Ambient stabilization of mrna-hybrid lipid nanocapsules in sugar glass

Abstract

An exemplary embodiment of the present disclosure provides nucleic acid delivery system comprising a nucleic acid therapeutic agent, a hybrid lipid nanocapsule comprising a fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate capable of condensing with the nucleic acid therapeutic agent, and a sugar glass. Also disclosed herein is a hybrid lipid nanocapsule for delivery of a nucleic acid into a cell and a method for delivering a nucleic acid into a cell.

Description

[0001] AMBIENT STABILIZATION OF mRNA-HYBRID LIPID NANOCAPSULES IN SUGAR GLASS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] [1] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 662,499, filed on June 21,2024, which is incorporated herein by reference in its entirety as if fully set forth below.

[0004] FIELD OF THE DISCLOSURE

[0005] [2] The various embodiments of the present disclosure relate generally to ambient stabilization of nucleic acid-hybrid lipid nanocapsules in sugar glass.

[0006] BACKGROUND

[0007] [3] The success of the mRNA vaccine in combating the COVID-19 pandemic has opened a new era of mRNA-based therapeutics. Furthermore, it is emerging as a novel modality in cancer therapy, showing considerable promise in reshaping the approach to address malignancies. Harnessing the body's innate molecular machinery to synthesize therapeutic proteins, mRNA therapies offer unparalleled precision and versatility. However, translating this promise into therapeutic reality hinges on achieving an optimal balance between the stability and efficient delivery of mRNA cargo. Various nanoparticles have been investigated for mRNA delivery; however, challenges remain with mRNA formulation's instability and achieving efficient delivery. Moreover, the immune response to the delivery vehicle is one of the major concerns. Therefore, there is a critical need for a robust and promising nanocarrier to stabilize and deliver the mRNA cargo while circumventing the immune response.

[0008] [4] Among many mRNA nanocarriers, lipid nanoparticles (LNPs) consist of phospholipids and ionizable polymer, at least one pegylated phospholipid has shown promising results in the delivery of mRNA therapeutics. Despite the promising output of lipid nanoparticles, they are unable to stabilize the mRNA at ambient temperature, which requires ultra-cold storage. Moreover, LNPs consist of PEGylated phospholipid which may induce the immune response. For decades, polyethylene glycol has stood as the cornerstone for imbuing nanoparticles with stabilizing and stealth properties, enabling their prolonged circulation and enhancing their ability to bypass immune recognition. Yet, the prevailing PEGylation strategy is not without its caveats, raising questions about potential immunogenicity, and concerns about its influence on the efficacy of delivered mRNA therapeutics. These challenges beckon researchers to seek alternative, innovative delivery solutions.

[0009] BRIEF SUMMARY

[0010] [5] An exemplary embodiment of the present disclosure provides a nucleic acid delivery system comprising: a nucleic acid therapeutic agent, a hybrid lipid nanocapsule comprising a fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate capable of condensing with the nucleic acid therapeutic agent, and a sugar glass.

[0011] [6] In any of the embodiments disclosed herein, the fatty acid-polyamine conjugate can comprise a naturally occurring polyamine.

[0012] [7] In any of the embodiments disclosed herein, the naturally occurring polyamine can be selected from the group consisting of putrescine, spermidine, spermine, agmatine, and cadaverine.

[0013] [8] In any of the embodiments disclosed herein, the fatty acid-polypeptide conjugate can comprise a polypeptide selected from the group consisting of poly-L-histidine, poly-L-lysine, and poly-L-arginine.

[0014] [9] In any of the embodiments disclosed herein, the fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate can comprise a fatty acid with a carbon chain length between 8 and 18.

[0015]

[0010] In any of the embodiments disclosed herein, the fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate can comprise a fatty add selected from the group consisting of palmitic acid, oleic acid, stearic acid, butyric acid, and mixtures thereof.

[0016]

[0011] In any of the embodiments disclosed herein, the nucleic acid therapeutic agent can comprise mRNA.

[0017]

[0012] In any of the embodiments disclosed herein, the mRNA can be a vaccine related to infectious disease, metabolic disease, or cancer.

[0018]

[0013] In any of the embodiments disclosed herein, the sugar glass can comprise a glass-former with a glass transition temperature greater than the temperature at which the nucleic acid delivery system is stored.

[0019]

[0014] In any of the embodiments disclosed herein, the glass-former can comprise at least one of an anti-plasticizer, a sugar, a sugar alcohol, a polymer, and / or a combination thereof.

[0020]

[0015] In any of the embodiments disclosed herein, the sugar glass can further comprise a buffering agent.

[0016] In any of the embodiments disclosed herein, the polymer can be selected from the group consisting of polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), a natural polysaccharide, and mixtures thereof.

[0021]

[0017] In any of the embodiments disclosed herein, the natural polysaccharide can be selected from the group consisting of dextran, hydroxyethyl starch, pullulan, chitosan, alginate, hyaluronic acid, and inulin.

[0022]

[0018] In any of the embodiments disclosed herein, the sugar can be selected from the group consisting of trehalose, sucrose, stachyose, lactose, fructose, glucose, and mixtures thereof.

[0023]

[0019] In any of the embodiments disclosed herein, the sugar alcohol can be selected from the group consisting of xylitol, sorbitol, erythritol, mannitol, glycerol, and mixtures thereof.

[0024]

[0020] In any of the embodiments disclosed herein, the system can inhibit mRNA degradation at temperatures about or below 50°C.

[0025]

[0021] In any of the embodiments disclosed herein, the system can inhibit mRNA degradation at temperatures between about 15°C and about 50°C, about 20°C and about 45°C, or about 25 °C and about 40°C.

[0026]

[0022] Another exemplary embodiment of the present disclosure provides a hybrid lipid nanocapsule for delivery of a nucleic acid into a cell, the hybrid lipid nanocapsule comprising: at least one fatty acid ester, at least one lipophilic surfactant, at least one non-ionic hydrophilic surfactant, and at least one fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate, wherein the hybrid lipid nanocapsule is modified with an amino acid, a peptide, a polypeptide, a protein, an antibody, a nucleic acid, or polyethylene glycol.

[0027]

[0023] In any of the embodiments disclosed herein, the at least one fatty acid-polyamine conjugate can comprise a naturally occurring polyamine.

[0028]

[0024] In any of the embodiments disclosed herein, the naturally occurring polyamine can be selected from the group consisting of putrescine, spermidine, spermine, agmatine, and cadaverine.

[0029]

[0025] In any of the embodiments disclosed herein, the at least one fatty acid-polyamine conjugate can comprise a polymer.

[0030]

[0026] In any of the embodiments disclosed herein, the polymer can comprise polyethyleneimine.

[0031]

[0027] In any of the embodiments disclosed herein, the at least one fatty acid-polypeptide conjugate can comprise a polypeptide selected from the group consisting of polyhistidine, poly- L-lysine, and poly-L-arginine.

[0028] In any of the embodiments disclosed herein, wherein the at least one fatty acid- polyamine conjugate or fatty acid-polypeptide conjugate can comprise a fatty acid with a carbon chain length between 8 and 18.

[0032]

[0029] In any of the embodiments disclosed herein, the at least one fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate can comprise a fatty acid selected from the group consisting of palmitic acid, oleic acid, stearic acid, butyric acid, and mixtures thereof.

