Lipid nanoparticle delivery systems with enhanced stability

Abstract

A lipid nanoparticle delivery system; it has a cargo molecule; and a lipid nanoparticle encapsulating the cargo molecule, the lipid nanoparticle comprising ionizable lipids; helper lipids; sterol; and acid-containing lipids that are crosslinked; and methods of use thereof.

Description

LIPID NANOPARTICLE DELIVERY SYSTEMS WITH ENHANCED STABILITY

[0001] The present application claims priority from U.S. provisional patent application No. 63 / 666,915 filed on July 2, 2024, incorporated herein by reference.Technical Field

[0002] The present disclosure relates to systems for delivering compounds, for triggering an immunological response or for treatment, to a subject, and more particularly to lipid nanoparticle delivery systems for delivering compounds to a subject.Background

[0003] Despite the encouraging clinical outcomes of lipid nanoparticles (LNP) for RNA delivery, several limitations associated with LNP remain. First, current LNP formulations lack good stability during lyophilization and storage, and thus require cold-chain storage and transportation, which significantly hinders the wide distribution and applications. Second, current LNP formulations exhibit insufficient in vivo stability, due to the fact that LNPs are formed by self-assembly of multiple molecules through weak interactions, such as hydrophobic and electrostatic interactions, which can be easily disrupted by many factors in vivo, including dilution, interactions with biomolecules, and physical changes (pH, ionic strength) in surrounding environments. Third, there is no active mechanism in the current LNP formulations to release cargos to function.

[0004] Therefore, strategies to enhance the stability of LNP while promoting cargo release are heavily needed.

[0005] The present disclosure relates to an advanced LNP formulation with enhanced stability and responsive cargo release for gene delivery. Specifically, an acid-modified lipid (e.g. phenylboronic acid (PBA)-modified lipid) is included to serve as the anchoring site for polyvinyl alcohol (PVA) coating through phenylboronate ester bonds, which endues the covalent crosslinking of a LNP (denoted as cLNP), thus increasing the LNP structure rigidity. Of note, the PVA coating in cLNP does not hinder the release of cargos because phenylboronate ester bonds can be easily cleaved in endosomes with a pH of 5.5-6.5. Importantly, glutathione (GSH)-cleavable lipids are also incorporated in the cLNP, which can facilitate the degradation of cLNPs in the GSH- rich cytosol to release cargos. cLNPs display higher gene transfection efficiency than the noncoating counterparts for various RNA cargoes (including siRNA, mRNA, and anti-virus5’pppRNA molecules) at different storage conditions. The cLNPs displayed an excellent lung targeting capability after intravenous administration, which resulted in a remarkable anti-virus effect and significantly prolonged survival in an influenza A virus (lAV)-infected mice model with 5’pppRNA loading.

[0006] A broad aspect of the present disclosure is a lipid nanoparticle delivery system including one or more cargo molecules; and a lipid nanoparticle encapsulating the one or more cargo molecules, the lipid nanoparticle comprising: 40 mol % to 60 mol % of ionizable lipids; 5 mol % to 20 mol % of helper lipids; 30 mol % to 50 mol % of sterol; and 5 mol % to 20 mol % of acid-containing lipids, wherein crosslinking is present on a surface of the lipid nanoparticle.

[0007] In some embodiments, the acid-containing lipids may be phenylboronic acidcontaining lipids.

[0008] In some embodiments, the phenylboronic acid-containing lipids may be crosslinked following exposure to polyvinyl alcohol to result in the crosslinking.

[0009] In some embodiments, the ionizable lipid may include a glutathione-cleavable bond.

[0010] In some embodiments, the acid-containing lipid may include a glutathione-cleavable bond.

[0011] In some embodiments, the ionizable lipid may be of formula (I):(formula I), wherein R may be an amine, and R’ may be an alkyl.

[0012] In some embodiments, R’ of formula (I) is -(CH2)nCH3, wherein n may be an integer between 4 and 20.

[0013] In some embodiments, n of formula (I) may be an integer between 6 and 12.

[0014] In some embodiments, n of formula (I) may be an integer between 5 and 14.

[0015] In some embodiments, R’ of formula (I) may be selected from the group consisting of:-(CH2)5CH3;-(CH2)7CH3;-(CH2)9CH3; and-(CH2)nCH3.

[0016] In some embodiments, R of formula (I) may be selected from the group consisting of:

[0017] In some embodiments, the ionizable lipid may be:

[0018] In some embodiments, the acid-containing lipid may be of formula (II):wherein R may be an amine and R’ may be an alkyl.

[0019] In some embodiments, R’ of formula (II) is -(CH2)nCH3, wherein n is an integer between 4 and 20.

[0020] In some embodiments, n of formula (II) may be an integer between 6 and 12.

[0021] In some embodiments, n of formula (II) may be an integer between 5 and 14.

[0022] In some embodiments, R’ of formula (II) may be selected from the group consisting of: -(CH2)5CH3;-(CH2)7CH3;-(CH2)9CH3; and-(CH2)nCH3.

[0023] In some embodiments, R of formula (II) may be selected from the group consisting of:

[0024] In some embodiments, the acid-containing lipid may be:

[0025] In some embodiments, the helper lipid may be one or more of DSPC, DOPE, DMPC and DOTAP.

[0026] In some embodiments, the one or more cargo molecules may include mRNA.

[0027] In some embodiments, the one or more cargo molecules may include an anti-viral treatment agent.

[0028] Another broad aspect is a method for administrating the one or more cargo molecules to a subject, comprising introducing into the subject the lipid nanoparticle delivery system as described herein to the subject.

[0029] In some embodiments, the introducing may be performed via injection.

[0030] In some embodiments, the introducing may be performed via parenteral intramuscular injection.

[0031] In some embodiments, the introducing may be performed via inhalation wherein the lipid nanoparticle delivery system may be located in an aerosolized solution.

[0032] Another broad aspect is use of the lipid nanoparticle delivery system as described herein for introducing the cargo molecule into the subject.

[0033] In some embodiments, the introducing may be performed via injection.

[0034] In some embodiments, the injection may be parenteral intramuscular injection.

[0035] In some embodiments, the introducing may be performed via inhalation wherein the lipid nanoparticle delivery system may be located in an aerosolized solution.

[0036] Another broad aspect is a kit for forming a lipid nanoparticle delivery system, including a lipid nanoparticle encapsulating one or more cargo molecules, the lipid nanoparticle comprising: 40 mol % to 60 mol % of ionizable lipids; 5 mol % to 20 mol % of helper lipids; 30 mol % to 50 mol % of sterol; and 5 mol % to 20 mol % of phenylboronic acid-containing lipids; and a hydroxylcontaining polymer for crosslinking the phenylboronic acid-containing lipids.

[0037] Another broad aspect is a method of manufacturing a lipid nanoparticle delivery system. The method includes adding a hydroxyl-containing polymer to a lipid nanoparticle, containing one or more cargo molecules, the lipid nanoparticle comprising: ionizable lipids; helper lipids; sterol; and phenylboronic acid-containing lipids, to crosslink the phenylboronic acidcontaining lipids.

[0038] In some embodiments, the lipid nanoparticle may include 40 mol % to 60 mol % of ionizable lipids; 5 mol % to 20 mol % of helper lipids; 30 mol % to 50 mol % of sterol; and 5 mol % to 20 mol % of phenylboronic acid-containing lipids.

[0039] Another broad aspect is a lipid nanoparticle delivery system including one or more cargo molecules; a lipid nanoparticle containing the one or more cargo molecules, comprising: ionizable lipids; helper lipids; sterol; and phenylboronic acid-containing lipids, wherein crosslinking occurs across a surface of the lipid nanoparticle.

[0040] Another broad aspect is a lipid nanoparticle delivery system including one or more cargo molecules; a lipid nanoparticle containing the one or more cargo molecules, comprising: ionizable lipids; helper lipids; sterol; and acid-containing lipids, wherein crosslinking occurs across a surface of the lipid nanoparticle.Brief Description of the Drawings

[0041] The invention will be better understood by way of the following detailed description of embodiments of the invention with reference to the appended drawings, in which:

[0042] Figure l is a diagram illustrating the internalization and cargo release of an exemplary lipid nanoparticle delivery system in accordance with the present teachings;

[0043] Figure l is a diagram illustrating an exemplary method of preparing an exemplary lipid nanoparticle delivery system;

[0044] Figure 3 is a 'H-NMR spectrum of an exemplary ionizable lipid;

[0045] Figure 4 is a 'H-NMR spectrum of an exemplary PBA lipid;

[0046] Figure 5 is a graph of an ARS assay of un-cLNPs and cLNPs. Statistical analysis was performed by ordinary one-way ANOVA, *P < 0.05;

[0047] Figure 6A is a graph of dynamic light scattering (DLS) characterization;

[0048] Figure 6B are TEM images of un-cLNPs and cLNPs (Scale bar, 200 nm);

[0049] Figure 7 are graphs illustrating the (A) mRNA, (B) siRNA, and (C) 5’pppRNA transfection by un-cLNPs and cLNPs;

[0050] Figure 8A are graphs of the siGFP transfection efficiency of lyophilized un-cLNP and lyophilized cLNP after 4week-storage under different storage conditions. Statistical analysis was performed by ordinary one-way ANOVA, ***p < 0.001, ns means no significant difference;

[0051] Figure 8B are graphs of the siGFP transfection efficiency of lyophilized un-cLNP and lyophilized cLNP after 2 month-storage under different storage conditions. Statistical analysis was performed by ordinary one-way ANOVA, ****p < 0.0001, ns means no significant difference;

[0052] Figure 9A illustrates in vivo luciferase-mRNA transfection of cLNP via intravenous injection in mice (luc-mRNA dose, 0.5 mg / kg);

[0053] Figure 9B is a graph showing the quantification of luciferase expression in heart, liver, spleen, lung, and kidney;

[0054] Figure 10A is a flowchart diagram illustrating how mice were treated with 5’pppRNA- loaded LNPs (Day 1) after IAV infection (Day 0);

[0055] Figure 10B is a graph illustrating percent survival of mice that was monitored until Day 14. Statistical analysis was performed by log-rank (Mantel-Cox) test, *P < 0.05, **P < 0.01;

[0056] Figure 10C is a graph illustrating a percent weight loss curve; and

[0057] Figure 10D are graphs illustrating individual weight loss of mice in different groups were recorded.

