Ionizable and cationic lipid nanoparticles and intracellular drug delivery thereof

Abstract

The present invention provides a formulation method for encapsulating a negatively charged or hydrophobic target agent by lipid nanoparticles (LNPs). The formulation method comprises impinging an organic phase solution having an ionizable or cationic lipid and an aqueous phase solution. The target agent may be in the organic or aqueous phase solution. The formulation method further comprises self-assembling LNPs, each of which comprises a lipid membrane formed having the ionizable or cationic lipid, whereby the target agent is entrapped by the ionizable or cationic lipid and encapsulated by the LNPs. Also provided is an LNP comprising two or more negatively charged or hydrophobic target agents entrapped by an ionizable or cationic lipid and encapsulated by the LNP. Further provided are the uses of LNPs for intracellular delivery and for stimulating an immune response.

Description

[0001]0074539-000157 -1- IONIZABLE AND CATIONIC LIPID NANOPARTICLES AND INTRACELLULAR DRUG DELIVERY THEREOF CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to United States Provisional Application No. 63 / 512,423, filed July 7, 2023, and the contents of which are incorporated herein by reference in their entireties for all purposes. REFERENCE TO U.S. GOVERNMENT SUPPORT This invention was made with government support under grant number 2P20GM121322-06 awarded by National Institutes of Health National Institute of General Medical Sciences (NIH-NIGMS) Tumor Microenvironment Centers of Biomedical Research Excellence (TME CoBRE). The United States has certain rights in the invention. FIELD OF THE INVENTION The invention relates to lipid nanoparticles and uses thereof for intracellular delivery of a target agent. BACKGROUND OF THE INVENTION Targeted intracellular drug delivery systems are especially advantageous because of their ability to release target agents into more specific and targeted locations such as the cytosol, mitochondria, or a variety of cellular organelles for prophylactic and therapeutic applications. LNPs that are sensitive to acidic conditions will enable the precise release of target agents such as nucleic acid, adjuvants, and chemotherapeutics from endolysosomes into the cytoplasm and also prevent leakage of these agents at neutral pH (blood or extracellular spaces). Ionizable and cationic lipid nanoparticles (LNPs) have emerged as a powerful tool for the intracellular delivery of nucleic acids. Following the recent success of LNP-based siRNA therapeutics and mRNA vaccines, the use of ionizable lipids for nucleic acid delivery has tremendously increased. Besides conventional ethanol injection and bulk mixing methods, microfluidic techniques utilizing rapid mixing are widely utilized to formulate LNPs. Rapid mixing using microfluidic devices creates a supersaturated condition of lipid molecules that helps in generating self-assembled LNPs. T junction mixing, Microfluidic Hydrodynamic Flow Focusing (HHF), and Staggered Herringbone Mixing (SHM) are the most commonly studied microfluidic techniques for LNP fabrication. T-junction mixer has been used to produce stable LNPs encapsulating DNA and siRNA on a large scale. In a T-junction mixer, organic and aqueous solutions are mixed under turbulent conditions where lipids self-assemble to form an emulsion, followed by diffusion-based dilution of organic solvents 0074539-000157 -2- that help LNPs form at the liquid-liquid HHF or SHM involves solvent mixing in a laminar flow environment, resulting in mass transfer through diffusion. In the case of the HHF, the center flow is compressed due to the greater flow velocity compared to the outer layered fluid, resulting in reduced solvent diffusion length. The SHM’s structure allows two different fluids to wrap around each other, amplifying the fluid-fluid interface exponentially and rapidly mixing the organic and aqueous solvents. Overall, the microfluidic platforms provide several advantages over conventional ethanol injection or bulk mixing methods, such as the high encapsulation efficiency of nucleotides, and controllable particle size with high monodispersity, reproducibility, and scalability. However, some of the major drawbacks of microfluidic techniques include their difficulty in producing LNPs in small volumes for testing in the discovery phase, their ready availability to researchers in an academic environment, and their high cost. Confined impingement jet (CIJ) mixers allow the rapid, millisecond mixing of aqueous and water-miscible organic solvents under a turbulent flow that induces high supersaturation and nucleation rates to form monodisperse nanoparticles. CIJ mixers have been widely employed to stabilize hydrophobic drugs and produce morphologically diverse nanoparticles for different biomedical applications. Similarly, multi-inlet vortex mixer (MIVM) mediated flash nanoprecipitation has been also reported to rapidly fabricate different types of nanoparticles where a supersaturation environment is achieved by mixing using multiple inlets of organic and aqueous solvents, which provides a higher degree of manipulation of the liquid stream composition. However, the complexity of turbulent flow due to multiple inlets, makes it difficult to predict the mixing parameters requiring optimization of several process variables. There remains a need for cost-effective alternative fabrication techniques suitable for the discovery phase and scalable production of lipid nanoparticles (LNPs) for intracellular delivery of a target agent. SUMMARY OF THE INVENTION The present invention relates to lipid nanoparticles (LNPs) for intracellular delivery of a target agent. The inventors have surprisingly discovered a flash nanoprecipitation (FNP) approach using a confined impingement jet (CIJ) mixer to stably self-assemble ionizable LNPs loaded with a target agent. The present invention provides a formulation method for encapsulating one or more target agents by lipid nanoparticles (LNPs). The one or more target agents comprise a negatively charged target agent, a hydrophobic target agent, or a combination thereof. The formulation method comprises providing an organic phase solution comprising a lipid 0074539-000157 -3- mixture and an organic solvent and an phase solution comprising a first aqueous solvent having a first volume. The lipid mixture comprises an ionizable or cationic lipid. Where the one or more target agents comprise the negatively charged target agent, the lipid mixture is dissolved in the organic solvent, and the negatively charged target agent is dissolved in the first aqueous solvent. Where the one or more target agents comprise the hydrophobic target agent, the hydrophobic agent and the lipid mixture are dissolved in the organic solvent. Where the one or more target agents comprise the negatively charged and the hydrophobic target agent, the hydrophobic agent and the lipid mixture are dissolved in the organic solvent, and the negatively charged target agent is dissolved in the first aqueous solvent. The formulation method further comprise impinging the organic phase solution and the aqueous phase solution into a second aqueous solvent having a second volume at least three times of the first volume of the first aqueous solvent, whereby an impingement mixture is formed; self-assembling LNPs in the impingement mixture, wherein each LNP comprises a lipid membrane formed by the lipid mixture and a space enclosed by the lipid membrane, wherein the one or more negatively charged or hydrophobic target agents are entrapped by the ionizable or cationic lipid and encapsulated by the LNPs; and removing organic solvent from the impingement mixture, whereby the LNPs are obtained. The impinging may comprise mixing the organic phase solution and the aqueous phase solution for no more than 200 milliseconds. The impingement mixture may have a total lipid concentration of no more than 50 mM. The impingement mixture may have an aqueous volume of no more than 3 mL. The LNPs may have a polydispersity index of less than 0.3. The LNPs may have a particle size of less than 200 nm. At least 50% of the one or more negatively charged or hydrophobic target agents may be encapsulated by the LNPs. The ionizable lipid may be selected from the group consisting of SM102, ALC-0315, DLin-MC3-DMA, C12-200, cKK-E12, C14-4, L-319, YSK12-C4, and DLin-KC2-DMA. The cationic lipid may be selected from the group consisting of 1,2-dioleoyl-3- trimethylammonium propane (DOTAP), N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), dimethyldioctadecylammonium (bromide) (DODAB), and 1,2-stearoyl-3-trimethylammoniumpropane (chloride) (DSTAP). Each of the one or more target agents may be selected from the group consisting of a nucleic acid, an adjuvant, and a hydrophobic drug. 0074539-000157 -4- The formulation method may further lyophilizing the LNPs, whereby a powder of LNPs is obtained. The formulation method may further comprise rehydrating the LNP powder with a third aqueous solvent such that an aqueous solution of the LNPs is obtained. The formulation method may further comprise interacting the LNPs with cells, and delivering the LNPs into the cells, whereby the one or more target agents are in the cells. The one or more target agents in the cells may be biologically active. The formulation method may further comprise releasing the one or more target agents in the cells from the LNPs. The released one or more target agents may be biologically active. The formulation method may further comprise stimulating an immune receptor in or on the cells. The cells may be in a subject. Where the cells are in a subject, the formulation method may further comprise stimulating an immune response in the subject. The present invention provides the lipid nanoparticles (LNPs) produced in accordance with the formulation method of the present invention. A composition comprising the LNPs is also provided. The present invention provides a lipid nanoparticle (LNP) comprising two or more negatively charged or hydrophobic target agents, a lipid membrane formed by a mixture of lipids, and a space enclosed by the lipid membrane. The mixture of lipids comprises an ionizable or cationic lipid. Each of the two or more negatively charged or hydrophobic target agents is selected from the group consisting of a nucleic acid, an adjuvant, and a hydrophobic drug. The ionizable lipid may be selected from the group consisting of SM102, ALC-0315, DLin-MC3-DMA, C12-200, cKK-E12, C14-4, L-319, YSK12-C4, and DLin-KC2- DMA. The cationic lipid may be selected from the group consisting of 1,2-dioleoyl-3- trimethylammonium propane (DOTAP), N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), dimethyldioctadecylammonium (bromide) (DODAB), and 1,2-stearoyl-3-trimethylammoniumpropane (chloride) (DSTAP). A composition comprising a plurality of the LNPs is provided. The present invention provides a method for intracellular delivery. The delivery method comprises interacting the LNPs of the present invention with cells, and delivering the LNPs into the cells, whereby at least one of the target agents is in the cells. The at least one target agent in the cells may be biologically active. The delivery method may further comprise releasing the at least one target agent in the cells from the LNPs. The at least one released target agent may be biologically active. The delivery method may further comprise stimulating an immune cell specific receptor in or on the cells. The cells may be 0074539-000157 -5- in a subject. The delivery method may stimulating an immune response in the subject. The present invention further provides a method for stimulating an immune response. The stimulation method comprises administering to a subject an effective amount of the LNPs of the present invention or the composition of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS FIGS.1A-E show fabrication and physicochemical characterization of lipid nanoparticles. A) Schematic showing the preparation of nucleic acid (RNA)-loaded Lipid Nanoparticle (LNP). B) Negative stained transmission electron microscope (TEM) images for the SM102, ALC0315, and DLin-MC3-DMA ionizable LNPs. The TEM image scale is 100 nm. C) Particle size of LNPs was measured using dynamic light scattering, and the hydrodynamic diameter is reported as a Z average and reported in nm. D) Zeta potential was measured by electrophoretic light scattering and reported in millivolt (mV). E) Encapsulation efficiency of LNP-loaded mRNA or siRNA was measured using the QuantiFluor® RNA System. Data represented in C, D, and E are mean ± SD (n=3). The image was created with BioRender.com. FIGS.2A-B show cellular uptake of ionizable lipid nanoparticles by RAW 264.7 macrophages. A) Time-dependent cellular uptake of SM102, ALC0315, and DLin-MC3- DMA LNPs after 2, 6, and 24 h of incubation with RAW 264.7 cells. Data represented as mean ± SD (n=4). Significant differences between each timepoint were determined by ANOVA with Tukey’s multiple comparison tests, **p = 0.0029, ****p < 0.0001. B) Intracellular localization of LNPs in RAW 264.7 cells. The scale bar is 10 μm. FIGS.3A-C shows intracellular delivery of mRNA using LNPs in DC 2.4 immortalized murine dendritic cells: A) SM102 and ALC0315 LNPs encapsulated with mCherry mRNA and incubated for 24 h with DC 2.4 cells, B) Schematic showing functional SIINFEKL assay, and C) The expression level of the H-2kb antibody-bound OVA257-264 (SIINFEKL). FIGS.4A-C show in vivo delivery of mRNA using LNPs in C57BL / 6 mice. A) Schematic representation of firefly luciferase mRNA loaded LNPs, route of administration, and IVIS imaging of lymphoid organs. B) IVIS bioluminescent images of draining inguinal lymph nodes and the spleens of SM102 LNPs or ALC0315 LNPs or PBS only administered mice. C) Bioluminescence is represented as average radiance in draining lymph nodes and spleens of mice administered with SM102 and ALC0315 LNPs. Data represented as mean ± SD (n=3). 