[0033]

[0030] In any of the embodiments disclosed herein, the at least one fatty acid-polyamine conjugate can comprise one or more of: a palmitic acid-derived spermine, an oleic acid-derived spermine, or a mixture thereof.

[0034]

[0031] In any of the embodiments disclosed herein, the at least one fatty acid-polyamine conjugate can comprise one or more of: a palmitic acid-derived polyethyleneimine, an oleic acid-derived polyethyleneimine, or a mixture thereof.

[0035]

[0032] In any of the embodiments disclosed herein, the at least one fatty acid-polypeptide conjugate can comprise one or more of: a palmitic acid-derived poly L-lysine, an oleic acid- derived poly L-lysine, an oleic acid-derived poly-L-histidine, a palmitic add-derived poly-L- histidine, an oleic acid-derived poly-L-arginine, a palmitic acid-derived poly-L-arginine, or a mixture thereof.

[0036]

[0033] In any of the embodiments disclosed herein, the at least one fatty acid ester can comprise a medium-chain triglyceride and / or a long chain triglyceride.

[0037]

[0034] In any of the embodiments disclosed herein, the at least one fatty acid ester can comprise one or more of: caprylic acid triglyceride, capric acid triglyceride, decanoic acid triglyceride, oleic acid triglyceride, stearic acid triglyceride, a polyethylene ester of a fattyacid, or a mixture thereof.

[0038]

[0035] In any of the embodiments disclosed herein, the at least one lipophilic surfactant can comprise a phospholipid.

[0039]

[0036] In any of the embodiments disclosed herein, the at least one lipophilic surfactant can comprise l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0040]

[0037] In any of the embodiments disclosed herein, the at least one non-ionic hydrophilic surfactant can comprise one or more of: polyethylene glycol (15)-hydroxystearate, polyethylene glycol (PEG) derived decanoic acid, PEG derived lauric acid, PEG derived stearic acid, PEG derived palmitic acid, or a mixture of thereof.

[0041]

[0038] In any of the embodiments disclosed herein, the at least one non-ionic hydrophilic surfactant can comprise polyethylene glycol (15)-hydroxystearate.

[0039] In any of the embodiments disclosed herein, the hybrid lipid nanocapsule can be modified with a zwitterionic amino acid and / or polyethylene glycol (PEG).

[0042]

[0040] In any of the embodiments disclosed herein, the zwitterionic amino acid can be L- histidine, tricine, arginine, lysine, or a mixture thereof.

[0043]

[0041] Another exemplary embodiment of the present disclosure provides nucleic acid delivery system comprising: a nucleic acid therapeutic agent, the hybrid lipid nanocapsule as disclosed herein, and a sugar glass.

[0044]

[0042] In any of the embodiments disclosed herein, the nucleic acid therapeutic agent can comprise mRNA.

[0045]

[0043] In any of the embodiments disclosed herein, the mRNA can be a vaccine related to infectious disease, metabolic disease, or cancer.

[0046]

[0044] In any of the embodiments disclosed herein, the sugar glass can comprise a glass-former with a glass transition temperature greater than the temperature at which the nucleic acid deliver system is stored.

[0047]

[0045] In any of the embodiments disclosed herein, the glass-former can comprise at least one of an anti-plasticizer, a sugar, a sugar alcohol, a polymer, and / or a combination thereof.

[0048]

[0046] In any of the embodiments disclosed herein, the sugar glass can further comprise a buffering agent.

[0049]

[0047] In any of the embodiments disclosed herein, the polymer can be selected from the group consisting of polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), a natural polysaccharide, and mixtures thereof.

[0050]

[0048] In any of the embodiments disclosed herein, the natural polysaccharide can be selected from the group consisting of dextran, hydroxyethyl starch, pullulan, chitosan, alginate, hyaluronic acid, and inulin.

[0051]

[0049] In any of the embodiments disclosed herein, the sugar can be selected from the group consisting of trehalose, sucrose, stachyose, lactose, fructose, glucose, and mixtures thereof.

[0052]

[0050] In any of the embodiments disclosed herein, the sugar alcohol can be selected from the group consisting of xylitol, sorbitol, erythritol, mannitol, and glycerol.

[0053]

[0051] In any of the embodiments disclosed herein, the system can inhibit mRNA degradation at temperatures about or below 50°C.

[0054]

[0052] In any of the embodiments disclosed herein, the system can inhibit mRNA degradation at temperatures between about 15°C and about 50°C, about 20°C and about 45°C, or about 25 °C and about 40°C.

[0053] Another exemplary embodiment of the present disclosure provides method for delivering a nucleic acid into a cell, comprising: administering the nucleic acid delivery system of the present disclosure to a subject, such that the system enters a cell of the subject.

[0055]

[0054] These and other aspects of the present disclosure are described in the Detailed Description below and the accompanying drawings. Other aspects and features of embodiments will become apparent to those of ordinary skill in the art upon reviewing the following description of specific, exemplary embodiments in concert with the drawings. While features of the present disclosure may be discussed relative to certain embodiments and figures, all embodiments of the present disclosure can include one or more of the features discussed herein. Further, while one or more embodiments may be discussed as having certain advantageous features, one or more of such features may also be used with the various embodiments discussed herein. In similar fashion, while exemplary embodiments may be discussed below as device, system, or method embodiments, it is to be understood that such exemplary embodiments can be implemented in various devices, systems, and methods of the present disclosure.

[0056] BRIEF DESCRIPTION OF THE DRAWINGS

[0057]

[0055] The following detailed description of specific embodiments of the disclosure will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, specific embodiments are shown in the drawings. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0058]

[0056] FIG. 1 provides particle size and zeta potential of the hybrid lipid nanocapsules (hLNCs), in accordance with an exemplary embodiment of the present disclosure. hLNCs-1 and hLNCs-3 represent the hLNCs that contain mRNA condensing agents such as oleic-PEI and oleic-spermine, respectively. The hLNCs-2 and hLNCs-4 represent the hLNCs that contain mRNA condensing agent as oleic-PEI, but it was further modified with L-histidine and Polyethylene glycol (PEG), respectively. Particle size of all different hLNCs is close to 40+3 nm. However, zeta potential depends on the fatty acid-polymer conjugate or their surface modification, which ranges from negative 3 to positive 25 mV.

[0059]

[0057] FIGs. 2A-2B provide stability of OVA-mRNA loaded on hLNCs stabilized in sugar glass and stored at 25 and 40°C, in accordance with an exemplary embodiment of the present disclosure. The mRNA stability was determined by transfecting the cells in vitro and separating the translated ovalbumin, followed by blotting. The integrated density value (OVA / GAPDH) was plotted from the blot. The W and M represent week and month, respectively. FIG. 2A provides the stability of OVA-mRNA loaded in hybrid lipid nanocapsules, while FIG. 2B provides the stability of OVA-mRNA loaded in surface-modified hybrid lipid nanocapsules with L-histidine.

[0060]

[0058] FIG. 3 provides radiance efficiency of luciferase after intramuscular (I.M.) or subcutaneous (S.C.) injection of firefly luciferase mRNA loaded on hybrid lipid nanocapsules or surface-modified hybrid lipid nanocapsules with L-histidine, in accordance with an exemplary embodiment of the present disclosure. The dose of mRNA / mouse is one pg.