[0058] Figure 11 is a schematic illustration of coated lipid nanoparticles (cLNPs) incorporating two lipids, including (1) the anchoring lipids that enable covalent PVA coating through phenylboronate ester bonds, thereby improving structural stability and providing a PEG- free stealth surface; (2) intracellularly degradable ionizable lipids that facilitate intracellular degradation of cLNPs and release of RNA by GSH. cLNPs displayed unique lung- selective RNA transfection, despite predominant accumulation in the liver. Abbreviations: RBC, red blood cell; PLT, platelet; PVA, polyvinyl alcohol;

[0059] Figure 12A is a schematic illustration of the preparation of unLNPs and cLNPs;

[0060] Figure 12B are graphs showing the fluorescence emission spectra of unLNPs and cLNPs analyzed by the ARS assay, with quantification of fluorescence intensity at 560 nm (n = 3);

[0061] Figure 12C includes c) TEM images of cLNPs (Scale bar, 500 nm), d) DLS characterization of unLNPs and cLNPs, e) Illustration of GSH-responsive LNP disassembly and RNA release, and f) TEM images of cLNPs after incubation with 2 mM GSH for 12h, showing structural changes (Scale bar, 500 nm);

[0062] Figure 12D includes graphs that show g) size changes of cLNPs and Modema-LNPs after incubation with varying GSH concentrations for 12h. In vitro GSH concentration-dependent RNA release from h) cLNPs and i) Moderna-LNPs under varying GSH concentrations. Transfection efficiency of GFP mRNA-loaded LNPs in j) B16F10 cells and k) 3T3 cells. 1) Antiviral efficacy of 5’pppRNA-loaded LNPs in Luc-expressing RSV-infected A549 cells, determined by luciferase activity, m) Fold changes in particle size of unLNPs and cLNPs after circulation in a microfluidic system over time (Flow rate: 4 ml / min; RBC concentration: 5 x 109 / ml; simulating blood flow). Data are presented as means ± SD (n = 3). Statistical analysis was performed by student- / test (g, m) or ordinary one-way ANOVA (b), ***p < 0.001, * < 0.05, ns means no significant difference;

[0063] Figure 13A includes a) a graph of serum radiant efficiency of Cy7 dye-loaded LNPs after intravenous injection (n = 6). Cy7 was used with reduced background signals in vivo, b) Bioluminescent imaging of luciferase mRNA transfection in C57BL / 6 mice and c) a graph showing quantification, following intravenous injection of Luc-mRNA loaded unLNPs or cLNPs(Luc-mRNA dose: 0.5 mg / kg). Organs were collected at 18h post-injection (n = 3). d) an image showing organ distribution and e) a graph showing quantification of Cy7 dye-loaded cLNPs, 18 h post-injection, measured by IVIS imaging. Cy7 was used with reduced background signals in vivo during IVIS imaging, (n = 4);

[0064] Figure 13B includes f) a drawing illustrating co-delivery of GFP-mRNA and Cy5- labeled RNA by cLNPs to assess colocalization of LNP accumulation and mRNA transfection in vivo (GFP-mRNA dose: 0.5 mg / kg; Cy5-labeled RNA, 0.5 mg / kg). GFP mRNA and Cy5 dye were selected to accommodate laser availability during flow cytometric analysis and fluorescence imaging, and g) representative fluorescence images of lung and liver tissue sections; Cy5 signal indicates LNP distribution, GFP signal indicates transfection (scale bar, 50 pm);

[0065] Figure 13C includes h) graphs showing cell-type-specific analysis of Cy5+and GFP+cells in lung tissues, including endothelial cells (CD45 CD31+), epithelial cells (CD45 CD3 F CD326+), and immune cells (CD45+) (n = 5). i) graphs showing cell-type-specific analysis of Cy5+and GFP+cells in the liver, including Kupffer cells (CD45+CD1 lb+F4 / 80+), Liver sinusoidal endothelial cells (LSECs) (CD45+CD31+CD146+), and hepatocytes (CD45 ASGR+) (n = 5). j) images of dose-dependent mRNA expression in vivo following intravenous injection of cLNPs loaded with Luc-mRNA at 0.1, 0.25, and 0.5 mg / kg. k) a graph showing organ-specific quantification of luciferase expression at each mRNA dose (n = 3). 1) a chart representing relative distribution of luciferase expression across major organs, highlighting lung-specific transfection with Luc-mRNA at 0.5 mg / kg. Data are presented as means ± SD. Statistical analysis was performed by student- / test (a, c), or ordinary two-way ANOVA (k), *** < 0.001, **P < 0.01, *P < 0.05, ns means no significant difference;

[0066] Figure 14A includes a) a graph showing IL-12 expression in the lungs and b) a graph showing IL-12 expression in the livers, 24h following systemic administration of IL-12 mRNA- loaded LNPs, including Moderna-LNPs, unLNPs, and cLNPs (n = 4), c) and a schematic of experimental design;

[0067] Figure 14B includes in vivo bioluminescence imaging of metastatic progression in mice bearing luciferase-expressing Bl 6F 10 melanoma cells during and after different treatments;

[0068] Figure 14C includes e) graphs illustrating quantification of pulmonary tumor burden based on bioluminescence intensity in different groups (n = 8). f) a graph showing survival of mice in different groups (n = 8). g) Representative lung images and H&E staining of lungs collected onDay 22 (scale bar, 500 m). Data are presented as means ± SD. Statistical analysis was performed using ordinary one-way ANOVA (a, b) or log-rank (Mantel-Cox) test (f), *** < 0.001, **P< 0.01, *P < 0.05;

[0069] Figure 15A includes a) a schematic of experimental timeline for lAV-infected mouse model. Mice were intranasally infected with IAV (Influenza A / PR / 8 / 34) on Day 0 and treated with 5’pppRNA-loaded cLNPs on Day 1, followed by survival monitoring, b) a graph showing survival of lAV-infected mice following treatment (n = 8). c) a graph showing average weight loss and d) graphs illustrating individual weight loss of lAV-infected mice (n = 8). The dashed line indicates the endpoint;

[0070] Figure 15B includes e) a schematic experimental timeline for RSV-infected mouse model. Mice were intranasally infected with RSV (Respiratory Syncytial Virus strain A2) on Day 0 and treated with 5’pppRNA-loaded cLNPs on Day 1, followed by survival monitoring; f) a graph showing survival of RSV-infected mice following treatment (n = 8); g) a graph showing average weight loss and h) graphs showing individual weight loss of RSV-infected mice (n = 8). The dashed line (representing a 20% weight loss) indicates the endpoint for euthanasia. Statistical analysis was performed by log-rank (Mantel-Cox) test (b, f), ***P < 0.001, **P < 0.01, * < 0.05, ns means no significant difference;

[0071] Figure 16 depicts a synthetic route of ionizable lipids and anchoring lipids;

[0072] Figure 17 is the1H-NMR spectrum of the ionizable lipid;

[0073] Figure 18 is the 'H-NMR spectrum of the anchoring PBA lipid;

[0074] Figure 19 is a TEM image of un-cLNPs (Scale bar, 500 nm);

[0075] Figure 20 are graphs showing internalization efficiency of FAM-RNA-loaded LNPs in a) B16F10 and b) 3T3 cells (n = 5);

[0076] Figure 21 is a graph showing transfection efficiency of GFP-targeting siRNA-loaded LNPs in GFP-expressing Hepal-6 cells (n = 3);

[0077] Figure 22 are graphs showing cytotoxicity of cLNPs in multiple cell lines (n = 6);

[0078] Figure 23 are images showing H&E staining of major organs collected from healthy mice receiving cLNP treatment and untreated mice. Scale bar, 100 pm;

[0079] Figure 24 are graphs showing hematological analysis of healthy mice 3 days post cLNP treatment and untreated mice (LNP dose: 20 mg / kg). Healthy mice with no treatment served as the control group. RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscularvolume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelets; WBC, white blood cell; ALT, alanine aminotransferase; AST, aspartate aminotransferase. Data are represented as mean± SD (ft = 3). Statistical analysis was performed by the Student- / test, ns indicated no significance;

[0080] Figure 25 includes a) bioluminescent imaging of Luc-mRNA transfection in C57BL / 6 mice and b) a graph showing a quantification, following intravenous injection of Luc-mRNA loaded Moderna-LNPs (Luc-mRNA dose: 0.5 mg / kg). Organs were collected at 18h post-injection. (n = 3);

[0081] Figure 26 is a graph showing fold change of luciferase expression in major organs compared to the liver. Data are presented as means ± SD (n = 3). Statistical significance was calculated via the Student’s / -test, ***p < 0.001;

[0082] Figure 27 includes a) bioluminescent imaging of luciferase mRNA expression in Balb / c mice and b) graphs showing quantification, following intravenous injection of Luc-mRNA loaded unLNPs or cLNPs (Luc-mRNA dose: 0.5 mg / kg). Organs were collected at 18h postinjection. (n = 3). c) Fold change of luciferase expression in major organs compared to the liver. Data are presented as means ± SD (n = 3);

[0083] Figure 28 includes representative flow cytometric analyses of a) Cy5+cells (gated on CD45+cells), b) GFP+cells (gated on CD45+cells), c) Cy5+cells (gated on CD45 CD31+cells), d) GFP+cells (gated on CD45 CD31+cells), e) Cy5+cells (gated on CD45 CD31 CD326+cells), and f) GFP+cells (gated on CD45 CD31 CD326+cells) in the lungs collected from the mice receiving Cy5-labeled RNA / GFP-mRNA co-loaded cLNPs and untreated mice;

[0084] Figure 29 includes representative flow cytometric analyses of a) Cy5+cells (gated on CD45 CD1 lb+F4 / 8+cells), b) GFP+cells (gated on CD45+CDl lb+F4 / 80+cells), c) Cy5+cells (gated on CD45+CD31+CD146+cells), d) GFP+cells (gated on CD45+CD31+CD146+cells), e) Cy5+cells (gated on CD45 ASGR+cells), and f) GFP+cells (gated on CD45 ASGR+cells) in the livers collected from the mice receiving Cy5-labeled RNA / GFP-mRNA co-loaded cLNPs and untreated mice;

[0085] Figure 30 is a graph showing relative distribution of luciferase expression across major organs from mice treated with Luc-mRNA loaded cLNPs, highlighting lung-specific transfection with Luc-mRNA at 0.5 mg / kg (n = 3). Data are presented as means ± SD. Statistical analysis was performed by ordinary two-way ANOVA, ***p < 0.001;

[0086] Figure 31 is a chart and a graph showing relative distribution of luciferase expression across major organs from mice treated with Luc-mRNA loaded cLNPs, highlighting lung-specific transfection with Luc-mRNA at 0.1 mg / kg (n = 3). Data are presented as means ± SD. Statistical analysis was performed by ordinary two-way ANOVA, ***P < 0.001; and

[0087] Figure 32 is a chart and a graph showing relative distribution of luciferase expression across major organs treated with Luc-mRNA loaded cLNPs, highlighting lung-specific transfection with Luc-mRNA at 0.25 mg / kg (n = 3). Data are presented as means ± SD. Statistical analysis was performed by ordinary two-way ANOVA, ***P < 0.001.Detailed Description

[0088] The present disclosure relates to lipid nanoparticle delivery systems that have an enhanced stability for storage and transportation, and with improved cargo release in the cell, thereby facilitating storage by reducing the need of maintaining significantly colder temperature for purposes of storage and transportation.