0074539-000157 -6- FIGS.5A-C show In vivo cellular of DiD dye-loaded LNP in C57BL / 6 mice. A) Schematic representation of DiD-loaded LNPs, route of administration, lymphoid organ harvesting, and flow cytometry analysis of the cells from the harvested organs. B) Heatmap of the DiD LNPs positive immune cells (CD 45+) and non-immune cells (CD45-) obtained from draining lymph nodes. C) Heatmap of the DiD LNPs positive immune cells (CD 45+) and non-immune cells (CD45-) obtained from spleen. Data represented as mean ± SD (n=3). FIGS.6A-D show intracellular delivery of siRNA using DLin-MC3-DMA LNPs. (A) Intracellular localization of Cy5-siRNA in RAW 264.7 cells and DC 2.4 cells. B) Schematic showing the siRNA-LNPs gene silencing functional assay through green fluorescent protein (GFP)-- expressing M.D. Anderson-Metastatic Breast-231 (MDA-MB-231) / Luc cells. C) GFP signal is represented as median fluorescent intensity (MFI) measured using flow cytometry. D) Percentage of knockdown efficiency was plotted against the control siRNA. FIG.7 shows Apparent pKa of the ionizable LNPs measured by TNS (6-p- Toluidino-2-naphthalenesulfonic acid) assay. FIGS.8A-C show stability of LNPs prepared through flash nanoprecipitation and stored at 4 °C. A) Particle size of the LNPs was measured over 28 days using dynamic light scattering, and the hydrodynamic diameter is reported as Z average (nm). B) Zeta potential of LNPs was measured over 28 days at different time intervals by electrophoretic light scattering, and the zeta potential is reported as millivolt (mV). C) Polydispersity index (PDI) was measured over 28 days using dynamic light scattering. Data represented as mean ± SD (n=3). FIG.9A-B show selection of water-miscible organic solvents for LNP fabrication via Flash nanoprecipitation (FNP). A) Solubility of the lipid mixture was tested in 1 mL of different organic solvents, only methanol and ethanol were able to dissolve the lipid components, making them suitable solvents for FNP. B) Table depicts the particle size, polydispersity index, and zeta potential of LNPs fabricated using methanol as the organic solvent. Data represented as mean ± SD (n=3). FIGS.10A-D show characterization of LNPs prepared with cationic lipid (DOTAP) and lipidoid (NT1-O14B): A) Positive stained TEM images for DOTAP containing LNPs, B) Particle size of LNPs was measured using dynamic light scattering, and the number average is reported as d.nm. to compare the particle size distribution of NT1-O14B and DOTAP formulations, C) The polydispersity index (PDI) was calculated based on Dynamic light scattering, and D) Zeta potential was measured by electrophoretic light scattering. 0074539-000157 -7- FIG.11 shows particle size, zeta and polydispersity index (PDI) of SM102, ALC0315, and DOTAP LNPs encapsulated with CpG ODN 2395. FIG.12A-C show negative stained TEM images of SM102, ALC0315, and DOTAP LNPs encapsulated with CpG ODN 2395 showed spherical morphology of the LNPs. Fig 13 shows encapsulation efficiency of the CpG ODN 2006 encapsulated into the SM102 and ALC0315 LNPs. FIGS.14A-B show secreted alkaline phosphatase (SEAP) assay using the A) SM102 and B) ALC0315 ionizable LNPs containing the CpG ODN 2395. RAW-Blue™ cells were treated with different concentrations of CpG ODN loaded LNPs for 24 h and 48 h. After that, the cell supernatant was collected to measure the secreted alkaline phosphatase through QUANTI-Blue™ reagent. FIG.15 shows an HEK blue assay using the SM102 loaded CpG ODN 2006, which triggers the human TLR9 receptors. hHEK blue TLR9 cells were incubated for 24 h and 48 h with different concentrations of the CpG ODN. As a positive control TNF-α was used. The data are presented as standard deviation (SD) ± 3. FIGS.16A-B show intracellular delivery of CpG ODN and cellular uptake through flow cytometry. A) Live cell confocal images of FITC CpG ODN 2395 loaded SM102 and ALC0315 LNPs. RAW 264.7 cells were treated for 24 h with SM102 and ALC0315 LNPs. Diffused fluorescence inside cells shows successful intracellular delivery of LNPs. B) Flow cytometry shows, successful cellular association of LNPs. Significant differences between each timepoint were determined by an unpaired t-test with Welch's correction. ***P < 0.0001 for ALC0315 vs. SM102. The data are presented as standard deviation (SD) ± 3. FIG.17 shows particle sizes of lyophilized SM102, ALC0315, and DOTAP LNPs. FIGS.18A-B show lyophilization study and immune cell activation assay of CpG ODN loaded SM102 LNPs in presence of 10% w / v sucrose. A) Particle size of the LNPs measured by the dynamic light scattering. B) Immune cell activation of the lyophilized LNPs. FIGS.19A-B show lyophilization study and immune cell activation assay of CpG ODN loaded ALC0315 LNPs in the presence of 10% w / v sucrose. A) Particle size of the LNPs measured by the dynamic light scattering. B) Immune cell activation of the lyophilized CpG 2006 loaded samples. FIG.20 shows the SEAP activity of CL429 LNPs in an immune cell activation assay through SEAP activity. FIGS.21A-B show morphological characterization of 50% SM102 loaded MPLA LNPs and immune cell activation assay. A) 50% SM102 ionizable lipid was used to prepare 0074539-000157 -8- MPLA-encapsulated LNPs. A) Particle size, index was measured using dynamic light scattering and electrophoretic light scattering was used to measure the zeta potential. B) SEAP study was used to check the immune cell response. FIGS.22A-B show particle characterization and immune cell activation assay of CpG ODN and MPLA LNPs. A) Particle size of SM102 loaded CpG 2395 and MPLA was measured by dynamic light scattering and zeta potential was measured by electrophoretic light scattering. B) SEAP study was used to check the immune cell response. FIGS.23A-C show surface marker expression of mouse BMDCs following adjuvant LNP treatment: A) CD40, B) CD80, and C) CD86. FIGS.24A-B show physicochemical characterization of CL347 LNPs with and without an ionizable lipid SM102. A) Particle size of the LNPs was measured using dynamic light scattering and the number average is reported as d.nm. B) Encapsulation efficiency of mCherry mRNA in CL347 LNPs and CL347-SM102 LNPs measured by QuantiFluor® RNA System. Data represented as mean ± SD (n=4). FIGS.25A-B show intracellular localization of CL347 LNPs in RAW 264.7 macrophages. FIGS.26A-B show intracellular delivery of mCherry mRNA in RAW 264.7 macrophages. FIGS.27A-B show in vitro functional assay of CL347 LNPs. A) Secreted embryonic alkaline phosphatase (SEAP) activity of CL347 LNPs after 48 hours of treatment with TLR7 specific hHEK-Blue™ Cells. B) DC2.4 cells were treated with OVA-mRNA loaded CL347- SM102 LNPs or CL347 admixed with OVA mRNA for 24 h, the dose of OVA-mRNA was 1 μg / mL in each well. FIGS.28A-C show in vivo delivery of mRNA using CL347 lipid nanoparticles in C57BL / 6 mice. A) Schematic representation of firefly luciferase mRNA loaded LNPs, route of administration, and IVIS imaging of lymphoid organs. B) IVIS bioluminescent images of axillary, brachial, and inguinal lymph nodes and the spleens of SM102-CL347 mRNA LNPs, only CL347 admixed with mRNA, and PBS control administered mice at mRNA concentration of 4 μg / mice and CL347 concentration of 12 μg / mice. C) The bioluminescence represented as average radiance in spleens of mice administered with CL347-SM102 LNPs loaded firefly luciferase mRNA, CL347-firefly luciferase mRNA mix, and PBS control. FIG.29 shows IgM antibodies specific to OVA following adjuvanted mRNA LNP vaccination. 0074539-000157 -9- FIGS.30A-B show intracellular of lymph and spleen cells. Th1 and Th2 Types of cytokine expression was reported for both A) lymph node and B) spleen cells. FIGS.31A-D show proinflammatory cytokines of human (peripheral blood mononuclear cells) PBMCs after treatment with CL347 loaded LNPs. A) Heatmap shows the expression of different cytokines. Significant differences were observed for B) IL-6, C) IL-10, and D) IFN-γ. FIG.32 shows in vitro functional activity of Imiquimod-loaded ionizable LNPs and Resiquimod-loaded ionizable LNPs. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to ionizable or cationic lipid nanoparticles (LNPs) for encapsulating a target agent, preparation and uses thereof. The present invention is based on the inventors’ discovery of a flash nanoprecipitation (FNP) technique using a confined impingement jet (CIJ) mixer as a simple, rapid millifluidic process for scalably producing the LNPs. The inventors have surprisingly discovered that a CIJ mixer containing only two inlets (one inlet for an aqueous phase and one inlet for an organic phase) allows the highest level of control over nanoparticle fabrication, and have established the CIJ mixer’s suitability to self-assemble ionizable lipid nanoparticles encapsulating nucleic acids such as mRNA or siRNA or DNA or plasmids, and immunological adjuvants or small molecule drugs. The present invention provides LNPs capable of encapsulating poorly soluble (hydrophobic) small-molecule drugs, which are tough candidates to formulate and administer. The inventors have demonstrated self-assembly of ionizable and cationic LNPs via the FNP technique utilizing a CIJ mixer. The FNP technique supports stable formation of clinically relevant LNPs containing an ionizable lipid such as SM102, ALC0315, and DLin- MC3-DMA or a cationic lipid such as DOTAP. The FNP technique allows encapsulation of nucleic acids, immunological adjuvants, and / or small molecule hydrophobic drugs. LNPs have been shown to precisely deliver nucleic acid payloads, adjuvants, and small molecule drugs into an intracellular compartment of cells with demonstrated functional efficiency. Adjuvants encapsulated into LNPs have been demonstrated to precisely stimulate immune cell specific receptors as compared to unencapsulated or free adjuvants (which are not encapsulated inside LNPs). The adjuvant-containing LNPs may be lyophilized into powders and, following rehydration of the LNP powders, the LNPs are stable and retain the functional adjuvant activity. Adjuvants co-encapsulated with mRNA vaccines have been shown to enhance immune responses both in vitro and in vivo indicating that adjuvants may be co-delivered with mRNA vaccines using ionizable LNPs. 0074539-000157 -10- The FNP approach may serve as an approach to reproducibly produce LNPs in smaller research setups where furnishing microfluidic devices is challenging or a smaller volume of LNPs is required to perform experiments in the discovery phase. The term “lipid nanoparticle (LNP)” as used herein refers to a nanoparticle composed of one or more lipids. The LNP may comprise a lipid membrane formed by the one or more lipids and a space enclosed by the lipid membrane. The LNP may comprise a carrier matrix in the space. The carrier matrix may be aqueous or non-aqueous. The LNP may be self-assembled by one or more lipids. Where the LNP comprises an ionizable or cationic lipid, the LNP may also be referred to as an ionizable or cationic LNP. The term “ionizable lipid” as used herein refers to a lipid that possesses a neutral charge at normal physiological pH, for example, a pH of about 6.5-8.0 or 7.0-7.5, but becomes positively charged when the pH is lowered to, for example, a pH of about 4-5. The term “cationic lipid” as used herein refers to a lipid carrying a permanent positive charge. The cationic lipid does not change its charge based on a change in an environmental pH. The term “amphiphilic lipid” as used herein refers to a lipid having both a hydrophilic (i.e., water-attracting) region and a hydrophobic (i.e., water-repelling) region and capable of interacting with both an aqueous environment and an organic environment. As a result, an amphiphile lipid may self-assemble into an organized structure, for example, LNP, when dispersed in an aqueous solution. An amphiphilic lipid helps in stabilizing LNPs in an aqueous environment. An amphiphilic lipid may be an ionizable lipid, a cationic lipid, a structural lipid, a helper lipid, or a PEG lipid. The terms “self-assembling” and “self-assembly” are used herein interchangeably and refer to spontaneous rearrangement of biological molecules into a desirable structure, for example, an LNP, in an aqueous environment without an external stimulus. Where the biological molecules comprise one or more lipids, this process is also known as "self- assembling of lipids." The self-assembly of a lipid may be driven by the amphiphilic nature of the lipid. When an amphiphilic lipid possessing hydrophobic tails and hydrophilic heads is placed in an aqueous environment, the hydrophobic tails orient themselves away from an aqueous environment while the hydrophilic heads interact with water such that the amphiphilic lipid may spontaneously form a lipid membrane enclosing a space without an external stimulus. Thus, an LNP comprising the lipid membrane and the space enclosed by the lipid membrane may be self-assembled. 