[0061]

[0059] FIG. 4 provides antibody titer of ovalbumin-specific antibody in the blood serum of mice after intramuscular injection of OVA-mRNA-hybrid lipid nanocapsules- sugar glass stored at different conditions, in accordance with an exemplary embodiment of the present disclosure. The dose of OVA-mRNA is five pg / mouse, and two doses of OVA-mRNA were given to each mouse on days 0 and 14 before blood collection.

[0062] DETAILED DESCRIPTION

[0063]

[0060] Although preferred exemplary embodiments of the disclosure are explained in detail, it is to be understood that other exemplary embodiments are contemplated. Accordingly, it is not intended that the disclosure is limited in its scope to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other exemplary embodiments and of being practiced or carried out in various ways. Also, in describing the preferred exemplary embodiments, specific terminology will be resorted to for the sake of clarity.

[0064]

[0061] To facilitate an understanding of the principles and features of the present disclosure, various illustrative embodiments are explained below. The components, steps, and materials described hereinafter as making up various elements of the embodiments disclosed herein are intended to be illustrative and not restrictive. Many suitable components, steps, and materials that would perform the same or similar functions as the components, steps, and materials described herein are intended to be embraced within the scope of the disclosure. Such other components, steps, and materials not described herein can include, but are not limited to, similar components or steps that are developed after development of the embodiments disclosed herein.

[0062] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0065]

[0063] Also, in describing the preferred exemplary embodiments, terminology will be resorted to for the sake of clarity. It is intended that each term contemplates its broadest meaning as understood by those skilled in the art and includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.

[0066]

[0064] Ranges can be expressed herein as from “about” or “approximately” one particular value and / or to “about” or “approximately” another particular value. When such a range is expressed, another exemplary embodiment includes from the one particular value and / or to the other particular value.

[0067]

[0065] Similarly, as used herein, “substantially free” of something, or “substantially pure”, and like characterizations, can include both being “at least substantially free” of something, or “at least substantially pure”, and being “completely free” of something, or “completely pure”.

[0068]

[0066] By “comprising” or “containing” or “including” is meant that at least the named compound, member, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, material, particles, method steps have the same function as what is named.

[0069]

[0067] Mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a device or system does not preclude the presence of additional components or intervening components between those components expressly identified.

[0070]

[0068] The materials described as making up the various members of the invention are intended to be illustrative and not restrictive. Many suitable materials that would perform the same or a similar function as the materials described herein are intended to be embraced within the scope of the invention. Such other materials not described herein can include, but are not limited to, for example, materials that are developed after the time of the development of the invention.

[0071]

[0069] Reference will now be made in detail to exemplary embodiments of the disclosed technology, examples of which are illustrated in the accompanying drawings and disclosed herein. Wherever convenient, the same references numbers will be used throughout the drawings to refer to the same or like parts.

[0070] The present disclosure provides ambient stabilization of mRNA vaccine composed of mRNA, sequestered in hybrid lipid nanocapsules and embedded in a sugar-based glass. Inhouse developed hybrid lipid nanocapsules (hLNCs) can be used as a delivery vehicle of mRNA and a sugar glass can be utilized to stabilize the whole system at room temperature. More specifically, the disclosure relates to the development of an mRNA-hLNCs-sugar glass system to store an mRNA vaccine at room temperature or higher temperature (>25°C) for an extended period while preserving the potency of the mRNA vaccine. Herein, hLNCs can be composed of a mixture of nonionic surfactant, medium chain triglyceride, phospholipids and either a poly (ethyleneimine) polymer-oleic acid conjugate or poly-L-lysine-oleic acid conjugate or Spermine-oleic acid conjugate or poly-L-histidine-oleic acid conjugate or poly- L-arginine-oleic acid conjugate. The particles may be surface-modified with antibodies, aptamers, or other affinity tags for targeting, polyethylene glycol, or amino acids such as L- histidine or L-lysine or L-arginine or poly peptide such as Poly L-Lysine, Poly L-histidine, Poly L-arginine. The hLNCs can be assembled using a solvent-free, emulsion phase inversion method. The sugar glass can be composed of sugars and a polymer, such as , polyvinyl alcohol (PVA), or polyvinylpyrrolidone (PVP), or inulin, and a buffering agent such as L-histidine or tricine.

[0072]

[0071] The hybrid lipid nanocapsules (hLNCs) were engineered for the co-delivery of hydrophobic small-molecule such as anticancer drugs and messenger RNA (mRNA), for synergistic therapeutic application. The hLNCs comprise a core-shell structure, wherein the lipophilic core consists of medium-chain triglycerides that encapsulate the hydrophobic drug / molecules, and the shell is formed by a blend of amphiphilic surfactants such as Kolliphor HS15 and phospholipids, including dioleoylphosphatidyl-ethanolamine (DOPE). For the condensation of mRNA, a conjugate of fatty acid-polyamine or fatty acid-polypeptide, such as poly-L-histidine, polyethylenimine (PEI), or oleic acid-spermine conjugates, was incorporated to electrostatically complex and condense the negatively charged mRNA. The dualcompartment design enables the simultaneous encapsulation and protection of chemically distinct payloads — hydrophobic drugs within the lipid core and nucleic acids on the particle surface — facilitating synchronized intracellular delivery. The hLNCs exhibit colloidal stability, high loading capacity, and enhanced endosomal release, making them suitable for intravenous or intratumoral administration for targeted combination therapy

[0073]

[0072] Some embodiments of the present disclosure provide an ambient stable mRNA-hybrid lipid nanocapsules-sugar glass system which has three individual components: the mRNA as a therapeutic agent, hybrid lipid nanocapsules (hLNCs) as an mRNA delivery vehicle, and a sugar glass to stabilize the whole system. The hLNCs of the present disclosure are robust and can withstand different temperature and medium with a wide pH range. The present disclosure provides the flexibility of medium and temperature for the loading of the desired amount of mRNA in hLNCs, and further stabilization of mRNA-HLNCs in sugar glass at >25°C for the long term. Moreover, the hLNCs of the present disclosure can feature flexible surface chemistry modifying it for long-circulating and tissue-specific targeting and delivery of mRNA vaccine.

[0074]

[0073] Current technology for mRNA vaccines requires costly storage of vaccines at -80°C. Further, with current technologies, loss of -80°C results in spoilage of the vaccine product. The present disclosure has the advantage that it enables ambient temperature (25 °C - 40°C) storage of mRNA vaccines without loss of activity, reducing storage and shipping cost, facilitating distribution and testing of candidate vaccines, and reducing the chance of product spoilage.

[0075]

[0074] An exemplary embodiment of the present disclosure provides a nucleic acid delivery system comprising a nucleic acid therapeutic agent, a hybrid lipid nanocapsule comprising a fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate capable of condensing with the nucleic acid therapeutic agent, and a sugar glass.

[0076]

[0075] In some embodiments, the fatty acid-polyamine conjugate can comprise a naturally occurring polyamine. Exemplary naturally occurring polyamines include, without limitation, putrescine, spermidine, spermine, agmatine, and cadaverine.

[0077]

[0076] In some embodiments, the fatty acid-polypeptide conjugate can comprise a polypeptide. Exemplary polypeptides include, without limitation, poly-L-histidine, poly-L-lysine, and poly- L-arginine.

[0078]

[0077] In some embodiments, the fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate comprises a fatty acid with a carbon chain length between 8 and 18. In some embodiments, the fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate comprises a fatty acid selected from the group consisting of palmitic acid, oleic acid, stearic acid, butyric acid, and mixtures thereof.