[0089] The lipid nanoparticle delivery systems in accordance with the present disclosure may be used for delivering gene encoding antigen, cytokine, or immune stimulus to a subject, for causing an immunological response. The lipid nanoparticle delivery systems in accordance with the present teachings may also be used for treatment of the subject, e.g. delivery of an antiviral to a subject.

[0090] The lipid nanoparticle delivery systems include an acid-containing lipid, such as a phenylboronic acid-containing lipid, that can be coated with hydroxyl groups containing polymer (e.g. polyvinyl alcohol) and polymer crosslinking will be formed on lipid nanoparticles for purposes of enhancing the stability of the lipid nanoparticle delivery system. Moreover, the ester bond (phenylboronate ester bond) that is formed via the crosslinking may be cleaved in the cell, in environments with a pH between 5.5 -6.5.

[0091] In some instances, glutathione-cleavable bonds may also be present in the ionizable lipids and acid-containing lipids of the lipid nanoparticle delivery systems for purposes of increasing degradation of the lipid nanoparticle delivery system in the cytosol, in order to promote the release of its contents.

[0092] Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is as “including, but not limited to.”

[0093] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0094] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0095] From the foregoing it will be appreciated that, although specific embodiments have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the teachings. Accordingly, the claims are not limited by the disclosed embodiments.

[0096] DEFINITIONS:

[0097] The term “alkyl”, as used herein, refers to a straight hydrocarbon chain radical consisting of carbon and hydrogen atoms, containing no unsaturation, having three to twenty carbons, and which is attached to the rest of the molecule by a single bond. Examples of alkyls include, but are not limited to: propyl, butyl, hexyl, heptyl, octyl, decyl, etc.

[0098] The term “alkene”, as used herein, refers to a straight or branched hydrocarbon chain radical consisting of carbon and hydrogen atoms, containing one or more double bonds, having three to twenty carbons, and which is attached to the rest of the molecule by a single bond. Examples of alkenes include, but are not limited to: butylene, hexylene, octylene, etc.

[0099] The term “alkyne”, as used herein, refers to a straight or branched hydrocarbon chain radical consisting of carbon and hydrogen atoms, containing one or more triple bonds, having three to twenty carbons, and which is attached to the rest of the molecule by a single bond. Examples of alkynes include, but are not limited to: propyne, hexyne, octyne, etc.

[0100] The term “compound”, as used herein, is meant to include all stereoisomers (geometric isomers, such as cis- and trans-i somers, enantiomers and diastereomers of a compound) and isotopes (atoms with the same atomic number, but different mass numbers resulting from a different number of neutrons in the nuclei) of the structure depicted.

[0101] The term “cycloalkyl” as used herein, refers to a five to ten membered monocyclic or polycyclic ring, saturated, partially saturated or unsaturated, where the ring atoms are selected from N, O, S and C. Examples of cycloalkyl include, but are not limited to, azetidinyl, tetrahydrofuran, dihydrofuran, dioxane, morpholine, etc. A cycloalkyl may be optionally substituted by one to five substituents independently selected from, for instance, the group consisting of hydroxy, thiol, cyano, nitro, a OH- alkyl, sulfonyl, halogen or amino.

[0102] The term “delivering”, as used herein, means providing an entity (a cargo molecule) to a destination. For instance, delivering a cargo molecule to a subject may involve administering a lipid nanoparticle including the cargo molecule contained therein to the subject.

[0103] The term “effective amount”, as used herein, with regard to an agent, is the amount sufficient to effect a beneficial or desired result. The “effective amount” depends on the context.

[0104] The term “alkoxy”, as used herein, refers to a radical of formula -ORa where Ra is an alkyl radical with three to twenty carbons as generally defined above. Examples of alkoxy include, but are not limited to: butoxy, pentoxy, hexoxy, etc.

[0105] The term “helper lipid”, as used herein, refers to a compound or molecule that includes a lipidic moiety (for insertion into a lipid layer, namely a lipid bilayer), and a polar moiety (for interaction with a physiological solution at the surface of the lipid layer). The helper lipid may be a phospholipid.

[0106] The term “nucleic acid sequence”, as used herein, as well as “nucleotide sequence”, “polynucleotide sequence”, may be used interchangeably herein and refer to a contiguous nucleic acid sequence. The sequence can be either single stranded or double stranded DNA or RNA, such as an mRNA.

[0107] The term “nucleic acid”, as used herein, refers to any compound that includes a polymer of nucleotides, and may be referred to as a polynucleotide. Exemplary nucleic acids include, but are not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), threose nucleic acids (TNA), glycol nucleic acids (GNA), peptide nucleic acids (PNA), locked nucleic acids (LNA), etc. The term includes triple-, double- and single- stranded deoxyribonucleic acids (DNA), and triple- , double- and single-stranded ribonucleic acid (RNA). The term also includes optionally a modification by, for instance, capping, alkylation, etc.

[0108] The term “OH- alkyl”, as used herein, refers to an alkyl radical, where one or one of the hydrogen atoms of the alkyl is replaced by “OH”. Exemplary OH- alkyl include, but are notlimited to, 2-hydroxy -butyl, 1 -hydroxy-hexyl, 1-hydroxy-oxyl, etc.

[0109] The term “amino- alkyl”, as used herein, refers to an alkyl radical, where one of the hydrogen atoms of the alkyl is replaced by an amino group. Exemplary amino- alkyls include, but are not limited to, 2-amino-butyl, 1 -amino-hexyl, 1-amino-oxyl, 1 -amino-decyl, etc.

[0110] The term “halogen”, as used herein, refers to bromo, chloro, fluoro or iodo.

[0111] The term “halogen- alkyl”, as used herein, refers to an alkyl, where one or more of the hydrogens are replaced by a halogen.

[0112] The term “lipid nanoparticle”, as used herein, refers to any lipid composition that can be used to deliver a cargo molecule, such as vaccine antigens, or a treatment agent, such as an antiviral. A lipid nanoparticle may be a liposome or a vesicle, where an aqueous volume is encapsulated by amphipathic lipid bilayers (e.g., single, unilamellar, multiple or multilamellar).

[0113] The term “pharmaceutical composition”, as used herein, refers to a compound in accordance with the present disclosure, or a pharmaceutically acceptable salt thereof, in a form suitable for, e.g., oral administration (e.g. through inhalation) or parenteral administration.

[0114] The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient”, as used herein, refers to a substance useful in the preparation of a pharmaceutical composition and includes, for instance, one or more of diluents, surfactants, preservatives, buffering agents, isotonic agents, salts, excipients, lubricants, wetting agents, flavoring, etc. Exemplary excipients include, but are not limited to, calcium carbonate, calcium phosphate, citric acid, hydroxyl methyl cellulose, gelatin, maltitol, providone, palmitate, sorbitol, starch, stearic acid, vitamins, etc.

[0115] The term “pharmaceutically acceptable salts”, as used herein, refers to derivatives of the disclosed compounds by converting an acid or base moiety to its salt form (e.g. by reacting a base group with a suitable organic acid).

[0116] The term “prevent”, “preventing” or “prevention”, as used herein, refers to the prophylactic treatment of a disease or disorder, or delaying the onset or progression of a disease or disorder.

[0117] In the present disclosure, by “subject”, it is meant a mammal, such as a human. The term “subject” should not bring on any limitations as to the sex or age.

[0118] The term “treat”, “treating” or “treatment”, as used herein, refers to alleviating or improving the outcome of the subject with regard to a given disease or disorder, which may bequantifiable by improving at least one physical parameter, ailment or biomarker of the subj ect with the disease or disorder.

[0119] EXEMPLARY LIPID NANOPARTICLE DELIVERY SYSTEMS:

[0120] As illustrated in Figure 1, advanced LNPs with enhanced stability and responsive cargo release for polynucleotide delivery are described. PBA lipids integrated in LNPs serve as the anchoring sites for PVA coating through a pH-responsive phenylboronate ester bond, forming multiple covalent crosslinking outlayer on LNP (cLNP), thereby ensuring the structural stability of LNPs. The PVA coating can be stripped as the phenylboronate ester bonds are pH-cleavable in endosomes (pH of 5.5-6.5). Furthermore, GSH-cleavable ionisiable lipids are also incorporated in the cLNP, which can facilitate the degradation of cLNPs in the GSH-rich cytosol, thus enabling rapid release of payloads to function.

[0121] The lipid nanoparticle delivery systems of the present disclosure include a lipid nanoparticle composed of ionizable lipids, acid-containing lipids, helper lipids and sterols, wherein crosslinking occurs on the lipid nanoparticle delivery system to improve stability (where acidcontaining lipids are crosslinked using hydroxyl-group-containing polymers). The lipid nanoparticle encloses a cargo molecule.

[0122] Ionizable lipids can affect the surface charge of the lipid in a pH dependent manner. Their head group has a weak basicity, rendering them positively charged in in an acidic pH environment, but charge neutral or close to charge neutral at physiological pH. Ionizable lipids are characterized by monovalent or multivalent charges on their headgroups. The ionizable lipids are capable of complexing with negatively charged bioactive molecules, promoting their cell internalization and endosome escape.

[0123] The ionizable lipids may include a glutathione-cleavable bond, which is cleavable in the cytosol of a cell.

[0124] Exemplary ionizable lipids are described herein may be of formula (I):(formula I) wherein R is an amine, and R’ is an alkyl.

[0125] R may correspond to one of the following substitute groups:

[0126] R’ may be a carbon chain with four to twelve carbons. R’ may be -(CH^sCHa; - (CH2)?CH3; -(CH2)9CH3; or -(CH2)nCH3. R’ may be a carbon chain with 8 carbons. R’ may be a carbon chain with 9 carbons. R’ may be a carbon chain with 10 carbons. R’ may be a carbon chain with 11 carbons. R’ may be a carbon chain with 12 carbons.