0074539-000157 -11- The term "impingement" as used to a process in which two high- velocity phase solutions, an organic phase solution and an aqueous phase solution, collide with each other within a confined chamber. The term “entrapped” as used herein refers to a target agent being held by an ionizable or cationic lipid by interaction between the target agent and the ionizable or cationic lipid so that the target agent cannot be freed from the ionizable or cationic lipid. Where the ionizable or cationic lipid is in a carrier matrix of an LNP, the target agent may be incorporated into the carrier matrix. The term “encapsulation” as used herein refers to the process of incorporating a target agent (e.g., nucleic acid) within the structure of an LNP. A hydrophilic target agent (e.g., nucleic acid) may be in the space enclosed by the lipid membrane. A negatively charged target agent (e.g., nucleic acid) may electrostatically interact with a positively charged ionizable or cationic lipid, and form an aqueous channel inside the LNP. A hydrophobic target agent may be attached to the lipid membrane. An amphipathic target agent (e.g., peptides) may be in the space enclosed by the lipid membrane and attached to the lipid membrane. The term “vaccine” as used herein refers to a composition used to stimulate immunity to a pathogen or infectious disease. The vaccine may be formulated using a live attenuated pathogen, whole killed pathogen, protein or subunit derived from a pathogen, or nucleic acid. Examples of vaccines include influenza vaccine, hepatitis vaccine, and measles-mumps-rubella (MMR) vaccine. Where vaccine is prepared using a genetic material, for example, DNA or RNA from a target pathogen, the vaccine may be referred to as a nucleic acid vaccine. Examples of nucleic acid vaccines include the Comirnaty Pfizer- BioNTech made against COVID-19 and the Spikevax Moderna made against COVID 19. The term “adjuvant” as used herein is an ingredient in a composition, which comprises a therapeutic drug or a prophylactic vaccine, to enhance the efficacy of the therapeutic drug or the potency of the prophylactic vaccine by increasing an immune response to an antigen in a subject. The antigen may be from a pathogen. The term “immune receptor” as used herein refers to a receptor that, upon binding a ligand, causes an immune response. The immune receptor may be on a cell membrane of an immune cell or in the cytoplasm, endoplasmic reticulum and endosome of an immune cell. The ligand may be specific for a pathogen such that, upon binding the ligand, the immune receptor causes an immune response to the pathogen. Examples of immune receptors include Toll Like Receptors (TLRs) (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9), Nod like Receptors (NLRs) (e.g., NOD1, NOD2, NLRC4, and 0074539-000157 -12- NLRP3), C-type Lectin Receptors (CLRs) 1 and Dectin-2), and Stimulator of Interferon Genes (STING). The term “subject” as used herein refers to an animal. The subject may be a human, mouse, non-human primate (NHP) (e.g., rhesus macaques, cynomolgus monkeys, African green monkeys, and baboons), ferret, guinea pig, hamsters (e.g., Syrian golden hamster, and Chinese hamster), rabbit, pig, dog, cat, sheep or goat. The term “target Agent” as used herein refers to any desirable agent. The target agent may be a compound, a biological molecule or a combination thereof. The term “cells” as used herein refers to any cells. The cells may be in a subject. The cells may be isolated from a subject or from a cell line. The cells may be selected from the group consisting of murine RAW 264.7 macrophages, murine bone marrow-derived dendritic cells (BMDCs), murine bone marrow-derived macrophages (BMDMs), human peripheral blood mononuclear cells (PBMCs), and non-human primate cells (e.g., T cells, B cells, natural killer (NK) cells, and hepatocytes). The present invention provides a formulation method for encapsulating one or more target agents by lipid nanoparticles (LNPs). The one or more target agents comprise a negatively charged target agent, a hydrophobic target agent, or a combination thereof. The formulation method comprises providing an organic phase solution and an aqueous phase solution. The organic phase solution comprises a lipid mixture and an organic solvent. The lipid mixture comprises an ionizable or cationic lipid. The aqueous phase solution comprises a first aqueous solvent. The first aqueous solvent may have a first volume. Where the one or more target agents comprise the negatively charged target agent, the lipid mixture is dissolved in the organic solvent, and the negatively charged target agent is dissolved in the first aqueous solvent. Where the one or more target agents comprise the hydrophobic target agent, the hydrophobic agent and the lipid mixture are dissolved in the organic solvent. Where the one or more target agents comprise the negatively charged and the hydrophobic target agent, the hydrophobic agent and the lipid mixture are dissolved in the organic solvent, and the negatively charged target agent is dissolved in the first aqueous solvent The formulation method further comprises impinging the organic phase solution and the aqueous phase solution into a second aqueous solvent so that an impingement mixture is formed. The second aqueous solvent may have a second volume that is at least about 2, 3, 4, 5, 10 or 20 times of the first volume of the first aqueous solvent. The formulation method further comprises self-assembling LNPs in the impingement mixture. Each LNP comprises a lipid membrane formed by the lipid mixture and a space enclosed by the lipid membrane. The one or more target agents are 0074539-000157 -13- entrapped by the ionizable or cationic lipid by the LNPs. The formulation method further comprises removing organic solvent from the impingement mixture. As a result, the LNPs are obtained. In one embodiment, the impinging is performed in a confined impinging jet (CIJ) mixer. The organic phase solution and an aqueous phase solution are directed towards each other in a confined chamber, resulting in a collision within microseconds. This collision generates high kinetic energy, which helps in the mixing of the two phase solutions. After the mixing of the two phase solutions, turbulent eddies and vortices are generated, leading to a rapid turbulent mixing phase, for example, within 10 to 100 milliseconds. This phase promotes extensive contact among the molecules in the two phase solutions, thereby resulting in a homogenized mixture of lipids and the target agent, also known as a cargo (e.g., drugs, adjuvants and nucleic acids). As a supersaturated condition is achieved due to the rapid turbulent mixing, the mixer then initiates the nucleation process. In this process, the solute molecules (e.g., lipids and cargo) dispersed in the solvents start to form nuclei, for example, within about 10 to 50 milliseconds. These nuclei then grow into larger particles. The growth phase may last about 100-300 milliseconds, for example, about 200 milliseconds. After the nucleation and growth processes have been completed, the mixed liquid formed by both the organic phase solution and the aqueous phase solution is directed into a reservoir that contains a large volume of a second aqueous solvent to form an impingement mixture. The second aqueous solvent may be the same as or different from the first aqueous solvent. Consequently, the nucleation and growth process cease in the impingement mixture. Upon removal of the organic solvent from the impingement mixture, a suspension of LNPs is created. This collision may result in rapid solvent mixing owing to the high turbulence and generated shear stress. Each of the organic phase solution and the aqueous phase solution may have a velocity of not exceeding about 5 m / s, for example, about 1-2, 1-3, 1- 4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5 or 4-5 m / s. The turbulence may have a Reynolds number of about 102-104, for example, about 103. The shear stress may be about 10-10,000, 10- 1,000, 100-10,000, 100-1,000, 500-10,000 or 500-1,000 Pa. The impinging may comprise mixing the organic phase solution and the aqueous phase solution for no more than about 1,000, 500, 200 or 100 milliseconds, or about 100- 1,000, 100-500, 100-300, 100-200 or 100-150 milliseconds. The impingement mixture may have a total lipid concentration of no more than about 100, 50, 40, 30, 20 or 10 mM, or about 1-100, 1-50, 1-40, 1-30, 1-20 or 1-10 mM. A 0074539-000157 -14- total lipid concentration exceeding the limit affect the particle size, monodispersity, and stability of the LNPs. The impingement mixture may have an aqueous volume of no more than about 1, 2, 3, 4, 5 or 10 mL, or about 0.1-1, 0.1-2, 0.1-3, 0.1-4, 0.1-5, or 0.1-10 mL. The LNPs may have a polydispersity index of less than about 0.1, 0.2, 0.3, 0.4, 0.5 or 1.0, or about 0.01-0.1, 0.01-0.2, 0.01-0.3, 0.01-0.4, 0.01-0.5 or 0.01-1.0. Samples with a polydispersity index of less than 0.3 are generally considered monodisperse. The LNPs may have a particle size of less than about 10, 20, 50, 100, 200, 300, 400, 500 or 100 nm, or about 1-1000, 1-500, 1-400, 1-300, 1-200, 1-100 or 1-10 nm. The LNPs may have a particle size less than about 10, 20, 50, 100, 200, 300, 400, 500 or 100 nm, or about 1-1000, 1-500, 1-400, 1-300, 1-200, 1-100 or 1-10 nm, and a polydispersity index of less than about 0.1, 0.2, 0.3, 0.4, 0.5 or 1.0, or about 0.01-0.1, 0.01- 0.2, 0.01-0.3, 0.01-0.4, 0.01-0.5 or 0.01-1.0. For example, the LNPs have a particle size less than about 200 nm and a polydispersity index of less than about 0.3. At least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 0.1-10%, 0.1-20%, 0.1-30%, 0.1-40%, 0.1-50%, 0.1-60%, 0.1-70%, 0.1- 80%, 0.1-90%, 0.1-95%, 0.1-99% or 0.1-100% of the one or more target agents may be encapsulated by the LNPs. Where the one or more target agents are negatively charged, for example, nucleic acids, the LNPs may have an encapsulation rate greater than about 70%, 80% or 90%, or about 0.1-70%, 0.1-80%, 0.1-90%, 0.1-95%, 0.1-99% or 0.1-100%. Where the one or more target agents are hydrophobic, the LNPs may have an encapsulation rate greater than about 40%, 50%, 60%, 70%, 80% or 90%, or about 0.1- 40%, 0.1-50%, 0.1-60%, 0.1-70%, 0.1-80%, 0.1-90%, 0.1-95%, 0.1-99% or 0.1-100%. The ionizable lipid has a hydrophobic tail and a hydrophilic head. The ionizable lipid may form a lipid membrane. The ionizable lipid may have a hydrophobic tail and a hydrophilic head containing an amine group capable of gaining or losing a proton in a pH- dependent environment. For instance, at a neutral pH, the head group remains neutral; however, in an acidic environment, the head group becomes protonated. Therefore, an ionizable lipid may facilitate escape of a loaded cargo from an endosome, in which a lower endosomal pH enables the ionizable lipid to become positively charged and disrupt the negatively charged endosomal membrane. Examples of ionizable lipids include SM102, ALC-0315, DLin-MC3-DMA, C12-200, cKK-E12, C14-4, L-319, YSK12-C4, and DLin-KC2- DMA. The cationic lipid has a hydrophobic tail and a hydrophilic head. The cationic lipid may form a lipid membrane. The cationic lipid may have a hydrophilic head group whose 0074539-000157 -15- charge does not change depending on an pH. Examples of cationic lipids include 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-[1-(2,3- Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), dimethyldioctadecylammonium (bromide) (DODAB), and 1,2-stearoyl-3- trimethylammoniumpropane (chloride) (DSTAP). The lipid mixture may further comprise one or more additional lipids. The lipid mixture may further comprise a structural lipid, a helper phospholipid and / or a PEG lipid. The PEG lipid may be replaced by another amphiphilic lipid. The structural lipid may be an amphiphilic lipid. The helper phospholipid may be an amphiphilic lipid. The lipid mixture may further comprise an additional amphiphilic lipid. The structural lipid provides rigidity, fluidity, and strength to the lipid membranes in the LNPs. The structural lipid maintains the overall structure of an LNP. The structural lipid may form a lipid membrane. Examples of a structural lipid include cholesterol, cholesterol- thiol, β-sitosterol, 3β-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC- Cholesterol), cholesterol hemisuccinate (CHEMS), and cholesteryl hexaethylene glycol (CHEG). The helper phospholipid aids in the stability of LNPs and enhances the delivery efficiency of encapsulated target agents. The helper phospholipid may have a high phase transition temperature such that it enhances the membrane stability of the LNP. Examples of the helper lipids include 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), 1,2-Distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-Dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), 1,2-Dipalmitoyl- sn-glycero-3-phospho-L-serine (DPPA), sphingomyelin, oleic acid, palmitic acid, distearoylphosphatidylcholine (DSPC), and dipalmitoylphosphatidylcholine (DPPC). The polyethylene glycol (PEG) lipid is a PEG derivative containing lipid. The PEG lipid may provide an amphiphilic protective coating to an LNP surface and prevent rapid clearance of the LNP by the immune system in a subject. Additionally, a PEG lipid may reduce nanoparticle aggregation. Examples of the PEG lipids include of 1,2-distearoyl-sn- glycero-3-phosphoethanolamine with conjugated methoxyl poly(ethylene glycol) (DSPE- mPEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG2000), DSPE-PEG2000-mannose, DSPE-PEG2000-maleimide, DSPE- PEG2000-thiol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG- PEG2000), DMG-PEG2000-maleimide, DMG-PEG2000-thiol, and 