[0079]

[0078] In some embodiments, the nucleic acid therapeutic agent can comprise mRNA. In any of the embodiments disclosed herein, the ambient stable system can be applied to any nucleic acid vaccine. In some embodiments, the mRNA can be a vaccine related to infectious disease, metabolic disease, or cancer.

[0079] In any of the embodiments disclosed herein, a sugar glass containing mRNA-hLNCs can stabilize a mRNA-hLNCs vaccine.

[0080]

[0080] In any of the embodiments disclosed herein, the sugar glass can comprise a glass-former with a glass transition temperature greater than the temperature at which the nucleic acid delivery system is stored. For example, in some embodiments, the nucleic acid delivery system can be stored at a temperature of 4°C to 40°C, and thus, the glass former can have a glass transition temperature of 45 °C to 100°C.

[0081]

[0081] In any of the embodiments disclosed herein, the glass former can comprise at least one of an anti-plasticizer, a sugar, a sugar alcohol, a polymer, and / or a combination thereof. Exemplary sugars include, without limitation, trehalose, sucrose, stachyose, or mixtures thereof. Exemplary sugar alcohols can include, without limitation, xylitol, sorbitol, erythritol, mannitol, glycerol, or mixtures thereof. Exemplary polymers include, without limitation, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), naturally occurring polysaccharides, such as dextran, hydroxyethyl starch, pullulan, chitosan, alginate, hyaluronic acid, inulin, or mixtures thereof. Exemplary anti-plasticizer include, without limitation, sorbitol, glycerol, ethelyne glycol, propylene carbonate or mixtures thereof.

[0082]

[0082] In any of the embodiments disclosed herein, the sugar glass can further comprise a buffering agent. For example, in some embodiments, the sugar glass can comprise a sugar, a polymer, and a buffering agent. Exemplary buffering agents include, without limitation, tricine and / or histidine.

[0083]

[0083] In any of the embodiments disclosed herein, the system can inhibit mRNA degradation at temperatures about or below 50°C. For example, in some embodiments, the system can inhibit mRNA degradation at temperatures between about 15°C and about 30°C, about 15°C and about 40°C, about 15°C and about 50°C, about 20°C and about 30°C, about 20°C and about 40°C, about 20°C and about 45°C, about 20°C and about 50°C, about 25°C and about 30°C, about 25°C and about 35°C, about 25°C and about 40°C, about 25°C and about 45°C, or about 25 °C and about 50°C.

[0084]

[0084] Another embodiment of the present disclosure provides a hybrid lipid nanocapsule for delivery of a nucleic acid into a cell. The hybrid lipid nanocapsule can comprise at least one triglyceride, at least one lipophilic surfactant, at least one non-ionic hydrophilic surfactant, and at least one fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate, where the hybrid lipid nanocapsule can be modified with an amino acid, a peptide, a polypeptide, a protein, an antibody, a nucleic acid, or polyethylene glycol.

[0085] In any of the embodiments disclosed herein, the at least one fatty acid-polyamine conjugate can comprise a naturally occurring polyamine. Exemplary naturally occurring polyamines include, without limitation, putrescine, spermidine, spermine, agmatine, and cadaverine. In some embodiments, the at least one fatty acid-polyamine conjugate comprises one or more of: a palmitic acid-derived spermine, an oleic acid-derived spermine, or a mixture thereof.

[0085]

[0086] In some embodiments, the at least one fatty acid-polyamine conjugate can comprise a polymer. In some embodiments, the polymer can comprise polyethyleneimine. Polyethyleneimine can be branched or linear chain polymers with an average molecular weight of approximately 800 - 150,000 Da. In some embodiments, the at least one fatty acid-polyamine conjugate comprises one or more of: a palmitic acid-derived polyethyleneimine, an oleic acid- derived polyethyleneimine, or a mixture thereof.

[0086]

[0087] In any of the embodiments disclosed herein, the at least one fatty add polypeptide conjugate can comprise a polypeptide. Exemplary polypeptides include, without limitation, polyhistidine, poly-L-lysine, and poly-L-arginine. Poly L-lysine can have an average molecular weight of about 15,000- 150,000 Da. In some embodiments, the at least one fatty acid- polpeptide conjugate comprises one or more of: a palmitic acid-derived poly L-lysine, an oleic acid-derived poly L-lysine, an oleic acid-derived poly-L-histidine, a palmitic acid-derived poly-L-histidine, an oleic acid-derived poly-L-arginine, a palmitic acid-derived poly-L- arginine, or a mixture thereof.

[0087]

[0088] In any of the embodiments disclosed herein, the at least one fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate can comprise a fatty acid with a carbon chain length between 8 and 18. In some embodiments, the at least one fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate can comprise a fatty acid selected from the group consisting of palmitic acid, oleic acid, stearic acid, butyric acid, and mixtures thereof.

[0088]

[0089] In any of the embodiments disclosed herein, the at least one triglyceride can comprise a medium-chain triglyceride and / or a long chain triglyceride. In some embodiments, the at least one triglyceride can comprise one or more of: caprylic acid triglyceride, capric acid triglyceride, decanoic acid triglyceride, oleic acid triglyceride, stearic acid triglyceride, or a mixture thereof.

[0089]

[0090] In any of the embodiments disclosed herein, in-house developed hybrid lipid nanocapsules (hLNCs) can be used as a delivery vehicle of mRNA and a sugar glass stabilizes the whole system at room temperature or above. The hybrid lipid nanocapsules (hLNCs) are a delivery vehicle with a core-shell structure. The hLNC core can be made of triglycerides (medium-chain triglyceride) and the shell can be made of a hydrophilic head of phospholipids and a nonionic surfactant. To condense mRNA, an oleic-polyamine such as polyethyleneimine, spermine or oleic-poly peptide conjugate can be incorporated. The lipophilic oleic acid is intact with the triglyceride core and the polyamine or poly-peptide is oriented towards the outer part of the hLNCs. Further, the surface of the hLNCs may be modified with agents to extend their circulation, including L-histidine, polyethylene glycol, or modified with antibodies for tissue targeting or modified with poly-peptide to enhance the transfection efficiency. The inner oily core comprises at least one triglyceride or its derivative (Propylene glycol dicaprolate / dicaprate) which amount can be varied from 30-70 % w / w, 35-65 % w / w, 40-60 % w / w, 45-55 % w / w, or 50 %, w / w. At least one lipophilic surfactant, which can be a phospholipid, and hydrophilic surfactant make the solid shell of hLNCs.. The lipophilic, and nonionic hydrophilic surfactants can be lecithin, phospholipids, and Polyethylene glycol (15)- hydroxy stearate, respectively. The lipophilic surfactant comprises 2-20 % w / w, however most preferably 10 % w / w. The nonionic hydrophilic surfactant comprises 30-60 % w / w, most preferably 50 %, w / w. The oleic-PEI or oleic-poly L-lysine comprises 1-40 % w / w, however most preferably 20 % w / w. In some embodiments, the disclosure provides hLNCs containing at least one triglyceride or its derivatives such as a mixture of propylene glycol esters of caprylic (C8) and capric (CIO) acids, medium-chain triglycerides of caprylic (C8) and capric (CIO) adds), decanoic acid triglyceride, oleic acid triglyceride, stearic acid triglyceride and their PEG derivatives and mixtures thereof, or any other medium and long chain triglycerides and their derivatives.