[0127] In some instances, the ionizable lipid may be:

[0128] In some instances, the ionizable lipid may compose from 0.1% mol to 99.90 mol % of the total lipid present in the lipid nanoparticle delivery system. In some instances, the ionizable lipid may compose from 80% mol to 99.9% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the ionizable lipid may compose from 2% mol to 70% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the ionizable lipid may compose from 5% mol to 60% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the ionizable lipid may compose from 30% mol to 40% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the ionizable lipid may compose from 20% mol to 40% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the ionizable lipid may compose from 40% mol to 60% molof the total lipid present in the lipid nanoparticle delivery system. In some instances, the ionizable lipid may compose from 50% mol to 60% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the ionizable lipid may compose from 40% mol to 50% mol of the total lipid present in the lipid nanoparticle delivery system.

[0129] The acid-containing lipid may be a phenylboronic acid-containing lipid, or a phenylboronic acid derivative-containing lipid with one to two substitutions on the phenyl ring joined to the Boron atom.

[0130] Exemplary phenylboronic acid derivative heads for acid-containing lipids include, but are not limited to, the following:

[0131] The acid-containing acid may contain a glutathione-cleavable bond, which is cleavable in the cytosol of a cell.

[0132] The acid-containing lipid may correspond to formula (II):(formula II), wherein R is an amine and R’ is an alkyl.

[0133] R may correspond to one of the following substitute groups:

[0134] R’ may be a carbon chain with four to twelve carbons. R’ may be -(Clfc^CHs; - (CH2)?CH3; -(CH2)9CH3; or -(CH2)nCH3. R’ may be a carbon chain with 8 carbons. R’ may be a carbon chain with 9 carbons. R’ may be a carbon chain with 10 carbons. R’ may be a carbon chain with 11 carbons. R’ may be a carbon chain with 12 carbons.

[0135] In some instances, the acid-containing lipid may be:

[0136] In some instances, the acid-containing lipid may compose from 0.1% mol to 99.90 mol % of the total lipid present in the lipid nanoparticle delivery system. In some instances, the acidcontaining lipid may compose from 80% mol to 99.9% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the acid-containing lipid may compose from 1% mol to 40% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the acid-containing lipid may compose from 5% mol to 40% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the acid-containing lipid may compose from 5% mol to 20% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the acid-containing lipid may compose from 5% mol to 10% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the acid-containinglipid may compose from 10% mol to 20% mol of the total lipid present in the lipid nanoparticle delivery system.

[0137] Crosslinking may be present across the surface of the lipid nanoparticle delivery system to increase stability of the lipid nanoparticle delivery system. For instance, the acid-containing lipids are crosslinked to form ester bonds. The crosslinking may be performed by exposing the lipid nanoparticle to a polymer containing a hydroxyl group, such as polyvinyl alcohol. Other exemplary hydroxyl-containing hydroxyl groups may be, but are not limited to:

[0138] The helper lipid is a neutral uncharged, zwitterionic or anionic lipid suitable for producing a stable complex. Exemplary helper lipids include, but are not limited to, phospholipids, fatty acids, sterols and any combination thereof. Exemplary helper lipids include, but are not limited to phosphotidylcholine (DSPC, DMPC, DLPC, DDPC, DPPC, POPC, DEPC, etc ), phosphatidylethanolamine (DOPE, DMPE, DPPE, etc.), l,2-dioleoyl-3 -trimethylammonium - propane (DOTAP), etc.

[0139] In some instances, the helper lipid may compose from 0.1% mol to 99.90 mol % of the total lipid present in the lipid nanoparticle delivery system. In some instances, the helper lipid may compose from 80% mol to 99.9% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the helper lipid may compose from 1% mol to 40% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the helper lipid may compose from 5% mol to 40% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the helper lipid may compose from 5% mol to 20% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the helper lipid may compose from 10% mol to 20% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, the helper lipid may compose from 5% mol to 10% mol of the total lipid present in the lipid nanoparticle delivery system.

[0140] The sterol may be one or more of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, stigmastanol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, etc. In one embodiment, the sterol is preferably cholesterol.

[0141] In some instances, sterol may compose from 0.1% mol to 99.90 mol % of the total lipid present in the lipid nanoparticle delivery system. In some instances, sterol may compose from 80% mol to 99.9% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, sterol may compose from 20% mol to 70% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, sterol may compose from 30% mol to 60% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, sterol may compose from 20% mol to 50% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, sterol may compose from 30% mol to 50% mol of the total lipid present in the lipid nanoparticle delivery system. In some instances, sterol may compose from 40% mol to 50% mol of the total lipid present in the lipid nanoparticle delivery system.

[0142] In one embodiment, a lipid nanoparticle includes 40 mol % to 60 mol % of ionizable lipids, 5 mol % to 20 mol % of helper lipids, 30 mol % to 50 mol % of cholesterol, and 5 mol % to 20 mol % of acid-containing lipids that are crosslinked.

[0143] The cargo molecule may be an antigen. The antigen may be a protein (such as a recombinant protein), polypeptide, or peptide, such as polypeptides suitable to induce an immune response against cancer cells, polypeptides suitable to induce an immune response against an infectious disease, polypeptides suitable for inducing an immune response against allergens, etc.

[0144] The cargo molecule may be a nucleic acid, such as RNA, for example a single stranded or double stranded RNA, such as dsRNA. The cargo molecule may be a microRNA (miRNA). MiRNAs are small noncoding RNA molecules that are capable of causing post-transcriptional silencing of specific genes in cells such as by inhibition of translation or through degradation of the targeted mRNA.

[0145] The cargo molecule may be a molecule for purposes of treating a subject, such as an antibiotic, an antiviral, an antifungal, a chemotherapeutic agents, etc.

[0146] In some embodiments, more than one type of cargo molecule may be included in a lipid nanoparticle delivery system (e.g. 2, 4, 5, 8, 10, etc. cargo molecule types in a lipid nanoparticle delivery system). In some instances, a single cargo molecule type may be included in a lipid nanoparticle delivery system.

[0147] A pharmaceutical composition comprising the lipid nanoparticle delivery systems as described herein may include other excipients such as viscosity modulators, preservatives, solubilizers, anti-flocculants, stabilizers, etc.

[0148] In some instances, the pH of the solution containing the lipid nanoparticle delivery systems may be maintained (e.g. between 6 pH and 8 pH) to improve stability.

[0149] METHOD OF ADMINISTERING A CARGO MOLECULE USING THE LIPID NANOPARTICLE DELIVERY SYSTEMS:

[0150] The present disclosure further describes a method of administering a cargo molecule using the lipid nanoparticle delivery systems, as described herein, where the cargo molecule contained in the lipid nanoparticle delivery systems is delivered for causing an immunological response in the subject, for treatment of the subject, etc.

[0151] A composition containing the lipid nanoparticle delivery systems may be administered via injection or via parenteral administration. Liquid dosage forms for parenteral or injectable administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, suspensions, syrups, etc. The composition may include an inert diluent such as water or other solvents, solubilizing agents, emulsifiers, such as ethyl alcohol, isopropyl alcohol, propylene glycol, glycerol, fatty acid esters, etc., and mixtures thereof. Compositions for oral administration may include wetting agents, sweeteners, flavouring, perfuming agents, etc.

[0152] A composition containing the lipid nanoparticle may be administered via inhalation into the respiratory tract. These compositions for inhalation are solid or liquid preparations including the lipid nanoparticle delivery systems, the cargo molecule contained in the lipid nanoparticle delivery systems, alone or together with one or more drugs. The size of the particles intended to be inhaled depends on their destination for purposes of deposition or distribution, and controlled by methods suitable for determining particle size (e.g. vapors, aerosols via nebulization, powders, as are known in the art).

[0153] In some instances, the inhaled particle size is between 1 pm and 10 pm, between 2 pm and 8 pm, between 3 pm and 5 pm, etc. The size may be suitable for alveolar deposition and lung retention.

[0154] The compositions as described herein may be formulated into a dosage form as described herein, such as a topical, intranasal, intratracheal, or injectable dosage form.

[0155] The delivery of the lipid nanoparticle delivery systems may be for causing an immunological response (a vaccine) or for treatment of a subject, with respect to a virus, bacteria, a fungus, a cancer. Exemplary viruses may be, for instance, SARS-CoV-2, HIV, Epstein-Barr virus, Herpes Simplex, Dengue virus, influenza (such as H5N1), RSV, etc. Exemplary bacteriamay be, for instance, Borrelia bacteria, streptococcus, Neisseria meningitidis, Haemophilus influenzae, Staphylococcus aureus, Klebsiella pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, etc. Exemplary cancers may be, for instance, cervical cancer, breast cancer, colon cancer, liver cancer, pancreatic cancer, prostate cancer, lymphoma, lung cancer, etc.

[0156] Treatments offered via the delivery of the lipid nanoparticle delivery systems as described herein may be for certain autoimmune diseases such as Addison disease, Graves disease, Hashimoto thyroiditis, multiple sclerosis, myasthenia gravis, interstitial lung disease, autoimmune connective tissue disease, etc.

[0157] Treatments offered via the delivery of the lipid nanoparticle delivery systems as described herein may be for certain chronic illnesses, such as post-acute sequelae of SARS-CoV- 2 infection (PASC), Myalgic encephalomyelitis / chronic fatigue syndrome, shingles, diabetes, Lyme disease, thyroid disorders, etc.

[0158] It will be understood that the nature of the disease or disorder that is targeted by the lipid nanoparticle delivery system depends on the nature of the cargo molecule contained therein.

[0159] The present disclosure also relates to a method of storage of compositions containing the lipid nanoparticle delivery systems as described herein. For instance, the lipid nanoparticle delivery systems of the present disclosure may be stored, in some instances, anywhere from - 200C to room temperature (e.g. -200C, 40C, 200C).

[0160] Compositions with the lipid nanoparticle delivery system may include one or more surfactants (an emulsifier or emulsifying agent reducing the surface tension of the different phases required for manufacturing the lipid nanoparticle delivery systems). The surfactants may be cationic, ionic or non-ionic. An exemplary surfactant may be cetrimide, docusate sodium, sodium lauryl sulfate, etc.

[0161] EXEMPLARY SYNTHESIS - IONIZABLE LIPID AND PBA LIPID:

[0162] Starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and so forth, that are used to synthesize the present compounds may either be commercially available or produced using known techniques of organic synthesis.

[0163] As used herein, the term “salt” or “salt” refers to an acid salt or base salt of a compound of the present disclosure. The term “pharmaceutically acceptable salts” refers to salts that retain the biological function and properties of the compounds of the present disclosure. For instance, the compounds of the present disclosure are capable of forming acid and / or base salts as a resultof ammo and / or carboxyl groups (or similar groups).

[0164] A pharmaceutically acceptable acid salt can be formed using an inorganic acid (e.g. hydrochloric acid, sulfuric acid, nitric acid, etc.) and / or an organic acid (e.g. acetic acid; oxalic acid; succinic acid; etc.).