1,2-Dioleoyl-sn-glycero- 3-phosphoethanolamine-poly(ethylene glycol) (DOPE-PEG) and 0074539-000157 -16- α-[2-(ditetradecylamino)-2-oxoethyl]-ω- poly(oxy-1,2-ethanediyl) (ALC-0159). In case of DSPE-PEG, it may integrate into lipid bilayers, where the DSPE part embeds into the lipid core, and the PEG chain provides steric stabilization. The amphiphile may be selected from the group consisting of D-α-tocopheryl polyethylene glycol succinate (Vitamin E TPGS) and 1,2-dimyristoyl-sn-glycero-3-succinyl- N-polysarcosine (DMG-pSar). Each target agent may be selected from the group consisting of a nucleic acid, an adjuvant, and a hydrophobic drug. A hydrophobic drug may have a molecular weight of less than 5000 Da. The one or more target agents may be a combination of two adjuvants, or a combination of one adjuvant and a nucleic acid. The nucleic acid may be a plasmid, siRNA, DNA or mRNA. The nucleic acid may be miRNA or circular RNA (circRNA), guide RNA (gRNA), single guide RNA (sgRNA), or Antisense oligonucleotides (ASOs). For example, an mRNA may be encapsulated by LNPs comprising an ionizable lipid such as SM102, ALC-0315 or DLin-MC3-DMA. An siRNA may be encapsulated by LNPs comprising an ionizable lipid such as DLin-MC3-DMA. An adjuvant may be a substance added to a vaccine to stimulate and increase the magnitude and durability of the immune response. Adjuvants can boost immunization effectiveness by acting as agonists for pattern-recognition receptors (PRRs), which stimulate innate immune cells. These receptors are strategically located within the cell. They can be present on the cell surface to identify extracellular pathogens like bacteria or fungi, in the endosomes to detect intracellular intruders like viruses, and finally in the cytoplasm to detect intracellular invaders like viruses. The toll-like receptor (TLR) is the most commonly researched adjuvant target among pattern recognition receptors that sense danger signals. The activation of intracellular toll-like receptors (TLRs) TLR7, TLR8, TLR9, and TLR4 found in lysosomes, as well as NLRs and stimulator of interferon genes (STING), found in the cytoplasm of antigen-presenting cells (APCs), is considered a challenging task. This is because adjuvants should reach APC intracellular compartments at activatable quantities without degradation after in vivo administration. For example, CpG oligonucleotides, which activate the TLR9 receptor in the endosome are often prone to degradation due to harsh in vivo conditions. Furthermore, adjuvants' variable physicochemical properties and pharmacokinetics limit their delivery to intracellular compartments. These issues can be addressed by adopting nanoparticulate delivery technologies, which not only enhance adjuvant solubility but also improves pharmacokinetic properties and cellular delivery of adjuvants. APCs frequently phagocytose nanoparticles, localizing in the endolysosome and releasing adjuvant 0074539-000157 -17- payloads in both endosomes and the Furthermore, nanoparticles allow simultaneous encapsulation and cellular delivery of two or more adjuvants. The adjuvant may be selected from the group consisting of imiquimod (TLR7 adjuvant), imidazoquinoline (resiquimod; TLR7 / 8 adjuvant), CL075 (human TLR8 adjuvant), CpG ODN (1018, 2216, 2395, 2006, 2007, 2336, D-SL01 (TLR9 adjuvants), QS- 21 (NOD agonist), cyclic [G(2’,5’)pA(3’,5’)p] (2’3’-cGAMP) (a cyclic dinucleotide (CDN); STING agonist), CL429 (TLR2 / NOD2 agonist), CL347 (TLR7 agonist), DMXAA (STING ligand), ADU-S100 (STING ligand), and trehalose-6,6-dibehenate (TDB) (Mincle agonist). The hydrophobic drug may be selected from the group consisting of aldoroxubicin, monomethyl auristatin E (MMAE), daunorubicin, doxorubicin, irinotecan, cytarabine, paclitaxel, amphotericin B, rapamycin, imiquimod, and resiquimod. In one embodiment, the target agent comprises a nucleic acid vaccine (e.g., mRNA vaccine) and an adjuvant. An LNP may have a positive or negative surface charge depending on the lipid components in the lipid membrane and the nature of the target agent. For example, a LNP formulated with a cationic lipid such as DOTAP may have a positive surface charge while a LNP formulated with an ionizable lipid may be mostly neutral at pH 7.4. Where the target agent is a vaccine formulated with a peptide or protein subunits, the resulting LNP may have a different charge depending on the nature of the amino acids in the target agent and the environmental pH. The organic solvents are often used individually. Nevertheless, in certain situations, to dissolve a drug or the adjuvant, a mixer of water-miscible solvents mentioned above may be employed. The organic solvent may be selected from the group consisting of methanol, ethanol, tetrahydrofuran (THF), and a combination thereof. The organic solvent may be a mixture of ethanol and methanol at a ratio, for example, from 50:50 to 90:10, a mixture of methanol, ethanol and tetrahydrofuran (THF) at a ratio, for example, from 2:2:1 to 4:4:1, a mixture of ethanol and THF at a ratio, for example, from 50:50 to 90:10, or a mixture of methanol and THF at a ratio, for example, from 50:50 to 90:10. The aqueous solvent may be any aqueous solution or buffer. The aqueous solvent may be 5-50 mM sodium citrate buffer (pH 4-5), 10-100 mM sodium acetate buffer (pH 4- 5), 0.9% normal saline (NaCl) (pH 7.4), water for injection (WFI), phosphate buffered saline (PBS) (pH 7.4), HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), and MES Buffer (2-(N-morpholino)ethanesulfonic acid)). A pH of 4-5 is critical for encapsulating nucleic acids in the presence of ionizable lipid. Ionizable lipids become cationic at low pH 0074539-000157 -18- aqueous solvent thereby allowing them to interact with negatively charged nucleic acid. The formulation method may further comprise lyophilizing the LNPs, whereby a powder of LNPs is obtained. The LNP powder may comprise a cryoprotectant (e.g., sucrose, mannitol and trehalose) at a concentration of about 5-15% or 10% w / v. In one embodiment, the first step of lyophilization of the LNPs includes freezing of the LNPs at -20oC over 2 h in presence of a cryoprotectant followed by lyophilization using a lyophilization apparatus. In the lyophilization apparatus, freezing may result in the formation of ice, which may cause the destruction of LNPs. However, the presence of a cryoprotectant prevents the growth of ice crystals and protects the LNPs by forming a glassy matrix around the nanoparticles. During the primary drying step at a low pressure, the ice sublimes, leaving the intact structure of the LNPs in the presence of the cryoprotectant. Then, during the secondary drying phase, any remaining water is removed through desorption. In essence, the cryoprotectant forms a protective layer around the LNPs, which helps maintain their structural integrity. The LNPs may remain functionally active. The formulation method may further comprise rehydrating the LNP powder with a third aqueous solvent so that an aqueous solution of the LNPs is obtained. The resulting LNPs may remain functionally active. The third aqueous solvent may be the same as or different from the first or second aqueous solvent. The formulation method may further comprise interacting the LNPs with cells, and delivering the LNPs into the cells such that the one or more target agents are in the cells. The one or more target agents in the cells may be biologically active. The LNPs may be delivered into an intracellular compartment (e.g., intracellular endosomal compartment or cellular lysosomes) in the cells. The formulation method may further comprise releasing at least one of the one or more target agents from the LNPs in the cells, for example, released into cytosol, mitochondria or cellular lysosomes. The at least one released target agent may be biologically active. Upon delivery of the LNPs into the cells, the formulation method may further comprise stimulating an immune receptor in or on the cells. The cells may be in a subject, the LNPs may be delivered into a lymphoid organ (e.g., lymph node or spleen) in the subject. The method may further comprise stimulating an immune response (e.g., cellular or humoral response) in the subject. 0074539-000157 -19- The present invention provides the (LNPs) produced in accordance with the method of the present invention. A composition comprising the LNPs is provided. The LNPs may be in an amount effective for stimulating an immune response (e.g., cellular or humoral response) in a subject or for stimulating an immune cell specific receptor. The present invention provides a lipid nanoparticle (LNP) comprising two or more target agents, a lipid membrane, and a space enclosed by the lipid membrane. The lipid membrane is formed by a mixture of lipids. The mixture of lipids comprises an ionizable or cationic lipid. The two or more target agents are negatively charged or hydrophobic. Each target agent is selected from the group consisting of a nucleic acid, an adjuvant, and a hydrophobic drug. The hydrophobic drug may have a molecular weight of less than 5000 Da. The two or more target agents may be a combination of two adjuvants, or a combination of one adjuvant and a nucleic acid. The ionizable lipid may selected from the group consisting of SM102, ALC-0315, DLin-MC3-DMA, C12-200, cKK-E12, C14-4, L-319, YSK12-C4, and DLin-KC2-DMA. The cationic lipid may be selected from the group consisting of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-[1-(2,3- Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), dimethyldioctadecylammonium (bromide) (DODAB), and 1,2-stearoyl-3- trimethylammoniumpropane (chloride) (DSTAP). Also provided is a composition comprising a plurality of these LNPs. The LNPs may have a particle size less than about 10, 20, 50, 100, 200, 300, 400, 500 or 100 nm, or about 1-1000, 1-500, 1-400, 1-300, 1-200, 1-100 or 1-10 nm, and / or a polydispersity index of less than about 0.1, 0.2, 0.3, 0.4, 0.5 or 1.0, or about 0.01-0.1, 0.01-0.2, 0.01-0.3, 0.01-0.4, 0.01-0.5 or 0.01-1.0. For example, the LNPs may have a particle size less than about 200 nm and a polydispersity index of less than about 0.3. For the LNPs of the present invention, a method for intracellular delivery. The method is provided. The method comprises interacting the LNPs with cells, and delivering the LNPs into the cells such that at least one of the target agents is in the cells. The at last one target agent in the cells may be biologically active. The LNPs may be delivered into an intracellular compartment such as endosomal compartment or cellular lysosomes. The method may further comprise releasing the at least one target agent in the cells from the LNPs. The at least one released target agent may be biologically active. The target agent may be released into cytosol, mitochondria or cellular lysosomes. The method may further comprise stimulating an immune cell specific receptor in or on the cells. The cells may be in a subject. The LNPs may be delivered into a lymphoid organ (e.g., lymph node or 0074539-000157 -20- spleen) in the subject. The method may stimulating an immune response (e.g., cellular or humoral response) in the subject. The present invention provides a method for stimulating an immune response (e.g., cellular or humoral response). The method comprises administering to a subject an effective amount of the LNPs of the present invention. The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate. Example 1. Fabrication and physicochemical characterization of lipid nanoparticles In this study, LNPs were prepared by the Flash Nanoprecipitation (FNP) technique using a confined impingement jet (CIJ) mixer in accordance with the schematic illustration in FIG.1A. Lipids were dissolved in the organic phase (ethanol), and the nucleic acids, such as mRNA / siRNA were dissolved in the aqueous phase, 50 mM sodium citrate buffer (pH 4). The aqueous and organic phases were impinged against each other and collected in a reservoir containing 2 mL of sodium citrate buffer pH 4. Ethanol was removed from the formulation by dialyzing against PBS buffer (pH 7.4) using a 10 kDa dialysis membrane overnight. The LNP was composed of a helper phospholipid, PEG lipid, and structural lipid Cholesterol. Nucleic acids are entrapped with the help of the ionizable lipid. The SM102, ALC0315, and DLin-MC3-DMA ionizable LNPs were characterized (FIGS.1B-E). This study demonstrates that Flash nanoprecipitation method can be used to prepare ionizable LNPs encapsulating nucleic acids such as mRNA or siRNA. These LNPs are stable and spherical in morphology, which are critical characteristics for a nanoparticle platform. Example 2. Cellular uptake of ionizable lipid nanoparticles by RAW 264.7 macrophages The cellular update of ionizable lipid nanoparticles by RAW 264.7 macrophages was studied. A model small molecule fluorophore, DiD dye, was loaded into LNPs, and cellular uptake was measured using flow cytometry and represented as Median Fluorescent intensity (MFI). FIG.2A shows time-dependent cellular uptake of SM102, ALC0315, and DLin-MC3-DMA LNPs after 2, 6, and 24 h of incubation with RAW 264.7 cells. Data represented as mean ± SD (n=4). Significant differences between each timepoint were determined by ANOVA with Tukey’s multiple comparison tests, **p = 0.0029, ****p < 0.0001. An overlayed confocal image was obtained after 24 hours of incubation of DiD- loaded LNPs with RAW 264.7 cells to show intracellular localization of LNPs in RAW 264.7 0074539-000157 -21- cells. Live-cell images were taken using a