[0090]

[0091] In any of the embodiments disclosed herein, the at least one lipophilic surfactant can comprise a phospholipid. In some embodiments, the at least one lipophilic surfactant can comprise l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In any of the embodiments disclosed herein, the hLNCs can contain at least one lipophilic surfactant or phospholipids such as Lipoid S-75 (consists of soya lecithin with approximately 69% of phosphatidylcholine and 9% of phosphatidyl ethanol amin), l,2-Didecanoyl-sn-glycero-3-phosphocholine, 1,2- Dierucoyl-sn-glycero-3-phosphate, 1 ,2-Dierucoyl-sn-glycero-3-phosphocholine, 1 ,2-

[0091] Dierucoyl-sn-glycero-3-phosphoethanolamine, l,2-Dilinoleoyl-sn-glycero-3-phosphocholine,

[0092] 1.2-Dilauroyl-sn-glycero-3-phosphate, 1 ,2-Dilauroyl-sn-glycero-3-phosphocholine, 1 ,2- Dilauroyl-sn-glycero-3-phosphoethanolamine, l,2-Dimyristoyl-sn-glycero-3-phosphoserine,

[0093] 1.2-Dioleoyl-sn-glycero-3-phosphate, 1 ,2-Dioleoyl-sn-glycero-3-phosphocholine, 1 ,2- Dioleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-Dioleoyl-sn-glycero-3-phosphoserine, 1 ,2- Dipalmitoyl-sn-glycero-3-phosphoethanolamine, l,2-Distearoyl-sn-glycero-3- phosphoethanolamine, Egg-PC, Hydrogenated Egg PC, Hydrogenated Soy PC, 1- Myristoylsnglycero-3-phosphocholine, l-Palmitoyl-sn-glycero-3-phosphocholine, 1-Stearoyl- sn-glycero-3-phosphocholine, l-Myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine, 1- Myristoyl-2-stearoylsnglycero-3-phosphocholine, l-Palmitoyl-2-myristoyl-sn-glycero-3- phosphocholine, 1 -Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1 -Palmitoyl-2-oleoyl-sn- glycero-3-phosphoethanolamine, l-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1- Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine, l-Stearoyl-2-palmitoyl-sn-glycero-3- phosphocholine 1, natural phospholipid, such as RBCs membrane phospholipid or cell membrane phospholipid or other phospholipid derivative of fatty add having a carbon chain of 10-18 carbons, or a mixture of phospholipids thereof.

[0094]

[0092] In any of the embodiments disclosed herein, the at least one non-ionic hydrophilic surfactant can comprise one or more of: polyethylene glycol (15)-hydroxystearate, polyethylene glycol (PEG) derived decanoic acid, PEG derived lauric acid, PEG derived stearic acid, PEG derived palmitic acid, or a mixture of thereof. In some embodiments, the hLNCs can contain at least one hydrophilic surfactant such as Solutol HS-15 (polyoxyethylated 12- hydroxy stearic acid, wherein it comprises around 15 PEG chain moieties), or a fatty acid derived non-ionic surfactant, such as PEG derived decanoic acid, lauric acid, stearic acid, palmitic acid, mixtures thereof; wherein the number of carbon chain of fatty is 10-18 in number and PEG moiety is 10-100 number. As used herein, “Solutol HS-15” is used interchangeably with “Kolliphor HS-15.”

[0095]

[0093] In any of the embodiments disclosed herein, the hybrid lipid nanocapsule can be modified with an amino acid and / or polyethylene glycol (PEG). In some embodiments, the hybrid lipid nanocapsule can be modified on its surface with zwitterionic amino acids, such as L-histidine, tricine, arginine, or lysine, and / or polyethylene glycol (PEG). In some embodiments, the hybrid lipid nanocapsule can comprise polyethyleneimine modified with zwitterionic amino acids, such as L-histidine or tricine, and / or polyethylene glycol (PEG).

[0096]

[0094] Another exemplary embodiment of the present disclosure provides a nucleic acid delivery system comprising a nucleic acid therapeutic agent, the hybrid lipid nanocapsule as disclosed herein, and a sugar glass as disclosed herein.

[0095] Another exemplary embodiment of the present disclosure provides method for delivering a nucleic acid into a cell. The method can comprise administering the nucleic acid delivery system as disclosed herein to a subject, such that the system enters a cell of the subject.

[0097]

[0096] Some embodiments of the present disclosure provide a breakthrough strategy that merges the advantages of hybrid lipid nanocapsules (hybrid structure of polymer (oleic acid- PEI) and lipids) with the power of L-histidine, heralding a new era in mRNA therapy. This approach aims to usher in stealth properties, address the PEGylation dilemma, and stabilize the mRNA at elevated ambient temperature to fully realize the potential of mRNA delivery in the complex landscape of tumor therapy. The choice of L-histidine as a surface modification is driven by its versatility and inherent pH-responsive properties. L-histidine, a natural amino acid, offers an intriguing combination of biocompatibility and environmental sensitivity. At neutral pH, it remains neutral, minimizing unwanted protein adsorption and opsonization, which is essential for evading immune detection. Yet, in the slightly acidic microenvironment, L-histidine undergoes a remarkable transformation i.e. cationic nature, making the nanocapsules suitable for targeted mRNA delivery.

[0098]

[0097] Applicant recently developed robust hybrid lipid nanocapsules for mRNA delivery, addressing the challenge of mRNA stability at ambient / or elevated temperature (Yadava et al., “Hybrid Lipid Nanocapsules: A Robust Platform for mRNA Delivery,” ACS Appl. Mater. Interfaces, 16( 13): 15981- 15992 (2024)). The hLNCs can be composed of amixture of polymer (oleic acid-polyethyleneimine (OA-PEI) conjugate), lipids (triglyceride and phospholipid), and non-ionic surfactant. The cationic nature of hLNCs due to OA-PEI (further represented as hLNCs-PEI), may not be suitable for passive targeted delivery of mRNA to the tumor via intravenous administration. Addressing this challenge, the hLNCs-PEI surface was modified with L-histidine (hLNCs-Hist.) or PEG (hLNCs-PEG), rendering them neutral. The examples that follow illustrate their cellular uptake behavior in the presence or absence of serum, immunogenicity, transfection efficiency, passive tumor targeting property, mRNA stabilization at elevated temperature and potential to enhance mRNA delivery at tumor sites, culminating in the advancement of precision medicine.

[0099]

[0098] Robust hybrid lipid nanocapsules (hLNCs) incorporating polyethyleneimine (hLNCs- PEI) offer ambient stabilization and efficient mRNA delivery, but their cationic nature limits tumor targeting. To address this, hLNCs-PEI surfaces with histidine (hLNCs-Hist) or polyethylene glycol (hLNCs-PEG) were engineered to neutralize charge and enhance blood circulation, minimizing random protein binding or recognition by immune cells enabling passive tumor targeting. Characterization revealed size (40+2 nm) and zeta potential (16+3 mV for hLNCs-PEI) reduced to neutral for hLNCs-Hist and hLNCs-PEG. The random protein binding decreased post-engineering which resulted in less effect of serum concentration on cellular uptake of hLNCs-Hist and hLNCs-PEG compared to hLNCs-PEI. Histidine or PEG surface modification reduced cellular toxicity and immune recognition, promoting efficient tumor accumulation. The hLNCs-PEG induced immune response (secretion of IgM) and reduced tumor accumulation post-first injection (after secretion of IgM) while, hLNCs-Hist showed neither immune response nor reduced accumulation, highlighting its potential for passively targeted mRNA delivery. Like hLNCs-PEI, hLNCs-Hist maintains its robustness to stabilize mRNA at ambient or / elevated temperatures and efficient transfection efficiency, herefore, hLNCs-Hist is a promising nanoplatform for stabilization and tumor targeted delivery of mRNA therapeutics.