[0165] A pharmaceutically acceptable base salt can be formed using an inorganic base (e.g. sodium hydroxide, potassium hydroxide, magnesium hydroxide, etc.) and / or an organic base (e.g. isopropylamine, cholinate, lysine, etc.)

[0166] Any formula provided herein is intended to also represent unlabeled forms or isotopically labelled forms.

[0167] Intermediates and final produces can be purified using known methods (e.g. such as chromatographic methods; distribution methods; re-crystallization, etc.)

[0168] An exemplary synthesis route for an exemplary glutathione-responsive ionizable lipid and an exemplary PBA lipid is provided in Scheme 1 below:PBA lipidScheme 1: exemplary synthesis of an exemplary ionizable lipid and of an exemplary PB lipid.

[0169] The chemical structures of which were confirmed by 1H NMR in Figure 3 and 4. To validate the PVA coating, the Alizarin Red S (ARS) assay was performed. ARS could enhance its fluorescence emission (lex: 490 nm; lem: 560 nm) upon binding with PBA derivatives. ARS was dissolved in PBS at a concentration of 25 pg / mL and then mixed with un-cLNP and cLNP (containing 25 pg / mL PBA lipid) at room temperature for 10 min, followed by fluorescenceanalysis. The ARS fluorescence emission intensity of un-cLNP group increased by 4.57 folds than that in the control group, indicating the PBA distributed on the LNP surface. Of note, the cLNP group showed a significantly decreased intensity compared with the un-cLNP group, indicating the effective PVA coating on PBA-derived LNP (as shown in Figure 5).

[0170] The following exemplary studies are provided to enable the skilled person to better understand the present disclosure. As they are but illustrative and representative examples, they should not limit the scope of the present disclosure. They are only added for illustrative and representative purposes. It will be understood that other exemplary studies may be used to further illustrate and represent the present disclosure without departing from the present teachings.

[0171] EXEMPLARY STUDY 1:

[0172] The LNP without PVA crosslinking (denoted as un-cLNP) and PVA-coated LNP (denoted as cLNP) are prepared as illustrated in Figure 2. Briefly, ionizable lipids, helper lipids, cholesterol, and PBA lipids are dissolved in ethanol at a specific ratio. Ribonucleic acid, such as messenger RNA (mRNA), 5 ’-triphosphate containing RNA (5’pppRNA), or small interfering RNA (siRNA), is diluted by sodium acetate buffer (pH 4, 10 mM). Then the ethanol phase and water phase are mixed at a volume ratio of 1 :3 and standed for 20 minutes, followed by pH adjustment to pH 7.4 to prepare the un-cLNP. At last, PVA containing deionized water is added to the LNP solution and stands for 1 hour to prepare the cLNP.

[0173] EXEMPLARY STUDY 2:

[0174] The size and morphology of un-cLNPs and cLNPs were studied by dynamic light scattering (DLS) and transmission electron microscopes (TEM) (Figures 6A and 6B). The size of un-cLNPs and cLNPs were both -150 nm. The surface charge of un-cLNPs and cLNPs were both neutral (< +8 mV). Moreover, un-cLNPs and cLNPs showed monodispersed spherical morphologies.

[0175] EXEMPLARY STUDY 3:

[0176] Figure 7 illustrates the gene transfection efficiency of un-cLNPs and cLNPs. The efficiency of cLNPs was evaluated for mRNA delivery on murine melanoma B16F10 cells. The mRNA-loaded LNPs were prepared at lipid: RNA = 40: 1 (wt ratio). As shown in (A) of Figure 7, B16F10 cells treated with mRNA-loaded un-cLNPs and cLNPs displayed a stronger GFP fluorescence than those in the Moderna-LNP group, indicating the high mRNA transfection efficiency.

[0177] cLNPs were also used to deliver siRNA to murine hepatoma Hepal-6 cells. GFP- siRNA-loaded LNPs were prepared at lipid: RNA = 10: 1 (wt ratio). As shown in (B) in Figure 7, the cells treated with siRNA-loaded un-cLNPs and cLNPs showed reduced GFP expression, suggesting excellent siRNA transfection efficiency.

[0178] The human lung carcinoma epithelial A549 cells was used for 5’pppRNA transfection. The 5’pppRNA has been reported to induce the antiviral immune response of host cells and then prevent the luciferase-expressed respiratory syncytial virus (RSV) infection7. The 5’pppRNA- loaded LNPs were prepared at lipid:RNA = 40:1 (wt ratio). As shown in (C) of Figure 7, the cells treated with 5’pppRNA-loaded un-cLNPs and cLNPs displayed a significantly reduced luciferase signal from RSV, indicating high 5’pppRNA transfection efficiency.

[0179] EXEMPLARY STUDY 4:

[0180] To study the storage stability of cLNPs, GFP-siRNA-loaded LNPs were tested after storage at different conditions. For lyophilized LNPs, test groups include: fresh lyophilized LNPs, and lyophilized LNPs stored under 4 °C, or room temperature (RT) for 2 months. As shown in Figures 8 A, after 4w-storage, lyophilized un-cLNPs stored at RT displayed a 17.8% decrease in siGFP transfection, while cLNPs maintained a stable transfection efficiency at all storage conditions. After 2m-storage, lyophilized un-cLNPs stored under 4°C and RT displayed a 9.3% and 23.2% decrease in siGFP transfection, respectively. cLNPs still displayed a consistent transfection efficiency under 4 °C (Figure 8B). Additionally, across all conditions, cLNPs consistently showed better siRNA transfection efficiency than lyophilized un-cLNPs after being stored under various conditions.

[0181] EXEMPLARY STUDY 5:

[0182] The cLNP -mediated gene delivery efficacy in vivo was investigated. Luciferase- mRNA was used as a model mRNA molecule. Luciferace-loaded cLNPs were intravenously injected into C57BL / 6J mice at a dose of 0.5 mg / kg of mRNA. Major organs, including heart, liver, spleen, lung, and kidney, were collected for ex vivo bioluminescence imaging (Figures 9A and 9B). Surprisingly, cLNPs displayed an excellent lung-specific mRNA delivery, with about 100-fold higher luciferase expression in the lungs compared to the livers. Since the liver is the main organ that many nanoparticles accumulate after intravenous injection, this unexpected hepatic escape and superior lung targeting capabilities of cLNPs can be beneficial for many lung- associated disorders, such as lung infections and lung cancer.

[0183] EXEMPLARY STUDY 6:

[0184] Given the lung-targeting nature of cLNPs, anti-viral 5’pppRNA was loaded in LNPs and their ability for treating lung infections was studied. An influenza A virus (lAV)-infected mouse model was established (Figure 10A). Briefly, C57B1 / 6 mice (8 weeks-old, from Charles River Laboratories) were infected intra-nasally with IAV (Female, 500 plaque forming units (PFU); Male, 2000 PFU, respectively) and ramdonly devided into four groups, including saline, Modema LNPs, un-cLNPs and cLNPs. Mice received one treatment, followed by monitoring of body weight and survival until Day 14. All animal experimentations were performed according to the guidelines of the Canadian Council on Animal Care and approved by the McGill University Animal Care Committee. As shown in Figures 10A-10D, all mice in the saline group succumbed to infection (0 / 7 mice survived) with continous weight loss. Modema LNPs and cLNPs slightly increased the survival (25%, 2 / 8 mice survived). Excitingly, cLNPs treatment significantly prolonged the survial to 75% (6 / 8 mice survived). Additionally, while some weight loss was observed in all treatment groups, cLNPs-treated mice showed a faster weight recovery and overall less weight loss (Figures 10B and 10C).

[0185] In summary, an advanced LNP with enhanced stability by coating the PBA-modified LNP with PVA polymer through phenylboronate ester bonds was reported. The successful coating was confirmed by classic ARS assay. The proposed LNP showed a stable gene transfection efficiency when used as the carrier for siRNA, mRNA and 5’pppRNA. The data suggests that due to the covalent crosslinking of the polymer coating, the advanced LNP (cLNP) showed a better gene delivery effect than its counterpart (un-cLNP) after being stored under different conditions. Notably, cLNPs achieved unexpectedly excellent lung-targeting gene delivery via intravenous injection, which contributed to significantly prolonged survival of lAV-infected mice with 5’pppRNA loading.

[0186] EXEMPLARY STUDY 7:

[0187] Lipid nanoparticles (LNPs) have emerged as a transformative platform for the clinical delivery of nucleic acids. Despite these encouraging clinical outcomes, several key limitations hinder the broader application and therapeutic efficiency of LNPs. One concern is its stability. LNPs are formed via self-assembly of multiple components through physical interactions, such as hydrophobic-hydrophobic interactions and electrostatic interactions, which are highly susceptible to disruption by physiological factors, including dilution upon systemic administration,interactions with serum proteins and circulating cells, and changes in shear stress, pH, and ionic strength in different tissue or phathological environments. This structural fragility also poses challenges during storage and lyophilization, where ice crystal formation, temperature fluctuations, and mechanical stress can destabilize or damage the LNP architecture. While polyethylene glycol-conjugated lipids (PEG lipids) are commonly incorporated into LNPs to improve colloidal stability and prolong circulation by forming a hydrophilic, stealth corona that reduces immune clearance, PEG-lipids are typically anchored through weak hydrophobic interactions, rendering them vulnerable to rapid desorption from LNPs in the bloodstream upon interaction with serum proteins. Additionally, the emergence of anti-PEG antibodies (APAs) following repeated dosing has been shown to reduce therapeutic efficacy and raise safety concerns, thereby limiting the clinical use of PEGylated formulations. Another limitation of current LNP platforms is associated with cytosolic RNA release - LNP-mediated RNA release into the cytosol occurs passively and uncontrolled. This uncontrolled release mechanism is often inefficient and inconsistent, which can compromise the functional delivery of RNA therapeutics. Therefore, there is a critical need for LNP strategies that simultaneously enhance structural stability and enable controlled, stimuli-responsive cytosolic release of RNA payloads.

[0188] In addition to these physicochemical and functional challenges, traditional LNP formulations exhibit a strong hepatic tropism, predominantly accumulating in the liver following systemic administration. This limits their applicability to non-hepatic targets, such as the lungs, and constrains the therapeutic potential of LNP -based RNA therapies in treating extrahepatic diseases. In particular, targeting the lungs is highly desirable for diseases such as lung cancer, pulmonary metastases, and respiratory viral infections (e.g., Respiratory syncytial virus (RSV), coronaviruses, and influenza). Recent reports have identified several LNP formulations that show preferential accumulation in the lungs following systemic administration; despite low accumulation in the liver and spleen, off-target distribution in these organs remains, which can lead to dose-dependent toxicity, especially under repeated or dose escalation treatment regimens. Consequently, developing LNPs with lung-specific RNA transfection represents a promising alternative strategy for treating pulmonary diseases.