microscope with a 60X objective and DiD uptake inside the cells, as indicated by black arrows (FIG.2B). The scale bar is 10 μm. This study shows the successful encapsulation of a small molecule hydrophobic dye – DiD indicating the LNPs can be used to encapsulate hydrophobic drugs. Confocal and flow cytometry studies showed that the LNPs are successfully taken up by the immune cells and released small molecule DiD inside cells. This finding is a critical aspect for modulating immune responses in vaccine and drug delivery. Example 3. Intracellular delivery of mRNA using LNPs in DC 2.4 dendritic cells Intracellular delivery of mRNA using LNPs in DC 2.4 dendritic cells SM102 and ALC0315 was studied. LNPs were encapsulated with mCherry mRNA and incubated for 24 h with DC 2.4 cells. mCherry mRNA encoded into mCherry protein indicates successful cytoplasmic delivery of mRNA by LNPs. Live-cell images were taken using a confocal microscope with a 60X objective and punctae inside the cells indicated by black arrows (FIG.3A). The scale bar is 10 μm. The functional SIINFEKL assay is illustrated in FIG.3B. DC 2.4 cells were treated with OVA-mRNA loaded SM102 and ALC0315 lipid nanoparticles or OVA peptide as control for 24 h, the dose of the OVA-mRNA was 1 μg / mL in each well. The expression level of the H-2kb antibody-bound OVA257-264 (SIINFEKL) was reported (FIG.3C). Data represented as mean ± SD (n=3). Significant differences between each timepoint were determined by one-way ANOVA with Tukey’s multiple comparison test, ****p < 0.0001, **p = 0.002, *p = 0.0121 for ALC0315 LNPs and no treatment, and *p = 0.0214 for SM102 and ALC0315 LNP formulations. The findings of this study demonstrate that mRNA vaccines encapsulated inside LNPs can be efficiently delivered to immune cells with high precision. This finding shows that mRNA LNPs formulated using the FNP method are functional in vitro Example 4. In vivo delivery of mRNA using LNPs in C57BL / 6 mice In vivo delivery of mRNA using LNPs in C57BL / 6 mice was studied. FIG.4A illustrates firefly luciferase mRNA loaded LNPs, route of administration, and IVIS imaging of lymphoid organs. FIG.4B shows IVIS bioluminescent images of draining inguinal lymph nodes and the spleens of SM102 LNPs or ALC0315 LNPs or PBS only administered mice. FIG.4C shows bioluminescence represented as average radiance in draining lymph nodes and spleens of mice administered with SM102 and ALC0315 LNPs. Data represented as mean ± SD (n=3). The experimental results of this study show successful delivery of mRNA encapsulated LNPs into lymphoid organs, lymph nodes, and spleen. Additionally, the 0074539-000157 -22- mRNA is functionally active in vivo. This mRNA LNPs formulated using the FNP method can be used for vaccine delivery. Example 5. In vivo cellular uptake of DiD dye-loaded LNP in C57BL / 6 mice In vivo cellular uptake of DiD dye-loaded LNP in C57BL / 6 mice was studied. FIG. 5A is a schematic representation of DiD-loaded LNPs, route of administration, lymphoid organ harvesting, and flow cytometry analysis of the cells from the harvested organs. B) Heatmap of the DiD-loaded LNPs positive immune cells (CD 45+) and non-immune cells (CD45-) obtained from draining lymph nodes. C) Heatmap of the DiD loaded LNPs positive immune cells (CD 45+) and non-immune cells (CD45-) obtained from spleen. Data represented as mean ± SD (n=3). This study shows that LNPs formulated by the FNP method are taken up by immune cells in the lymphoid organs. This indicates that LNPs can be utilized to deliver drugs or vaccines to immune cells of interest. Example 6. Intracellular delivery of siRNA using DLin-MC3-DMA LNPs Intracellular delivery of siRNA using DLin-MC3-DMA LNPs was studied. Intracellular localization of Cy5-siRNA in RAW 264.7 cells and DC 2.4 cells is shown in FIG.6A. An overlayed confocal image was obtained after 24 h of incubation of the Cy5- siRNA-loaded LNPs with RAW 264.7 or DC 2.4 cells. Live-cell images were taken using a confocal microscope with a 60X objective and punctae (Cy5-siRNA) inside the cells, as indicated by black arrows. The scale bar is 10 μm. FIG.6B shows schematically the siRNA-LNPs gene silencing functional assay through green fluorescent protein (GFP)-- expressing MDA-MB-231 / Luc cells. GFP signal is represented as median fluorescent intensity (MFI) measured using flow cytometry (FIG.6C). GFP expressing MDA-MB- 231 / Luc cells were incubated with GFP siRNA LNPs or control siRNA LNPs for 48 h before measuring the MFI. Significant differences were determined using one-way ANOVA with Tukey's multiple comparisons test, **p = 0.0027 and ***p = 0.0001. Data are represented as mean ± SD (n=4). Additionally, D) Percentage of knockdown efficiency was plotted against the control siRNA. Significant differences were determined using unpaired t-test with Welch’s correction, *p = 0.0104. Data are represented as mean ± SD (n=4). The image was created with BioRender.com. This study shows that FNP method can be utilized to encapsulate siRNA therapeutics inside LNPs. siRNA therapeutics precisely delivered using LNPs are functionally active. Example 7. Fabrication of ionizable LNPs Ionizable LNPs were prepared. Table 1 shows the molar ratios of different lipid components used for the LNP fabrication through the FNP process. Table 1. LNP Formulations 0074539-000157 -23- Apparent pKa of the ionizable LNPs was measured by TNS (6-p-Toluidino-2- naphthalenesulfonic acid) assay. The pKa values of the LNPs were determined by extrapolating the pH value with 50% of the maximum normalized TNS fluorescent intensity (FIG.7). Data represented as mean ± SD (n=3). Example 8. Stability of LNPs The stability of LNPs prepared through flash nanoprecipitation were stored at 4 °C. Particle size of the LNPs was measured over 28 days using dynamic light scattering, and the hydrodynamic diameter is reported as Z average (nm) (FIG.8A). Zeta potential of LNPs was measured over 28 days at different time intervals by electrophoretic light scattering, and the zeta potential is reported as millivolt (mV) (FIG.8B). Polydispersity index (PDI) was measured over 28 days using dynamic light scattering (FIG.8C). Data represented as mean ± SD (n=3). This study shows that the LNPs prepared using the FNP method are stable for 30 days when stored at 4 °C. Example 9. Selection of water-miscible organic solvents for LNP fabrication via Flash nanoprecipitation (FNP) Water-miscible organic solvents were selected for LNP fabrication via Flash nanoprecipitation (FNP). Solubility of the lipid mixture was tested in 1 mL of different organic solvents, only methanol and ethanol were able to dissolve the lipid components, making them suitable solvents for FNP (FIG.9A). FIG.9B shows a table depicting the particle size, polydispersity index, and zeta potential of LNPs fabricated using methanol as the organic solvent. Data represented as mean ± SD (n=3). Only methanol and ethanol can be used as organic solvents to formulate LNPs. Example 10. Comparison of LNPs SM102, ALC0315, and DLin-MC3-DMA LNPs were prepared through ethanol injection and flash nanoprecipitation method. mRNA was used for formulations with SM102 and ALC0315 ionizable LNPs and siRNA was used for DLin-MC3-DMA ionizable LNPs. The particle size, polydispersity index (PDI), zeta potential, and encapsulation efficiency of the LNPs were shown in Table 2. Table 2. Particle sizes of LNPs 0074539-000157 -24- Table 2 shows a comparison of conventional ethanol injection and the FNP method to prepare LNPs. The Encapsulation efficiency of the RNA is higher in the FNP method compared to the ethanol injection method. The particle size of LNPs produced using the FNP method is consistent and reproducible. Example 11. Characterization of LNPs prepared with cationic lipid (DOTAP) and lipidoid (NT1-O14B) LNPs were prepared with cationic lipid (DOTAP) and lipidoid (NT1-O14B). Positive stained TEM images for DOTAP containing LNPs are shown in FIG.10A. TEM images scale bar is T100 nm. Particle size of LNPs was measured using dynamic light scattering, and the number average is reported as d.nm. to compare the particle size distribution of NT1-O14B and DOTAP formulations (FIG.10B). The polydispersity index (PDI) was calculated based on Dynamic light scattering (FIG.10C). Zeta potential was measured by electrophoretic light scattering (FIG.10D). FIGS.10B, 10C and 10D are represented as mean ± SD (n=3). This study shows that the lipidoid and the cationic LNPs can be prepared through the FNP technique. Lipidoids are cationic and synthetically prepared lipids, which can be used to entrap nucleic acids and have shown promising results in delivering cargo across the brain recently. Example 12. Formulations of LNPs encapsulated with CpG ODN, A hydrophilic adjuvant targeting TLR9 receptor CpG oligodeoxynucleotides (CpG ODN) are synthetic DNA containing the unmethylated CpG motifs which get recognized by the immune system as pathogen- associated molecular patterns (PAMPs). Hence, CpG ODN can act as an adjuvant to enhance the body’s immune response. Mechanistically, the CpG Adjuvants activate the immune system through Toll-like receptor 9 (TLR9), present in the intracellular endosomal compartment of the immune cells. Upon binding to the receptors, the CpG ODN triggers a signalling cascade leading to the production of different cytokines and chemokines associated with the immune response. Here we showed that the FNP method can be used to encapsulate CpG ODN (specific for humans and mice) into LNP formulations (Table 3). The LNP formulations precisely released CpG ODN inside immune cells and stimulated them. This indicates the 0074539-000157 -25- potential of delivering water-soluble DNA adjuvants into the intracellular compartments of the immune cells. It is important to note that CpG ODN are expensive and demonstrate poor stability in vivo requiring higher doses to administer to stimulate an immune response. Encapsulating CpG ODN adjuvants inside LNPs can effectively stimulate an immune response at lower doses than the free CpG ODN. Table 3. CpG ODN adjuvanted LNP formulations CpG ODN 2395 encapsulation into ionizable and cationic lipid LNPs did not change the physicochemical characteristics of LNPs (FIGS.11 and 12). Ionizable LNPs demonstrated greater than 90% encapsulation efficiency of CpG ODN adjuvants (FIG.13). The LNPs containing CpG ODN precisely stimulated TLR9 receptors present on the RAW Blue cells, which resulted in the activation of NF-ĸB pathway (FIGS.14A-B). The activation of NF-ĸB pathway leads to the transcriptional activation of the SEAP reporter gene, which in turn translates to the SEAP enzyme secreted in the cytoplasm. The enzyme can be detected through SEAP colorimetric assay. The HEK blue assay data shows successful intracellular delivery of CpG ODN- loaded LNPs to the human TLR9-specific cells (FIG.15). Successful cellular uptake and intracellular delivery of CpG ODN by SM102 and ALC0315 LNPs were observed (FIGS.16A-B). Example 13. Lyophilization of LNPs LNPs were tested in a lyophilization study and immune cell activation assay The SM102, ALC0315, and DOTAP LNPs were lyophilized with 10% w / v sucrose as a cryoprotectant. The samples were kept at 4oC till day 30 and thereafter stored at - 20 °C till day 180. The LNPs were mostly stable and particle size did not increase beyond 200 nm (FIG.17). The data are presented as standard deviation (SD) ± 3. 0074539-000157 -26- CpG ODN loaded ALC0315 LNPs with 10% w / v sucrose as a cryoprotectant. The particle size of the LNPs was measured by the dynamic light scattering (FIG.18A). In an immune cell activation study of the lyophilized LNPs, human HEK blue TLR9 cells were treated with lyophilized CpG 2006 loaded samples for 48 h, and then the immune cell activation was measured by HEK-BlueTMdetection reagent (FIG.18B). The data were presented as standard deviation (SD) ± 3. CpG ODN loaded ALC0315 LNPs were lyophilized with 10% w / v sucrose as a cryoprotectant. The particle size of the LNPs was measured by the dynamic light scattering (FIG.19A). In an immune cell activation study of the lyophilized LNPs, human HEK blue TLR9 cells were treated with lyophilized CpG 2006 loaded samples for 48 h, and then the immune cell activation was measured by HEK-BlueTMdetection reagent (FIG.19B). The data are presented as standard deviation (SD) ± 3. The data in this study shows that the adjuvanted LNPs can be lyophilized in the presence of sucrose as a cryoprotectant and can maintain the functional activity of the adjuvant. Example 14. LNPs encapsulated with CL429, a hydrophobic adjuvant Targeting TLR2 / NOD2 receptors CL429 is a hydrophobic adjuvant that targets Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2). CL429 is composed of murabutide (NOD2 ligand) covalently linked to Pam2C (TLR2 ligand). A formulation of CL429 LNPs was prepared for an immune cell activation assay through SEAP activity. SM102 was utilized as an ionizable lipid to formulate LNPs. Quanti- blue reporter gene assay was performed using the RAW Blue cells. Briefly, the RAW blue cells were treated with the CL429 loaded LNPs at different concentrations for 24 h and 48 h. As a control LPS and Non-treated cells were used. After 24 h and 48 h, QUANTI-Blue™ reagent was used to measure the SEAP activity of the cells in the presence of CL429 (FIG. 20). The data are presented as standard deviation (SD) ± 3. The data of this study shows the LNPs can encapsulate hydrophobic adjuvant CL429, and successfully TLR2 / NOD2 receptors to trigger the immune response. Example 15. MPLA- Hydrophobic adjuvant Targeting TLR4 receptors MPLA (Monophosphoryl Lipid A) is a hydrophobic adjuvant derived from the lipopolysaccharide (LPS) of the bacterial cell wall. MPLA stimulates the innate immune system by triggering the Toll-like receptor (TLR4) leading to the secretion of cytokines. MPLA is a component of marketed adjuvant and is used as an AS04 adjuvant system for HPV (human papillomavirus) vaccine Cervarix. 