[0100]

[0099] It is to be understood that the embodiments and claims disclosed herein are not limited in their application to the details of construction and arrangement of the components set forth in the description and illustrated in the drawings. Rather, the description and the drawings provide examples of the embodiments envisioned. The embodiments and claims disclosed herein are further capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purposes of description and should not be regarded as limiting the claims.

[0101]

[0100] Accordingly, those skilled in the art will appreciate that the conception upon which the application and claims are based may be readily utilized as a basis for the design of other structures, methods, and systems for carrying out the several purposes of the embodiments and claims presented in this application. It is important, therefore, that the claims be regarded as including such equivalent constructions.

[0102]

[0101] Furthermore, the purpose of the foregoing Abstract is to enable the United States Patent and Trademark Office and the public generally, and especially including the practitioners in the art who are not familiar with patent and legal terms or phraseology, to determine quickly from a cursory inspection the nature and essence of the technical disclosure of the application. The Abstract is neither intended to define the claims of the application, nor is it intended to be limiting to the scope of the claims in any way. EXAMPLES

[0103]

[0102] The following Examples are presented to illustrate various aspects of the present application, but are not intended to limit the scope of the claimed application.

[0104] Example 1 - Materials and Methods

[0105] Composition of Hybrid Lipid Nanocapsules

[0106]

[0103] Tables 1-4 provide exemplary compositions of hybrid lipid nanocapsules.

[0107] Table 1. Composition of HLNC-PEI (0.8 kDa)

[0108] Table 2. Composition of HLNC-PEI (1.8 kDa)

[0109] Table 3. Composition of HLNC-PEI (25 kDa)

[0110] Table 4. Composition of HLNC-Spermine

[0111] Composition of Sugar Glass Containing mRNA-Loaded Hybrid Lipid Nanocapsules

[0112]

[0104] Tables 5-9 provide exemplary compositions of the sugar glass containing mRNA- loaded hybrid lipid nanocapsules.

[0113] Table 5. Composition of mRNA-HPLC-PEI (0.8 kDa)

[0114] Table 6. Composition of mRNA-HPLC-PEI (1.8 kDa)

[0115] Table 7. Composition of mRNA-HPLC-PEI (25 kDa)

[0116] Table 8. Composition of mRNA-HPLC-PEI (1.8 kDa)-L-His

[0117] Table 9. Composition of mRNA-HPLC-Spermine

[0118] Methods

[0119] Preparation of oleic acid- polyethyleneimine (PEI) or spermine conjugate

[0120]

[0105] The Oleic acid-PEI / or spermine conjugate was synthesized using a DCC / NHS coupling reaction in anhydrous dichloromethane (DCM). Briefly, oleic acid (1 mmol) was dissolved in dry DCM, followed by the addition of DCC (1.2 mmol) and NHS (1.2 mmol) under stirring at room temperature for overnight to activate the carboxyl group of oleic acid. The reaction mixture was then centrifuged to remove dicyclohexylurea (DCU) byproduct. Separately, branched PEI (Mw: 0.8-25 kDa) / or spermine was dissolved in DCM and added dropwise to the activated oleic acid solution, followed by stirring for an additional 12-24 hours under an inert atmosphere. The product was precipitated using cold diethyl ether.

[0121] Preparation of mRNA-hybrid lipid nanocapsules-sugar glass

[0122]

[0106] Hybrid lipid nanocapsules (hLNCs) were prepared using the temperature phase inversion method. Briefly, the formulation consisted of a lipid phase containing medium-chain triglyceride (MCT), phospholipid, oleic acid conjugate with cationic polymer / molecules (such as polyethyleneimine, spermine, etc.), a surfactant, and an aqueous phase composed of water containing sodium chloride. The components were mixed and subjected to three successive temperature cycles between 60°C and 90°C, followed by rapid cooling to induce phase inversion and form hLNCs. After purification by dialysis, the hLNCs were dispersed in a solution of trehalose and polyvinyl alcohol, followed by the addition of mRNA at optimized mass ratios (e.g., hLNC-PEL0.8 kDa at 150:1, hLNC-PEL1.8 kDa at 50:1, or hLNC-PEL25 kDa at 25:1, hLNC-spermine at 100:1) to allow electrostatic complexation. The resulting mixture was dried under a hood, followed by desiccation to form an mRNA-hLNCs-sugar glass. Various mRNAs encoding different proteins have been used, including firefly luciferase, mCherry, ovalbumin, and hemagglutinin subtype 3 (HA3), to evaluate protein translation efficiency.

[0123] Surface engineering of hybrid lipid nanocapsules

[0124]

[0107] The surface engineering of hybrid lipid nanocapsules (hLNCs) with L-histidine or PEG was successfully achieved using EDC / NHS coupling, targeting the primary amine groups of PEI on the surface of the hLNCs. The activation of carboxyl-functionalized PEG or L-histidine was carried out in MES buffer (pH 5.5) using EDC and NHS, followed by conjugation to hLNCs through covalent bonding (pH 7.5). After 12-24 hours of reaction, the modified hLNCs were purified by dialysis to remove unreacted reagents.

[0125] Example 2 - Results and Discussion

[0126] Particle size and zeta potential

[0127]

[0108] Hybrid lipid nanocapsules (hLNCs) exhibit a tunable particle size and zeta potential, which are critical parameters influencing their stability, cellular uptake, and biodistribution. The particle size of hLNCs can be precisely controlled within the nanometer range, typically between 25 nm and 150 nm, depending on the formulation composition. The hLNCs usually have a positive surface charge, which facilitates the condensation of negatively charged nucleic acids. However, the zeta potential of hLNCs can be modulated by surface engineering with polyethylene glycol, L-histidine, etc., without hindering the condensation capacity of nucleic acids. These tunable physicochemical properties of hLNCs provide a versatile platform for the efficient and targeted delivery of therapeutic agents, making them highly suitable for advanced drug and gene delivery applications. The particle size and zeta potential of different hLNCs are provided in FIG. 1.