[0189] Here, an LNP formulation with improved stability is reported, responsive cytosolic cargo release, and enhanced lung-selective RNA transfection (Fig. 11). To improve the stability of LNPs, a phenylboronic acid (PBA)-modified lipid is incorporated, which serves as an anchoringlipid for polyvinyl alcohol (PVA) polymer coating on the surface of LNPs via the formation of phenylboronate ester bonds. This covalent crosslinking confers structural stability of LNPs, forming a coated LNP (cLNP) with improved colloidal stability. PVA is a biocompatible, water- soluble polymer widely used in pharmaceutical formulations and offers a PEG-free alternative, thus mitigating APA-related concerns. To enable efficient cytosolic RNA release, integrated glutathione (GSH)-cleavable ionizable lipids was further integrated into cLNP. These ionizable lipids are stable in the extracellular environment (low GSH concentration, -2-20 pM), but are degradable in the cytosol (high GSH, 2-10 mM), enabling responsive disassembly and rapid release of RNA in cells. The in vivo studies in mice demonstrated that the cLNPs exhibited strong lung-selective RNA transfection following intravenous administration, resulting in superior therapeutic effects in several preclinical models of pulmonary diseases, including pulmonary metastasis and viral infections.

[0190] EXEMPLARY STUDY 8:

[0191] cLNPs were prepared and characterized. The GSH-cleavable ionizable lipids and the anchoring lipid were synthesized through Michael addition, and their chemical structures were confirmed by 1H NMR (Figures 16-18). cLNPs were then formulated by the self-assembly of the ionizable lipid, anchoring lipid, cholesterol, and helper lipid at a molar ratio of 42 / 8 / 40 / 10, followed by PVA polymer coating through boronate ester formation between PBA and PVA (Figure 12A). To verify successful PVA coating, Alizarin Red S (ARS) was employed, a dye known to bind specifically to PBA moieties, generating fluorescence. When ARS was incubated with uncoated LNPs (unLNPs), fluorescence intensity increased by 4.57-fold, confirming the presence of PBA groups on the surface of unLNPs. In contrast, the fluorescence signal in the cLNP group was reduced, suggesting that surface PBA groups mostly reacted with PVA (Fig. 12B). The PVA coating process did not significantly affect the physical characteristics of LNPs. Transmission electron microscopy (TEM) revealed that both unLNPs and cLNPs maintained a uniform spherical morphology (Figure 12C, Figure 19). The hydrodynamic diameters of unLNPs and cLNPs remained consistent at approximately 150 nm with relatively neutral surface potential (Figure 12C, d).

[0192] Both the ionizable lipids and anchoring lipids contained disulfide bonds, enabling GSH-responsive cleavage. This design facilitates cytosolic LNP disassembly and payload release in response to elevated intracellular GSH concentrations (Fig. 12C, e). To study this, the stabilityand release profiles of cLNPs under a range of physiologically relevant GSH concentrations were systematically evaluated, spanning extracellular and circulatory levels (<0.02 mM) to intracellular cytosolic levels (2-10 mM). The size of the cLNPs remained unchanged under low GSH conditions (0.2 mM or lower). In contrast, at 2 mM and 10 mM GSH, the particle size increased (Fig. 12D, g), indicating structural changes of cLNPs. TEM imaging at 2 mM GSH further confirmed the disassembly, revealing irregular and non-uniform particle morphologies (Fig. 12C, f). Concurrently, in the RNA release study, approximately 75% of encapsulated RNA was released from cLNPs within 24 hours under high GSH conditions (2 and 10 mM), whereas a low level of RNA release was observed in conditions with low GSH levels (0 and 0.02 mM) (Fig. 12D, h). These results confirm the GSH-responsiveness of cLNPs and their capacity to responsively release RNA cargo in the cytosolic environment. In contrast, Modema-LNPs did not exhibit any GSH- dependent changes in particle size or payload release under identical conditions (Fig. 12D, i).

[0193] The RNA delivery efficiency by cLNPs was first evaluated in vitro. cLNPs loaded with GFP-encoding reporter mRNA were tested across B16F10 melanoma cells and 3T3 fibroblasts, all of which exhibited efficient GFP expression (Figs. 12D, j and k) after cell internalization (Figure 20). In addition, siRNA-loaded cLNPs demonstrated potent gene silencing efficiency in vitro (Figure 21). The application of cLNPs was extended to deliver 5 ’-triphosphate RNA (5'pppRNA), an antiviral RNA that possesses broad-spectrum antiviral efficacy demonstrated in previous studies. In the RSV-infected A549 lung epithelial cells, 5ppp’RNA-loaded cLNPs effectively suppressed the replication of RSV (Fig. 12D, 1). Across all in vitro settings, unLNPs showed comparable delivery efficacy to cLNPs, as nanoparticle stability was not compromised under static in vitro conditions. Taken together, cLNPs exhibited robust transfection efficiency across various nucleic acid types, including mRNA, siRNA, and 5’pppRNA, highlighting their versatility for a wide range of RNA-based therapeutic applications.

[0194] The in vitro and in vivo toxicity of cLNP was evaluated to assess their biocompatibility. The cLNPs showed no observable cytotoxicity across multiple cell lines (Figure 22). Furthermore, systemic administration of cLNPs in mice did not induce any signs of toxicity in major organs, as confirmed by histological analysis (Figure 23). Hematological parameters also remained within normal ranges, with no significant differences observed between cLNP -treated mice and untreated healthy mice (Figure 24). These results collectively demonstrate the favorable safety profile and biocompatibility of cLNPs for potential therapeutic applications.

[0195] EXEMPLARY STUDY 9:

[0196] Stability of cLNPs in circulation was then observed. To evaluate whether the introduction of PVA coating enhances the structural stability of LNPs, the behavior of unLNPs and cLNPs ex vivo was assessed using a microfluidic system that contains red blood cells (RBC; 5 * 109cell / ml) and a flow rate (4 ml / min) to mimic blood circulation. The particle size of unLNPs began to increase after 10 min of flow, and exhibited a 3 -fold increase by 30 minutes, indicative of structural destabilization. In contrast, cLNPs maintained a consistent particle size, suggesting improved mechanical stability under shear stress (Fig. 12D, m). While this represents a simplified ex vivo model, the in vivo stability of cLNPs was further assessed by measuring their blood circulation time. This was achieved by quantifying the fluorescence intensity of dye-loaded LNPs in the blood following systemic administration in mice. Encouragingly, cLNPs exhibited significantly prolonged circulation compared to unLNPs (Fig. 13A, a), further indicating their improved stability in vivo.

[0197] EXEMPLARY STUDY 10:

[0198] Lung-secific transfection of cLNPs was then performed. The in vivo transfection efficiency of cLNPs was evaluated in mice using luciferase-encoding reporter mRNA. As expected and consistent with previous studies, Moderna-LNPs induced mRNA transfection primarily in the liver in C57BL / 6 mice (Figure 25). In contrast, both unLNPs and cLNPs displayed lung-specific mRNA transfection, (Fig. 13A, b). Notably, cLNPs exhibited a higher transfection efficiency compared to unLNPs, probably attributed to the enhanced structural stability conferred by the polymer coating (Fig. 13A, c). Specifically, luciferase expression in the lungs was approximately 100-fold higher than in the liver for cLNPs, whereas this ratio was 38-fold for unLNPs (Figure 26). The lung-specific transfection pattern was also observed in Balb / c mice, suggesting that the property was not strain-dependent (Figure 27).

[0199] To investigate the potential mechanism underlying this lung-specific transfection, dye- labeled RNA was loaded into cLNPs for intravenous administration. Interestingly, similar to Moderna LNPs, cLNPs also accumulated mainly in the liver, with 9.1 -fold higher signal compared to the lungs (Figs. 13A, c, d, e). This suggests that hepatic accumulation by cLNPs did not result in effective RNA transfection. To further dissect this discrepancy between nanoparticle biodistribution and RNA expression, the GFP-mRNA and Cy5 dye-labeled RNA were co-loaded into the cLNPs, and cell-level analyses were performed (Fig. 13B, f). Consistent with IVIS results,while higher Cy5 fluorescence was observed in the liver sections, GFP expression was minimal. Conversely, despite decreased Cy5 intensity in the lungs, robust GFP expression was observed (Fig. 13B, g). Flow cytometric analyses revealed that GFP expression was detected in all Cy5- positive cell populations in the lungs (Figs. 13C, h, Fig. 28), indicating efficient translation following cellular uptake. In contrast, while approximately 45% of Kupffer cells internalized cLNPs, less than 2% exhibited GFP expression (Fig. 13C, i, Fig. 29, a and b). Additionally, 10% of the LSECs and 5% of the hepatocytes internalized cLNPs, but failed to express GFP (Fig. 29, c-f). These results indicate that despite predominant accumulation in the liver, cLNPs enabled lung- selective transfection.

[0200] To further validate this unique lung specific RNA transfection property, the mRNA dosages were increased (e.g., 0.1 to 0.5 mg / kg) and a dose-dependent increase in mRNA expression was found exclusively in the lungs, with no significant corresponding increase in other organs (Fig 13C, j, k). Quantitative analysis revealed that 94.9% of mRNA expression was located in the lungs (Fig. 13C, 1, Figs. 30-32). These results suggest that cLNPs can achieve potent and selective RNA transfection in the lungs, potentially minimizing off-target therapeutic effects and dose-dependent toxicities for RNA-based therapies targeting pulmonary diseases.

[0201] EXEMPLARY STUDY 11 :

[0202] cLNPs were then used for delivering IL-12 mRNA for lung metastasis inhibition. Lung metastasis is a leading cause of cancer-related deaths globally due to its high morbidity and poor prognosis. IL-12 immunostimulatory cytokine therapy holds significant promise due to its potent anti-tumor immune activation. However, their clinical application has been hampered by systemic toxicity resulting from off-target effects. Leveraging the lung- selectivity of cLNPs, it was investigated whether IL- 12 mRNA-loaded cLNPs could improve the treatment outcomes for lung metastases. The lung-specific IL-12 expression with intravenously administered IL-12 mRNA- loaded cLNPs was first evaluated. Both unLNPs and cLNPs induced IL- 12 expression primarily in the lungs, with cLNPs achieving significantly higher levels than unLNPs (Fig. 14A, a). In contrast, Modema-LNPs led to IL-12 expression mainly in the liver, consistent with their known liver tropism (Fig. 14 A, b).