0074539-000157 -27- 50% SM102 ionizable was used to MPLA-encapsulated LNPs. The particle size and polydispersity index were measured using dynamic light scattering and electrophoretic light scattering was used to measure the zeta potential (FIG.21A). A SEAP study was used to check the immune cell response to the 50% SM102 loaded MPLA LNPs (FIG.21B). RAW Blue cells were treated with the MPLA loaded SM102 LNPs for 24 and 48 h. After that the immune cell activation was measured by QUANTI-Blue™ reagent. The data are presented as standard deviation (SD) ± 3. The study shows that ionizable LNPs can encapsulate MPLA (a hydrophobic TLR4 adjuvant) and can deliver the adjuvant more efficiently into cells than an unencapsulated MPLA. Example 16. Dual adjuvanted LNPs Dual adjuvanted LNPs were prepared for CpG ODN- MPLA- (Hydrophilic- Hydrophobic) dual adjuvant delivery. Particle size of SM102 loaded CpG 2395 and MPLA was measured by dynamic light scattering and zeta potential was measured by electrophoretic light scattering (FIG. 22A). A SEAP study was used to check the immune cell response. RAW Blue cells were treated with the CpG ODN 2395 or CpG ODN 2395-MPLA or MPLA-loaded SM102 LNPs for 24 and 48 h. After that, the immune cell activation was measured by QUANTI-Blue™ reagent (FIG.22B). The data are presented as standard deviation (SD) ± 3. Surface marker expression of mouse BMDCs following adjuvant LNP treatment was studied.6-week-old male C57BL / 6 mice were sacrificed and bone marrow was harvested to collect dendritic cells (BMDCs). These cells were treated with CpG ODN 2395, MPLA, CpG-MPLA loaded LNPs and CpG LNPs + MPLA LNPs mixed at different concentrations. Cells were incubated for 24 h after that flow cytometry assay was performed to check the immune cell surface marker expressions A) CD40, where Significant differences between each timepoint were determined by one-way ANOVA with Tukey’s multiple comparison test. ***P = 0.0007 for SM102 CpG vs. SM102 CpG-MPLA 5 μg / mL, ***P = 0.0006 for SM102 CpG vs. MPLA LNPs + CpG LNPs Mix, ****P < 0.0001 for SM102 CpG-MPLA 10 μg / mL vs. SM102 CpG-MPLA 5 μg / mL, *P = 0.0261 for SM102 CpG-MPLA 10 μg / mL vs. MPLA 10 μg / mL, *P = 0.0314 for SM102 CpG-MPLA 10 μg / mL vs. MPLA LNPs + CpG LNPs Mix, **P = 0.0100 for SM102 CpG-MPLA 5 μg / mL vs. MPLA 10 μg / mL, ****P < 0.0001 for SM102 CpG-MPLA 5 μg / mL vs. MPLA LNPs + CpG LNPs Mix, and ****P < 0.0001 for MPLA 10 μg / mL vs. MPLA LNPs + CpG LNPs Mix. B) CD80, where Significant differences between each timepoint were determined by one-way ANOVA with Tukey’s multiple comparison test. ***P = 0.0004 for SM102 CpG vs. SM102 CpG-MPLA 10 μg / mL, 0074539-000157 -28- ****P < 0.0001 for SM102 CpG vs. MPLA CpG LNPs Mix, ****P < 0.0001 for SM102 CpG-MPLA 10 μg / mL vs. SM102 CpG-MPLA 5 μg / mL, *P = 0.0118 for SM102 CpG-MPLA 10 μg / mL vs. MPLA 10 μg / mL, **P = 0.0100 for SM102 CpG-MPLA 10 μg / mL vs. MPLA LNPs + CpG LNPs for SM102 CpG-MPLA 5 μg / mL vs. MPLA 10 μg / mL, ****P < 0.0001 for 5 μg / mL vs. MPLA LNPs + CpG LNPs Mix, and ****P < 0.0001 for MPLA 10 μg / mL vs. MPLA LNPs + CpG LNPs Mix. C) CD86, where Significant differences between each timepoint were determined by one-way ANOVA with Tukey’s multiple comparison test. *P = 0.0427 for SM102 CpG vs. SM102 CpG-MPLA 10 μg / mL, ****P < 0.0001 for SM102 CpG vs. SM102 CpG-MPLA 5 μg / mL, ***P = 0.0002 for SM102 CpG vs. MPLA LNPs + CpG LNPs Mix, ****P < 0.0001 for SM102 CpG-MPLA 10 μg / mL vs. SM102 CpG-MPLA 5 μg / mL, ****P < 0.0001 for SM102 CpG- MPLA 5 μg / mL vs. MPLA 10 μg / mL, ****P < 0.0001 for SM102 CpG-MPLA 5 μg / mL vs. MPLA LNPs + CpG LNPs Mix, and **P = 0.0018 for MPLA 10 μg / mL vs. MPLA LNPs + CpG LNPs Mix. The data are presented as standard deviation (SD) ± 4. This data in this study shows that ionizable LNPs can entrap both hydrophilic CpG ODN and hydrophobic MPLA concurrently and allow the synchronous release of two adjuvants inside immune cells. Moreover, Dual adjuvanted LNPs showed enhanced immune response compared to the single adjuvanted LNPs. Example 17. Ionizable LNPs for adjuvanted mRNA vaccines CL347 (Adifectin) is a hydrophobic adjuvant that targets the TLR 7 receptor inside the endosome to trigger the immune response. mRNA vaccine is co-encapsulated with CL347 inside LNPs to efficiently stimulate immune cells and concurrently deliver mRNA vaccines inside immune cells. CL347 LNPs with and without an ionizable lipid were characterized. The particle size of the LNPs was measured using dynamic light scattering and the number average and reported as d.nm (FIG.24A). Negative stained TEM images of CL347 LNPs and CL347-SM-102 LNPs encapsulated with mRNA are reported along with it. The scale is 100 nm. Encapsulation efficiency of mCherry mRNA in CL347 LNPs and CL347-SM102 LNPs was measured by QuantiFluor® RNA System (FIG.24B). Data represented as mean ± SD (n=4). The intracellular localization of CL347 LNPs in RAW 264.7 macrophages was observed. An overlayed confocal image was obtained after 24 hours of incubation of DiD- loaded CL347 and CL347-SM102 LNPs with RAW 264.7 cells. CL347-SM102 LNPs showed more uptake (FIG.25A) compared to the CL347 LNPs (FIG.25B). Live-cell images were taken using a confocal microscope with a 60X objective. DiD uptake inside the cells is 0074539-000157 -29- indicated as the red color and nuclei stain is as the blue color. Scale bar 10 μm. The intracellular delivery of mCherry mRNA in RAW 264.7 macrophages was observed. mCherry mRNA- loaded LNPs were successfully encoded into mCherry protein. CL347 mCherry mRNA LNPs was unable to transfect cells and no mCherry protein signal was evident (FIG.26A). CL347 with SM102 LNPs efficiently transfected cells and bright red fluorescence was indicative of mCherry protein (FIG.26B). Live-cell images were taken using a confocal microscope with a 60X objective. The scale bar is 10 μm. CL347 LNPs were characterized in an in vitro functional assay. Secreted embryonic alkaline phosphatase (SEAP) activity of CL347 LNPs was measured after 48 hours of treatment with TLR7 specific hHEK-Blue™ Cells (FIG.27A). Y-axis indicates absorption intensity at 640 nm, directly proportional to the SEAP activity of the samples containing CL347 in LNPs and CL347 control (5 μg / mL). Imiquimod only (5 μg / mL) and PBS were used as a positive and negative control, respectively. Data represented as mean ± SD (n=3). Significant differences between each timepoint were determined by one-way ANOVA with Tukey’s multiple comparison test, ****p < 0.0001. DC2.4 cells were treated with OVA-mRNA loaded CL347-SM102 LNPs or CL347 admixed with OVA mRNA for 24 h, the dose of OVA-mRNA was 1 μg / mL in each well. The expression level of the H-2kb antibody-bound OVA257-264 (SIINFEKL) peptide complex was measured by flow cytometry and the percentage of H-2kb antibody-bound SIINFEKL was reported (FIG. 27B). Data represented as mean ± SD (n=3). Significant differences between each timepoint were determined by one-way ANOVA with Tukey’s multiple comparison test, ***p = 0.0008, ***p = 0.0003, and *p = 0.0351. This study shows successful concurrent encapsulation of CL347 adjuvant and mRNA into LNPs, which is demonstrated by no change in the morphological characteristics of LNPs. These adjuvanted mRNA LNPs are efficiently taken up by immune cells and delivered mRNA inside immune cells. CL347 encapsulated LNPs stimulate TLR7 receptors inside immune cells and demonstrated functional mRNA activity in vitro. Example 18. Delivery of mRNA vaccine to lymphoid organs CL347 lipid nanoparticles were used for in vivo delivery of mRNA in C57BL / 6 mice. FIG.28A illustrates the firefly luciferase mRNA loaded LNPs, route of administration, and IVIS imaging of lymphoid organs. FIG.28B shows IVIS bioluminescent images of axillary, brachial, and inguinal lymph nodes and the spleens of SM102-CL347 mRNA LNPs, only CL347 admixed with mRNA, and PBS control administered mice at mRNA concentration of 4 μg / mice and CL347 concentration of 12 μg / mice. The bioluminescence is represented as 0074539-000157 -30- average radiance in spleens of mice with CL347-SM102 LNPs loaded firefly luciferase mRNA, CL347-firefly luciferase mRNA mix, and PBS control (FIG.28C). Significant differences were determined by an ordinary one-way ANOVA with Tukey’s multiple comparisons, *p = 0.0360, and *p = 0.0339 against spleen for CL347-SM102 mRNA LNPs, CL347 admixed mRNA and PBS respectively. Data are represented as mean ± SD (n=3). The image was created with BioRender.com. IgM antibodies specific to OVA were detected following adjuvanted mRNA LNP vaccination.6 weeks old C57BL / 6 mice were intramuscularly injected with OVA mRNA loaded SM102 LNPs and OVA mRNA CL347-SM102 LNPs. As a positive control and negative control, Alhydrogel- EndoFitTMOVA and PBS were injected. After 7 days, the mice were sacrificed, and serum was collected to analyze IgM antibodies specific to OVA (FIG. 29). Significant differences between each treatment were determined by one-way ANOVA with Tukey’s multiple comparison test, **p = 0.0072 was observed for PBS and SM102- OVA and **p = 0.0063 was observed for PBS and CL347-SM102-OVA mRNA. Data are represented as mean ± SD (n=4). Intracellular cytokine expression was observed in lymph node and spleen cells.6 weeks old Male C57BL / 6 mice were injected with the CL347-SM102 OVA mRNA and SM102 OVA mRNA treatments and the controls. After 7 days of injection, the mice were sacrificed, and the lymph nodes and spleens were harvested to perform the intracellular cytokine staining assay using flow cytometry. Th1 and Th2 Types of cytokine expression was reported for both Lymph node (FIG.30A) and spleen cells (FIG.30B). Data are represented as mean ± SD (n=4). Expression of proinflammatory cytokines was observed in human (peripheral blood mononuclear cells) PBMCs after treatment with CL347 loaded LNPs. PBMCs were incubated with CL347-SM102 LNPs (10 µg / mL), Imiquimod (10 µg / mL) for 24 h. The cell cytokines were measured through cytometric bead array. The heatmap shows the expression of different cytokines (FIG.31A). Significant differences between the groups were determined using a one-way ANOVA followed by Tukey’s multiple comparison test. Significant differences were observed for IL-6 (FIG.31B), with ****p < 0.0001 for the following comparisons: Cells Only vs. CL347-SM102 LNPs, Cells Only vs. Imiquimod, CL347-SM102 LNPs vs. SM102-Blank LNPs, and SM102-Blank LNPs vs. Imiquimod. Additionally, a significant difference was observed with *p = 0.0437 for the comparison CL347-SM102 LNPs vs. Imiquimod. No significant difference was observed between Cells Only and SM102-Blank LNPs. Significant differences were observed for IL-10 (FIG.31C), with **p = 0.0018 for the comparison Cells Only vs. CL347-SM102 LNPs, and ****p < 0074539-000157 -31- 0.0001 for the comparisons Cells Only vs. and SM102-Blank LNPs vs. Imiquimod. No significant differences were observed for the comparisons Cells Only vs. SM102-Blank LNPs and CL347-SM102 LNPs vs. SM102-Blank LNPs. Additionally, a non- significant difference (ns) was observed for the comparison CL347-SM102 LNPs vs. Imiquimod. D) Significant differences were observed for IFN-γ (FIG.31D), with ****p < 0.0001 for both comparisons: CL347-SM102 LNPs vs. SM102-Blank LNPs and CL347- SM102 LNPs vs. Imiquimod. Data are represented as mean ± SD (n=4). Thus, CL347 adjuvanted LNPs efficiently stimulate Human PBMCs and induce greater proinflammatory cytokines than non-adjuvanted LNPs. This study shows successful delivery of mRNA vaccine to lymphoid organs (Lymph nodes and the spleen) by mRNA-CL347 encapsulated LNPs. When loaded with the OVA mRNA vaccine, CL347 LNPs efficiently generated cellular and humoral responses in vivo that are comparable to clinical gold standard Alhydrogel adjuvant. Example 19. Encapsulation of imiquimod and resiquimod (Hydrophobic drugs) by ionizable LNPs Imiquimod and Resiquimod are primarily used to treat keratosis and genital warts caused by human papillomavirus (HPV). Imiquimod binds to TLR7 receptors on immune cells, leading to the production of cytokines that help fight pathogens and abnormal cell growth. Resiquimod, while similar in function, is more potent because it activates both TLR7 and TLR8 receptors, making it effective for antiviral and anti-cancer treatments. Imiquimod-loaded ionizable LNPs and Resiquimod-loaded ionizable LNPs were tested in an immune cell activation assay to detect their in vitro functional activities. Briefly, human HEK TLR7 cells were treated with different concentrations of Imiquimod and Resiquimod-loaded LNPs as well as soluble control (20 µg / mL). After 24 h of incubation, secreted alkaline phosphatase activity was measured using the HEK-BlueTMDetection reagent (FIG.32). Data represented as mean ± SD (n=3). This study shows that ionizable LNPs efficiently encapsulate hydrophobic drugs, imiquimod, and resiquimod. These LNPs precisely release drugs inside cellular lysosomes and stimulate TLR7 receptors inside human cells in vitro. All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and / or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