[0128] Stability

[0129]

[0109] Hybrid lipid nanocapsules (hLNCs) embedded in a sugar glass matrix demonstrate exceptional stability at elevated temperatures, ensuring prolonged shelf life and integrity of condensed mRNA. The optimized sugar glass formulation, primarily composed of trehalose and polyvinyl alcohol (PVA), effectively preserves the structural integrity of hLNCs, preventing degradation at 25°C and 40°C. Notably, mRNA-loaded hLNCs embedded in sugar glass maintain their stability for over twelve months at room temperature (25°C) and up to five months at 40°C without significant degradation (FIG. 2A), as the integrity of mRNA is confirmed by gel electrophoresis and successful translation of encoded protein. Moreover, surface engineering of PEI in the surface with L-histidine maintains the integrity of the hLNCs and provides mRNA stability (FIG. 2B). This stabilization is attributed to the protective properties of the sugar glass, which minimizes hydrolytic and oxidative damage while maintaining the lipid nanostructure. The combined effect of hLNCs and sugar glass ensures long-term mRNA preservation under non-refrigerated conditions, offering a robust solution for vaccine and gene therapy applications where cold-chain logistics are challenging. In vitro transfection efficiency

[0130]

[0110] The in vitro transfection efficiency of mRNA-loaded hybrid lipid nanocapsules (mRNA- hLNCs) remains highly effective, even after prolonged storage at elevated temperatures, demonstrating their superior stability for gene delivery applications. Freshly prepared mRNA- hLNCs exhibit high transfection efficiency in dendritic cells and other target cells, enabling robust protein expression. When stored in sugar glass at 25°C for over seven months, the mRNA-hLNCs retain their transfection efficiency with minimal loss in potency. Even after storage at 40°C for five months, mRNA-hLNCs demonstrate substantial transfection capability, confirming the protective role of the sugar glass in preserving mRNA integrity and delivery efficiency. These findings indicate that hLNC-based formulations offer a stable and effective platform for mRNA delivery, eliminating the need for ultra-cold storage and facilitating widespread vaccine and gene therapy distribution.

[0131] In vitro Antigen Presentation

[0132]

[0111] Antigen (Ovalbumin as an antigen) presentation by dendritic cells transfected with ovalbumin (OVA) mRNA was evaluated. Bone marrow-derived dendritic cells (BMDCs) were generated by culturing murine bone marrow cells with granulocyte-macrophage colonystimulating factor (GM-CSF) and interleukin-4 (IL-4) over a period of seven days, promoting their differentiation into immature dendritic cells. Subsequently, these BMDCs were transfected with OVA mRNA condensed with hybrid lipid-based nanocapsules (hLNCs), embedded in sugar glass, facilitating efficient intracellular delivery of the mRNA and subsequent translation of the OVA protein. After 24 hours, the dendritic cells were harvested and identified by the expression of CD11c, a specific surface marker for murine dendritic cells. To evaluate antigen presentation capabilities, the presence of the SIINFEKL peptide bound to MHC class I molecules (H-2KAb) on the surface of BMDCs was detected using the monoclonal antibody, which specifically recognizes the SIINFEKL-H-2KAb complex. This method confirms the effective processing and presentation of antigens by approximately 20% of dendritic cells, providing a robust platform for the development of dendritic cell-based vaccines and immunotherapies.

[0133] In vivo Transfection Efficiency

[0134]

[0112] The in vivo transfection efficiency of firefly luciferase (FLuc) mRNA-loaded hybrid lipid nanocapsules (mRNA-hLNCs) embedded in sugar glass was evaluated following intramuscular (LM.) and subcutaneous (S.C.) administration, demonstrating effective mRNA delivery and expression. Bioluminescence imaging confirmed that both routes resulted in significant luciferase expression, indicating enhanced mRNA uptake and translation in muscle tissue (FIG. 3). The S.C. route also demonstrated efficient transfection, resulting in localized protein expression suitable for vaccine applications and antigen presentation by immune cells. These findings underscore the stability and efficacy of hLNCs for mRNA delivery via different administration routes, making them a versatile platform for gene therapy and vaccination without reliance on cold-chain storage.

[0135] In vivo T cell Activation

[0136]

[0113] T cell activation was assessed following intramuscular (LM.) administration of ovalbumin (OVA) mRNA condensed with hybrid lipid-based nanocapsules (hLNCs), stabilized in sugar glass matrix. Upon intramuscular injection into female mice, the hLNCs facilitated efficient delivery of OVA mRNA, leading to efficient translation of the OVA protein. The endogenously produced OVA was processed and presented on major histocompatibility complex (MHC) class I molecules, specifically as the SIINFEKL peptide bound to H-2KAb, on the surface of antigen-presenting cells. This antigen presentation prompted the activation and expansion of T lymphocytes (CTLs). The activation of these CTLs was confirmed through flow cytometric analysis measuring the expression of activation markers such as CD69 / CD25 / CD44 in about 2-20% of T cells. This method demonstrates the efficacy of the OVA-mRNA-hLNCs-sugar glass formulation in inducing robust antigenspecific T cell responses, highlighting its potential for vaccine development and immunotherapeutic applications.

[0137] B cell activation by Quantifying Specific Antibody Against the Given mRNA

[0138]

[0114] The activation of B cells by OVA-mRNA-loaded hybrid lipid nanocapsules in sugar glass (OVA-mRNA-hLNCs) was evaluated by tracking OVA-specific antibody production over time. Following administration, serum samples were collected on days 7, 14, 21, and 28 to assess the humoral immune response. ELISA analysis confirmed a time-dependent increase in OVA-specific antibody titers, with a significant rise observed by day 14, which continued to peak through days 21 and 28 (FIG. 4). These results indicate robust B cell activation and sustained antigen-specific immune responses elicited by OVA-mRNA-hLNCs. Furthermore, formulations stored in sugar glass at 25°C for over twelve months and at 40°C for five months retained their ability to induce comparable antibody responses, demonstrating the stability and immunogenic potential of the mRNA-hLNC platform (FIG. 4). This highlights the suitability of hLNC-based mRNA vaccines for long-term storage and effective immune activation, even in the absence of cold-chain requirements.

Claims

What is claimed is:

1. A nucleic acid delivery system comprising: a nucleic acid therapeutic agent; a hybrid lipid nanocapsule comprising a fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate capable of condensing with the nucleic acid therapeutic agent; and a sugar glass.

2. The nucleic acid delivery system according to claim 1 , wherein the fatty acid-polyamine conjugate comprises a naturally occurring polyamine.

3. The nucleic acid delivery system according to claim 2, wherein the naturally occurring polyamine is selected from the group consisting of putrescine, spermidine, spermine, agmatine, and cadaverine.

4. The nucleic acid delivery system according to claim 1, wherein the fatty acid- polypeptide conjugate comprises a polypeptide selected from the group consisting of poly-L- histidine, poly-L-lysine, and poly-L-arginine.

5. The nucleic acid delivery system according to any one of claims 1-4, wherein the fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate comprises a fatty acid with a carbon chain length between 8 and 18.

6. The nucleic acid delivery system according to any one of claims 1-4, wherein the fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate comprises a fatty acid selected from the group consisting of palmitic acid, oleic acid, stearic acid, butyric acid, and mixtures thereof.

7. The nucleic acid delivery system according to any one of claims 1-6, wherein the nucleic acid therapeutic agent comprises mRNA.

8. The nucleic acid delivery system according to claim 7, wherein the mRNA is a vaccine related to infectious disease, metabolic disease, or cancer.

9. The nucleic acid delivery system according to any one of claims 1-8, wherein the sugar glass comprises a glass-former with a glass transition temperature greater than the temperature at which the nucleic acid delivery system is stored.

10. The nucleic acid delivery system according to claim 9, wherein the glass-former comprises at least one of an anti-plasticizer, a sugar, a sugar alcohol, a polymer, and / or a combination thereof.

11. The nucleic acid delivery system according to claim 9 or claim 10, wherein the sugar glass further comprises a buffering agent.

12. The nucleic acid delivery system according to claim 10, wherein the polymer is selected from the group consisting of polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), a natural polysaccharide, and mixtures thereof.

