 086558;and (3R,4R)-3,4-bis((Z)-hexadec-9-enyloxy)-1-methylpyrrolidine and N-methyl-N,N-bis(2-((Z)-octadec-6-enyloxy)ethyl)amine disclosed in WO 2011 / 13636, or any combination thereof.

26. 26. The composition of any one of claims 1 to 25, wherein the lipid particles have an average diameter of less than 1 micron, e.g., between 50 nm and 750 nm, between 50 nm and 250 nm, between 50 nm and 200 nm, between 50 nm and 150 nm, or between 50 nm and 100 nm.

27. 27. The composition of any one of claims 1 to 26, wherein the lipid particles have a polydispersity index (PDI) of less than 0.

4.

28. The composition of any one of claims 1 to 27, wherein the active agent comprises a nucleic acid.

29. 30. The composition of claim 28, wherein the nucleic acid comprises siRNA, mRNA, or any combination thereof.

30. 30. A method of delivering an active agent to a cell, comprising contacting said cell with a composition according to any one of claims 1 to 29.

31. 30. A method for delivering an active agent to a cell in vivo, comprising administering to a mammalian subject a composition according to any one of claims 1 to 29.

32. 32. The method of claim 31 , wherein the mammal is a human.

33. 33. The method of claim 31 or 32, wherein the administration is intravenous or intramuscular.

34. 1. A method of systemically administering an active agent to a subject in need thereof, said method comprising intravenously injecting a pharmaceutical composition comprising lipid particles encapsulating said active agent, said lipid particles comprising: 0.5 mol % to 8 mol % of one or more cationic lipids; 20 mol % to 65 mol % of one or more ionizable lipids; 35 mol% to 80 mol% of one or more neutral lipids, and The method comprises greater than 0 mol% to 5 mol% of one or more PEGylated lipids.

35. 35. The method of claim 34, wherein the active agent comprises an anti-cancer agent.

36. 1. A method of administering an active agent to the liver of a subject, the method comprising intravenously injecting a pharmaceutical composition comprising lipid particles encapsulating the active agent, the lipid particles comprising: 0.5 mol % to 3 mol % of one or more cationic lipids; 20 mol % to 65 mol % of one or more ionizable lipids; 35 mol% to 80 mol% of one or more neutral lipids, and The method comprises greater than 0 mol% to 5 mol% of one or more PEGylated lipids.

37. 37. The method of claim 36, wherein the active agent comprises an anti-cancer agent, for example, an active agent for the treatment of liver cancer.

38. 1. A method of administering an active agent to a solid tumor in a subject, the method comprising intravenously injecting a pharmaceutical composition comprising lipid particles encapsulating the active agent, the lipid particles comprising: 3 mol % to 6 mol % of one or more cationic lipids; 20 mol % to 65 mol % of one or more ionizable lipids; 35 mol% to 80 mol% of one or more neutral lipids, and The method comprises greater than 0 mol% to 5 mol% of one or more PEGylated lipids.

39. 37. The method of claim 36, wherein the active agent comprises an anti-cancer agent.

40. 40. The method of claim 38 or 39, wherein the method targets tumor angiogenesis.

41. 1. A method of administering an active agent to the lungs of a subject, the method comprising intravenously injecting a pharmaceutical composition comprising lipid particles encapsulating the active agent, the lipid particles comprising: 6 mol % to 10 mol % of one or more cationic lipids; 20 mol % to 65 mol % of one or more ionizable lipids; 35 mol% to 80 mol% of one or more neutral lipids, and The method comprises greater than 0 mol% to 5 mol% of one or more PEGylated lipids.

42. 42. The method of claim 41, wherein the active agent comprises an anti-cancer agent, e.g., an active agent for the treatment of lung cancer.

43. 1. A method of locally administering an active agent to a subject in need thereof, said method comprising intramuscularly injecting a pharmaceutical composition comprising lipid particles encapsulating said active agent, said lipid particles comprising: greater than 0 mol% to 5 mol% of one or more cationic lipids; 20 mol % to 65 mol % of one or more ionizable lipids; 35 mol% to 80 mol% of one or more neutral lipids, and The method comprises greater than 0 mol% to 5 mol% of one or more PEGylated lipids.

44. 44. The method of claim 43, wherein the active agent comprises a nucleic acid.

45. 45. The method of claim 44, wherein the nucleic acid comprises siRNA, mRNA, or any combination thereof.

46. 46. ​​The method of any one of claims 43 to 45, wherein the active agent comprises a vaccine.

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