0074539-000157 -32- WHAT IS CLAIMED:

1. A method for encapsulating one or more target agents by lipid nanoparticles (LNPs), wherein the one or more target agents comprise a negatively charged target agent, a hydrophobic target agent, or a combination thereof, said method comprising: (a) providing an organic phase solution comprising a lipid mixture and an organic solvent and an aqueous phase solution comprising a first aqueous solvent having a first volume, wherein the lipid mixture comprises an ionizable or cationic lipid, and where the one or more target agents comprise the negatively charged target agent, the lipid mixture is dissolved in the organic solvent, and the negatively charged target agent is dissolved in the first aqueous solvent, where the one or more target agents comprise the hydrophobic target agent, the hydrophobic agent and the lipid mixture are dissolved in the organic solvent, or where the one or more target agents comprise the negatively charged and the hydrophobic target agent, the hydrophobic agent and the lipid mixture are dissolved in the organic solvent, and the negatively charged target agent is dissolved in the first aqueous solvent; (b) impinging the organic phase solution and the aqueous phase solution into a second aqueous solvent having a second volume at least three times of the first volume of the first aqueous solvent, whereby an impingement mixture is formed; (c) self-assembling LNPs in the impingement mixture, wherein each LNP comprises a lipid membrane formed by the lipid mixture and a space enclosed by the lipid membrane, wherein the one or more target agents are entrapped by the ionizable or cationic lipid and encapsulated by the LNPs; and (d) removing organic solvent from the impingement mixture, whereby the LNPs are obtained.