13. The nucleic acid delivery system according to claim 12, wherein the natural polysaccharide is selected from the group consisting of dextran, hydroxyethyl starch, pullulan, chitosan, alginate, hyaluronic acid, and inulin.

14. The nucleic add delivery system according to claim 10, wherein the sugar is selected from the group consisting of trehalose, sucrose, stachyose, lactose, fructose, glucose, and mixtures thereof.

15. The nucleic acid delivery system according to claim 10, wherein the sugar alcohol is selected from the group consisting of xylitol, sorbitol, erythritol, mannitol, glycerol, and mixtures thereof.

16. The nucleic acid delivery system according to any one of claims 1-15, wherein the system inhibits mRNA degradation at temperatures about or below 50°C.

17. The nucleic acid delivery system according to any one of claims 1-16, wherein the system inhibits mRNA degradation at temperatures between about 15 °C and about 50°C, about 20°C and about 45°C, or about 25°C and about 40°C.

18. A hybrid lipid nanocapsule for delivery of a nucleic acid into a cell, the hybrid lipid nanocapsule comprising: at least one fatty acid ester; at least one lipophilic surfactant; at least one non-ionic hydrophilic surfactant; and at least one fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate, wherein the hybrid lipid nanocapsule is modified with an amino acid, a peptide, a polypeptide, a protein, an antibody, a nucleic acid, or polyethylene glycol.

19. The hybrid lipid nanocapsule according to claim 18, wherein the at least one fatty acid- polyamine conjugate comprises a naturally occurring polyamine.

20. The hybrid lipid nanocapsule according to claim 19, wherein the naturally occurring polyamine is selected from the group consisting of putrescine, spermidine, spermine, agmatine, and cadaverine.

21. The hybrid lipid nanocapsule according to claim 18, wherein the at least one fatty acid- polyamine conjugate comprises a polymer.

22. The hybrid lipid nanocapsule according to claim 21, wherein the polymer comprises polyethyleneimine.

23. The hybrid lipid nanocapsule according to claim 18, wherein the at least one fatty acid- polypeptide conjugate comprises a polypeptide selected from the group consisting of polyhistidine, poly-L-lysine, and poly-L-arginine.

24. The hybrid lipid nanocapsule according to any one of claims 18-23, wherein the at least one fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate comprises a fatty acid with a carbon chain length between 8 and 18.

25. The hybrid lipid nanocapsule according to any one of claims 18-23, wherein the at least one fatty acid-polyamine conjugate or fatty acid-polypeptide conjugate comprises a fatty acid selected from the group consisting of palmitic acid, oleic acid, stearic add, butyric acid, and mixtures thereof.

26. The hybrid lipid nanocapsule according to any one of claims 18-20, wherein the at least one fatty acid-polyamine conjugate comprises one or more of: a palmitic acid-derived spermine, an oleic acid-derived spermine, or a mixture thereof.

27. The hybrid lipid nanocapsule according to any one of claim 18, claim 21, or claim 22, wherein the at least one fatty acid-polyamine conjugate comprises one or more of: a palmitic acid-derived polyethyleneimine, an oleic acid-derived polyethyleneimine, or a mixture thereof.

28. The hybrid lipid nanocapsule according to claim 18 or claim 23, wherein the at least one fatty acid-polypeptide conjugate comprises one or more of: a palmitic acid-derived poly L-lysine, an oleic acid-derived poly L-lysine, an oleic acid-derived poly-L-histidine, a palmitic acid-derived poly-L-histidine, an oleic acid-derived poly-L-arginine, a palmitic acid-derived poly-L-arginine, or a mixture thereof.

29. The hybrid lipid nanocapsule according to any one of claims 18-28, wherein the at least one fatty acid ester comprises a medium-chain triglyceride and / or a long chain triglyceride.

30. The hybrid lipid nanocapsule according to any one of claims 18-29, wherein the at least one fatty acid ester comprises one or more of: caprylic acid triglyceride, capric acid triglyceride, decanoic acid triglyceride, oleic acid triglyceride, stearic acid triglyceride, a polyethylene ester of a fatty-acid, or a mixture thereof.

31. The hybrid lipid nanocapsule according to any one of claims 18-30, wherein the at least one lipophilic surfactant comprises a phospholipid.

32. The hybrid lipid nanocapsule according to claim 31 , wherein the at least one lipophilic surfactant comprises l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

33. The hybrid lipid nanocapsule according to any one of claims 18-32, wherein the at least one non-ionic hydrophilic surfactant comprises one or more of: polyethylene glycol (15)- hydroxystearate, polyethylene glycol (PEG) derived decanoic acid, PEG derived lauric acid, PEG derived stearic acid, PEG derived palmitic acid, or a mixture of thereof.

34. The hybrid lipid nanocapsule according to any one of claims 18-33, wherein the at least one non-ionic hydrophilic surfactant comprises polyethylene glycol (15)-hydroxystearate.

35. The hybrid lipid nanocapsule according to any one of claims 18-34, wherein the hybrid lipid nanocapsule is modified with a zwitterionic amino acid and / or polyethylene glycol (PEG).

36. The hybrid lipid nanocapsule according claim 35, wherein the zwitterionic amino acid is L-histidine, tricine, arginine, lysine, or a mixture thereof.

37. A nucleic acid delivery system comprising: a nucleic acid therapeutic agent; the hybrid lipid nanocapsule of any one of claims 18-36; and a sugar glass.

38. The nucleic acid delivery system according to claim 37, wherein the nucleic acid therapeutic agent comprises mRNA.

39. The nucleic acid delivery system according to claim 38, wherein the mRNA is a vaccine related to infectious disease, metabolic disease, or cancer.

40. The nucleic acid delivery system according to any one of claims 37-39, wherein the sugar glass comprises a glass-former with a glass transition temperature greater than the temperature at which the nucleic acid deliver system is stored.

41. The nucleic acid delivery system according to claim 40, wherein the glass-former comprises at least one of an anti-plasticizer, a sugar, a sugar alcohol, a polymer, and / or a combination thereof.

42. The nucleic acid delivery system according to claim 40 or claim 41, wherein the sugar glass further comprises a buffering agent.

43. The nucleic acid delivery system according to claim 41 , wherein the polymer is selected from the group consisting of polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), a natural polysaccharide, and mixtures thereof.

44. The nucleic acid delivery system according to claim 43, wherein the natural polysaccharide is selected from the group consisting of dextran, hydroxyethyl starch, pullulan, chitosan, alginate, hyaluronic acid, and inulin.

45. The nucleic add delivery system according to claim 41, wherein the sugar is selected from the group consisting of trehalose, sucrose, stachyose, lactose, fructose, glucose, and mixtures thereof.

46. The nucleic acid delivery system according to claim 41, wherein the sugar alcohol is selected from the group consisting of xylitol, sorbitol, erythritol, mannitol, and glycerol.

47. The nucleic acid delivery system according to any one of claims 37-46, wherein the system inhibits mRNA degradation at temperatures about or below 50°C.

48. The nucleic acid delivery system according to any one of claims 37-47, wherein the system inhibits mRNA degradation at temperatures between about 15 °C and about 50°C, about 20°C and about 45°C, or about 25°C and about 40°C.

49. A method for delivering a nucleic acid into a cell, comprising: administering the nucleic acid delivery system of any one of claims 1-17 or claims 37-48 to a subject, such that the system enters a cell of the subject.

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