[0203] To assess therapeutic efficacy, mice were injected intravenously with luciferaseexpressing Bl 6F 10 melanoma cells to establish a model of lung metastasis, where luciferase was used to track the growth of lung metastases. Mice were then treated with saline, IL- 12 mRNA-loaded Modema-LNPs, IL-12 mRNA-loaded unLNPs, or IL-12 mRNA-loaded cLNPs (Fig. 14A, c). Tumor burden was monitored by bioluminescence signals from tumor cells using IVIS (Fig. 14B) Saline-treated mice showed rapid tumor progression, with 100% mortality by Day 28. While all LNP-treated groups exhibited reduced tumor growth and extended survival, the cLNP group achieved the most pronounced therapeutic benefit (Fig. 14C, e, f). Ex vivo lung images and H&E staining confirmed the reduced metastatic nodules in cLNP-treated mice (Fig. 14C, g).

[0204] EXEMPLARY STUDY 12:

[0205] cLNPs were then used to deliver 5’pppRNA for treating respiratory viral infections. Respiratory viral infections, such as influenza, RSV, and coronaviruses (e.g., SARS-CoV-2), can spread rapidly. These infections often lead to severe complications, such as pneumonia and bronchitis, and can exacerbate pre-existing respiratory conditions, including asthma and chronic obstructive pulmonary disease. While mRNA vaccines have effectively mitigated the burden of SARS-CoV-2 and hold promise for emerging pathogens, key limitations remain, including waning immunity and the emergence of viral variants. Furthermore, not all individuals are eligible for vaccination due to underlying medical conditions, immunodeficiencies, or allergic reactions, leaving gaps in population-wide protection. These challenges underscore the urgent need for broad-spectrum antiviral therapeutics capable of rapidly countering respiratory viral infections and enhancing pandemic preparedness. To this end, 5 ’-triphosphate RNA (5’pppRNA) was selected, a potent anti-viral RNA known to activate type I and III interferons (IFNs) signaling and suppress viral replication as demonstrated in our previous studies, as a therapeutic RNA payload to evaluate the antiviral potential of cLNPs.

[0206] Aan influenza A virus (lAV)-infected mouse model was established and administered with a single dose of various LNP formulations one day after viral infection (Fig. 15A, a). All mice in the saline group succumbed to infection by Day 11 with a weight loss exceeding 20%. Treatment with unLNPs and Modema LNPs modestly improved survival to 25% (2 / 8 mice survived). Remarkably, a single administration of cLNPs achieved a 75% survival rate (6 / 8 mice survived), significantly outperforming other groups (Figs. 15A, b, c). While all treatment groups experienced some degree of initial weight loss, a common symptom of viral infection, cLNP treatment demonstrated faster recovery and reduced weight loss overall (Fig. 15A, d).

[0207] To further evaluate the generalizability of this approach, 5’ppp-RNA-loaded cLNPs in a respiratory syncytial virus (RSV)-infected mouse model was tested. Strikingly, cLNPs treatmentconferred complete protection, with 100% survival (8 / 8 mice), underscoring the broad antiviral potential of this platform for treating lung-respiratory viral infections (Figs. 15B, e-h). Collectively, these results support the unique cLNP -mediated lung- selective transfection for effective treatment of respiratory viral infections.

[0208] EXEMPLARY STUDY 12:

[0209] In this study, a coated LNP (cLNP) formulation was developed that showed improved structural stability, stimuli-responsive cytosolic RNA release, and lung-specific RNA transfection. To improve stability, an anchoring lipid was incorporated to enable covalent PVA polymer coating on the surface of LNPs. Additionally, both the ionizable and anchoring lipids contained GSH- cleavable disulfide bonds, facilitating controlled intracellular RNA release. In vitro studies demonstrated that cLNPs efficiently delivered a variety of RNA payloads, including mRNA, siRNA, and antiviral 5’pppRNA, across different cell types. Notably, cLNPs mediated potent and selective RNA transfection in the lungs, despite hepatic accumulation. While the mechanism underlying this lung- selective RNA transfection remains to be fully elucidated, the therapeutic potential of cLNPs was demonstrated in two representative pulmonary disease models, including a murine lung metastasis model where IL-12 mRNA-loaded cLNPs significantly reduced lung metastasis, and in models of respiratory viral infections (i.e., I AV and RSV) where 5’pppRNA- loaded cLNPs conferred superior anti-viral efficacy. Overall, these findings highlight the potential of cLNPs as a versatile delivery platform for RNA therapeutics in pulmonary diseases.

[0210] EXEMPLARY SYNTHESIS OF LIPIDS:

[0211] Pyridine disulfide (2.27 mmol) was dissolved in 4 ml of methanol with acetic acid (0.59 mmol). Dodecanethiol (1.13 mmol) was dissolved in 1.25 ml of dichloromethane and dropped into the former solution under a nitrogen atmosphere. The mixture was stirred at room temperature (RT) for 5 h and purified by column chromatography to obtain Compound 1. Compound 1 (1.70 mmol) was then dissolved in 10 ml of methanol / dichloromethane (1 : 1, v / v) with acetic acid (0.70 mmol). Mercaptoethanol (3.23 mmol) was dropped into the Compound 1 solution and stirred at RT for 3 h. Column chromatography was performed to obtain the purified Compound 2. Compound 2 (4.582 mmol) was dissolved in 50 ml of dichloromethane with triethylamine (6.88 mmol) and cooled down to 0 °C. Acryloyl chloride (4.92 mmol) was then dropped into the solution and stirred overnight. The mixture was washed with 0.1 M HC1 solution and dried with anhydrous Na2SC>4. The organic phase was evaporated, and the residue was purified by columnchromatography to obtain Compound 3. 3 -(Dimethylamino)- 1 -propylamine (0.41 mmol) and Compound 3 (0.90 mmol) were mixed and reacted at 65 °C for 48 h to obtain the ionizable lipids. 4-(Bromomethyl)phenylboronic acid (0.047 mmol) and ionizable lipid (0.039 mmol) were dissolved in 500 pl of acetonitrile and the reaction was conducted at 65 °C for 24 h to obtain the PBA-lipids.

[0212] EXEMPLARY LNP PREPARATION AND CHARACTERIZATION

[0213] LNPs were formulated using the standard ethanol dilution method. The ionizable lipids, DSPE, cholesterol, and PBA-lipids were dissolved in ethanol at a fixed molar ratio of 42 / 10 / 40 / 8. RNA molecules, including mRNA, 5’pppRNA, or siRNA, were dissolved in the sodium acetate buffer (pH 4, 10 mM). The two phases were rapidly mixed at a volume ratio of organic solvent to aqueous volume of 1 :3. The weight ratio of LNP to RNA was fixed at 40: 1. The mixture was stabilized at RT for 15 min before dialysis against PBS to obtain unLNPs. PVA solution was mixed with the unLNPs for 1 h to obtain the cLNP (PVA / PBA-lipid = 1 / 1.2, weight ratio). Standard Moderna LNPs were formulated with SMI 02, DSPE, cholesterol, and DMG- PEG2000 at a molar ratio of 50 / 10 / 38.5 / 1.5 using the same method. Dynamic light scattering (DLS, Malvern) was applied to measure the size and zeta potential of LNPs. Transmission Electron Microscopy (TEM, Thermo Scientific Talos F200X G2) was applied to observe the morphology of the LNPs. For lyophilization, the cryoprotectant (10% trehalose) was added to cLNP solution, and the mixture was frozen at -80 °C and lyophilized with a freeze dryer (Labconco™).

[0214] EXEMPLARY PREPARATION OF ALIZARIN RED S (ARS) BINDING ASSAY

[0215] ARS exhibits enhanced fluorescence emission (lex: 490 nm; lem: 560 nm) binding to PBA derivatives. ARS was dissolved in PBS at a concentration of 25 pg / ml and incubated with unLNP and cLNP (containing 25 pg / ml PBA-lipid) at RT for 10 min, followed by fluorescence measurement.

[0216] EXEMPLARY PREPARATION FOR MEASURING LNP STABILITY IN MICROFLUIDIC SYSTEM:

[0217] Mouse blood was collected and centrifuged (800 g, 5 min) at 4 °C to get the red blood cells. The cell pellet was resuspended in LNPs dispersed in PBS, resulting in an RBC concentration of 5 x 109cells / ml. The suspension was circulated through the microfluidic flowing system at a constant flow rate of 4 ml / min. LNPs were subjected to circulation for varying durations (0, 10,30, and 40 min). The size of the LNPs was measured using DLS.

[0218] EXEMPLARY GSH-RESPONSIVE LNP DISASSEMBLY AND PAYLOAD RELEASE:

[0219] The LNPs were incubated in PBS containing 0, 0.02, 2 mM, or 10 mM GSH for 48 h. Particle size and morphology of the LNPs before and after GSH incubation were analyzed using DLS and TEM, respectively.

[0220] Cy5-labelled RNA (Cy5-RNA) was encapsulated into LNPs using the same method as described above. LNP solutions (200 pl) were placed into a dialysis bag (MWCO, 300kD), which was then immersed in 60 ml of PBS containing 0, 0.02, 2 mM, or 10 mM GSH. The dialysis setup was maintained on a shaker at 100 rpm and 37 °C. At predetermined time points (0, 0.5, 1, 2, 3, 6, 12, and 24 h), 200 pl samples were withdrawn (ft = 3), and an equal volume of fresh PBS was added to maintain sink conditions. The amount of RNA released was quantified by measuring the fluorescence intensity of Cy5 (lex: 640 nm; lem: 665 nm).

[0221] EXEMPLARY ASSESSMENT OF IN VITRO CYTOTOXICITY:

[0222] Cells were seeded in 96-well plates and cultured for 24 h, followed by treatment with blank LNPs at various concentrations (0, 0.5, 1, 2.5, 5, 10, 15, and 20 pg / ml) for an additional 24 h. Cell viability was then assessed using the MTT assay (Invitrogen).

[0223] EXEMPLARY CELL INTERNALIZATION OF LNPS:

[0224] Cells were seeded in 96-well plates and cultured for 24 h, followed by treatment with F AM-labeled RNA-loaded LNPs (RNA dose: 0.1 pg / ml) for 2, 4, and 6 h. The internalization efficiency was then assessed using LSRII (IMED) flow cytometer (BD Biosciences).

[0225] EXEMPLARY IN VITRO TRANSFECTION:

[0226] In vitro mRNA transfection was conducted using 3T3 cells. Cells were seeded in a 96- well plate and cultured for 24 h. GFP-expressing mRNA-loaded LNPs (mRNA dose: 0.5 pg / ml) were then added and incubated with the cells for an additional 24 h. GFP-positive cells were analysed using an LSRII (IMED) flow cytometer (BD Biosciences).