2. The method of claim 1, wherein the impinging comprises mixing the organic phase solution and the aqueous phase solution for no more than 200 milliseconds.

3. The method of claim 1 or 2, wherein the impingement mixture has a total lipid concentration of no more than 50 mM.

4. The method of any one of claims 1-3, wherein the impingement mixture has an aqueous volume of no more than 3 mL.

5. The method of any one of claims 1-4, wherein the LNPs have a polydispersity index of less than 0.

3.

6. The method of any one of claims 1-5, wherein the LNPs have a particle size of less than 200 nm.0074539-000157 -33- 7. The method of any one of 1-6, wherein at least 50% of the one or more target agents are encapsulated by the LNPs.

8. The method of any one of claims 1-7, wherein the ionizable lipid is selected from the group consisting of SM102, ALC-0315, DLin-MC3-DMA, C12-200, cKK-E12, C14- 4, L-319, YSK12-C4, and DLin-KC2-DMA.

9. The method of any one of claims 1-7, wherein the cationic lipid is selected from the group consisting of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-[1- (2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), dimethyldioctadecylammonium (bromide) (DODAB), and 1,2-stearoyl-3- trimethylammoniumpropane (chloride) (DSTAP).

10. The method of any one of claims 1-9, wherein each of the one or more target agents is selected from the group consisting of a nucleic acid, an adjuvant, and a hydrophobic drug.

11. The method of any one of claims 1-10, further comprising lyophilizing the LNPs, whereby a powder of LNPs is obtained.

12. The method of claim 11, further comprising rehydrating the LNP powder with a third aqueous solvent, whereby an aqueous solution of the LNPs is obtained.

13. The method of any one of claims 1-12, further comprising interacting the LNPs with cells, and delivering the LNPs into the cells, whereby the one or more target agents are in the cells.

14. The method of claim 13, further comprising releasing the one or more target agents in the cells from the LNPs.

15. The method of claim 13 or 14, wherein the one or more target agents in the cells are biologically active.

16. The method of any one of claims 13-15, further comprising stimulating an immune receptor in or on the cells.

17. The method of any one of claims 13-16, wherein the cells are in a subject.

18. The method of claim 17, further comprising stimulating an immune response in the subject.

19. The lipid nanoparticles (LNPs) produced in accordance with the method of any one of claims 1-18.

20. A composition comprising the LNPs of claim 19.

21. A lipid nanoparticle (LNP) comprising two or more negatively charged or hydrophobic target agents, a lipid membrane formed by a mixture of lipids, and a space enclosed by the lipid membrane, wherein the mixture of lipids comprises an ionizable or0074539-000157 -34- cationic lipid, and wherein each of the two negatively charged or hydrophobic target agents is selected from the group consisting of a nucleic acid, an adjuvant, and a hydrophobic drug.

22. The LNP of claim 21, wherein the ionizable lipid is selected from the group consisting of SM102, ALC-0315, DLin-MC3-DMA, C12-200, cKK-E12, C14-4, L-319, YSK12-C4, and DLin-KC2-DMA.

23. The LNP of claim 21, wherein the cationic lipid is selected from the group consisting of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-[1-(2,3- Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), dimethyldioctadecylammonium (bromide) (DODAB), and 1,2-stearoyl-3- trimethylammoniumpropane (chloride) (DSTAP).

24. A composition comprising a plurality of the LNPs of any one of claims 21-23.

25. A method for intracellular delivery, comprising interacting the LNPs of any one of claims 19 and 21-23 with cells, and delivering the LNPs into the cells, whereby at least one of the target agents are in the cells.

26. The method of claim 25, further comprising releasing the at least one target agent in the cells from the LNPs.

27. The method of claim 25 or 26, wherein the at least one target agent in the cells is biologically active.

28. The method of any one of claims 25-27, further comprising stimulating an immune cell specific receptor in or on the cells.

29. The method of any one of claims 25-28, wherein the cells are in a subject.

30. The method of claim 29, further comprising stimulating an immune response in the subject.

31. A method for stimulating an immune response, comprising administering to a subject an effective amount of the LNPs of any one of claims 19 and 21-23 or the composition of claim 20 or 24.

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