[0227] In vitro 5’pppRNA transfection was conducted using A549 lung cells. Cells were seeded in a 24-well plate and cultured for 24 h. Following infection with luciferase-expressing respiratory syncytial virus (RSV), 5’pppRNA-loaded LNPs (5’pppRNA dose: 0.1 pg / ml) were added and incubated with the infected cells for an additional 24 h. Luciferase intensity was then measured using a Luciferase Reporter Assay, according to the manufacturer’s instructions(Promega).

[0228] In vitro siRNA transfection was conducted using GFP-expressing Hepal-6 cells. Cells were seeded in a 96-well plate and cultured for 24 h. GFP -targeting siRNA-loaded LNPs (siRNA dose: 0.6 pg / ml) were added and incubated with the cells for an additional 48 h. GFP-positive cells were then analysed using an LSRII (IMED) flow cytometer (BD Biosciences).

[0229] EXEMPLARY CHARACTERIZATION OF IN VIVO BIODISTRIBUTION AND CLEARANCE OF LNPS:

[0230] Mice were intravenously injected with Cy7-labeled nucleic acid-loaded LNPs (Cy7- nucleic acid, 0.5 mg / kg). For biodistribution analysis, mice were euthanized 18 h post-injection, and major organs were harvested for fluorescent imaging using an IVIS imaging system (Perkin Elmer). For clearance analysis, 30 pl of blood was collected from the orbital vein at designated time points (15 min, 45 min, 1.5 h, 3 h, 6 h, and 12 h). Fluorescence signals in the blood samples were captured and analyzed using an IVIS imaging system (Perkin Elmer).

[0231] EXEMPLARY IN VIVO MRNA TRANSFECTION:

[0232] Mice were intravenously injected with Luc-mRNA-loaded LNPs at doses of 0.1, 0.25, or 0.5 mg / kg. At 18 h post-injection, D-luciferin (15 mg / ml in PBS) was intraperitoneally injected. After 10 min, the mice were imaged using an IVIS imaging system (Perkin Elmer), followed by euthanasia and collection of major organs for ex vivo imaging.

[0233] EXEMPLARY TRANSFECTION AND INTERNALIZATION OF LNPS IN SPECIFIC CELLS

[0234] Mice were intravenously injected with LNPs co-loaded with Cy5-labeled RNA and GFP-mRNA (Cy5-labeled RNA, 0.5 mg / kg; GFP-mRNA, 0.5 mg / kg). At 18 h post-injection, mice were euthanized, and tissues (lungs and livers) were harvested and processed into single-cell suspensions. For lung samples, the cell suspensions were stained with Pacific Blue anti -mouse CD31 (PECAM-1) antibody (Biolegend, Cat. no. 160216(BLG)), PE anti-mouse CD326 (EpCAM) antibody (Biolegend, Cat. no. 119004), and APC / Cy7 anti-mouse CD45 antibody (Biolegend, Cat. no. 103116). For liver samples, the cell suspensions were stained with Pacific Blue anti-mouse CD31 (PECAM-1) antibody (Biolegend, Cat. no. 160216(BLG)), ASGR1 Polyclonal Antibody (Proteintech, Cat. no. 11739-1-AP), Goat anti-Rabbit IgG (H+L) Cross- Adsorbed Secondary Antibody, DyLight 594 (Invitrogen, Cat. no. 35561), PE anti-mouse CD146 antibody (Biolegend, Cat. no. 134703), Pacific Blue anti -mouse F4 / 80 antibody (Biolegend, Cat.no. 123123) and APC / Cy7 anti-mouse CD45 antibody (Biolegend, Cat. no. 103116). Frozen sections of the lungs and livers were also prepared and imaged using a Zeiss LSM 800 Confocal Microscope.

[0235] EXEMPLARY LUNG METASTASIS INHIBITION WITH IL-12-MRNA- LOADED LNPS:

[0236] To establish a melanoma lung metastasis mouse model, B 16F10-luc cells were injected intravenously into C57BL / 6 mice. After 4 days, mice were randomly divided into four groups and intravenously injected every three days with saline, Modema LNPs, unLNPs, or cLNPs (IL- 12 mRNA, 0.4 mg / kg), for a total of four doses. Bioluminescence imaging was performed on Day 7, 12, 17, and 22 using an IVIS imaging system. Body weight and survival were monitored. Animals showing signs of compromised health were euthanized. Lungs from each group were collected for imaging and fixed for hematoxylin and eosin (H&E) staning. IL- 12 expression in major organs and blood was measured by ELISA assay in healthy mice.

[0237] EXEMPLARY CHARACTERIZATION OF ANTI-VIRUS EFFICIENCY OF 5’ PPPRNA-LOADED LNPS:

[0238] C57BL / 6 mice were used to determine 5’pppRNA-loaded LNPs mediated antiviral response. Mice under 4% isoflurane anesthesia were infected intranasally with Influenza A / PR / 8 / 34 (both female and male, lethal dose of IAV (Influenza A / Puerto Rico / 8 / 34)) and randomly divided into four groups, including saline, Moderna LNPs, unLNPs, and cLNPs. At 24 h post-infection, all groups received a single intravenous dose of 5'pppRNA-loaded LNPs (5'pppRNA, 1 mg / kg). Body weight and survival were monitored daily. The respiratory syncytial virus (RSV)-infected mouse model (both female and male, lethal dose of RSV (Respiratory Syncytial Virus strain A2)) and treatment schedule followed the same procedure as described above. Animals were euthanized when they showed 20% weight loss or signs of compromised health were observed.

[0239] EXEMPLARY SAFETY EVALUATION:

[0240] Healthy mice received a single dose of cLNPs. Blood samples were collected for hemological analysis, including Red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet, white blood cell (WBC), and for serum biochemistry analysis, including creatinine, urea, alanine transaminase (ALT), and aspartate transaminase(AST). Major organs were also harvested for histological examination via H&E staining. Untreated healthy mice served as the control group.

[0241] Representative, non-limiting examples of the present invention were described above in detail with reference to the attached drawing. This detailed description is merely intended to teach a person of skill in the art further details for practicing preferred aspects of the present teachings and is not intended to limit the scope of the invention. Furthermore, each of the additional features and teachings disclosed above and below may be utilized separately or in conjunction with other features and teachings to provide useful lipid nanoparticle delivery systems and methods of using same within the subject.

[0242] Moreover, combinations of features and steps disclosed in the above detailed description, as well as in the experimental examples, may not be necessary to practice the invention in the broadest sense, and are instead taught merely to particularly describe representative examples of the invention. Furthermore, various features of the above-described representative examples, as well as the various independent and dependent claims below, may be combined in ways that are not specifically and explicitly enumerated in order to provide additional useful embodiments of the present teachings.

[0243] All features disclosed in the description and / or the claims are intended to be disclosed separately and independently from each other for the purpose of original written disclosure, as well as for the purpose of restricting the claimed subject matter, independent of the compositions of the features in the embodiments and / or the claims. In addition, all value ranges or indications of groups of entities are intended to disclose every possible intermediate value or intermediate entity for the purpose of original written disclosure, as well as for the purpose of restricting the claimed subject matter.

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Claims

What is claimed is:

1. A lipid nanoparticle delivery system comprising: one or more cargo molecules; and a lipid nanoparticle encapsulating the one or more cargo molecules, the lipid nanoparticle comprising:40 mol % to 60 mol % of ionizable lipids;5 mol % to 20 mol % of helper lipids;30 mol % to 50 mol % of sterol; and5 mol % to 20 mol % of acid-containing lipids, wherein crosslinking is present on a surface of the lipid nanoparticle.

2. The lipid nanoparticle delivery system according to claim 1, wherein the acid-containing lipids are phenylboronic acid-containing lipids.

3. The lipid nanoparticle delivery system according to claim 2, wherein the phenylboronic acid-containing lipids are crosslinked following exposure to polyvinyl alcohol to result in the crosslinking.

4. The lipid nanoparticle delivery system according to any one of claims 1 to 3, wherein the ionizable lipid comprises a glutathione-cleavable bond.

5. The lipid nanoparticle delivery system according to any one of claims 1 to 4, wherein the acid-containing lipid comprises a glutathione-cleavable bond.

6. The lipid nanoparticle delivery system according to any one of claims 1 to 5, wherein the ionizable lipid is of formula (I):(formula I), wherein R is an amine, and R’ is an alkyl.

7. The lipid nanoparticle delivery system according to claim 6, wherein R’ of formula (I) is - (CH2)nCH3, wherein n is an integer between 5 and 14.

8. The lipid nanoparticle delivery system according to claim 7, wherein n of formula (I) is an integer between 6 and 12.

9. The lipid nanoparticle delivery system according to claim 8, wherein R’ of formula (I) is selected from the group consisting of:-(CH2)5CH3;-(CH2)7CH3;-(CH2)9CH3; and -(CH2)nCH3.

10. The lipid nanoparticle delivery system according to any one of claims 6 to 9, where R of formula (I) is selected from the group consisting of:

11. The lipid nanoparticle delivery system according to any one of claims 1 to 10, where the ionizable lipid is:

12. The lipid nanoparticle delivery system according to any one of claims 1 to 11, wherein the acid-containing lipid is of formula (II):wherein R is an amine and R’ is an alkyl.

13. The lipid nanoparticle delivery system according to claim 12, wherein R’ of formula (II) is - (CH2)nCH3, wherein n is an integer between 5 and 14.

14. The lipid nanoparticle delivery system according to claim 13, wherein n of formula (II) is an integer between 6 and 12.

15. The lipid nanoparticle delivery system according to claim 14, wherein R’ of formula (II) is selected from the group consisting of:-(CH2)5CH3;-(CH2)7CH3;-(CH2)9CH3; and-(CH2)nCH3.

16. The lipid nanoparticle delivery system according to any one of claims 12 to 15, where R of formula (II) is selected from the group consisting of:

17. The lipid nanoparticle delivery system according to any one of claims 1 to 16, wherein the acid-containing lipid is:

18. The lipid nanoparticle delivery system according to any one of claims 1 to 17, wherein the helper lipid is one or more of DSPC, DOPE, DMPC and DOTAP.

19. The lipid nanoparticle delivery system according to any one of claims 1 to 18, wherein the one or more cargo molecules includes mRNA.

20. A method of manufacturing a lipid nanoparticle delivery system, comprising: adding a hydroxyl-containing polymer to a lipid nanoparticle, containing one or more cargo molecules, the lipid nanoparticle comprising: ionizable lipids; helper lipids; sterol; and phenylboronic acid-containing lipids, to crosslink the phenylboronic acid-containing lipids.

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