Targeted therapeutic delivery of lipid nanoparticles

Abstract

Compositions and methods are described for targeted therapeutic delivery. A composition of matter for targeted therapeutic delivery comprises one or more ionizable cationic lipids; a mixture of one or more helper lipids; and one or more lipids with ligands that target receptors for cells.

Description

TARGETED THERAPEUTIC DELIVERY OF LIPID NANOPARTICLESSTATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under Award No R01CA196266 and Award No R21CA283583 awarded by National Institutes of Health (NIH). The government has certain rights in the invention.BACKGROUND INFORMATION1. Field:

[0002] The present invention relates generally to the field of medicine and disease treatment. More particularly, it concerns methods and compositions for targeted therapeutic delivery of lipid nanoparticles.2. Background:

[0003] Therapeutic delivery refers the methods and technologies used to transport therapeutic agents such as drugs, proteins, genes, or cells to specific site within body where the therapeutic agents can exert intended effect. Therapeutic delivery can optimize the efficacy of treatments while minimizing side effects and damage to healthy tissues. One major approach in therapeutic delivery is the drug delivery systems. Such systems are designed to target specific tissues or cells and deliver the drugs in a controlled manner. For example, such systems can include liposomes, nanoparticles, polymeric micelles, and hydrogels that encapsulate drugs such that the drugs can be protected from degradation and released at target site in response to specific stimuli.

[0004] However, traditional drug delivery systems often lack specificity, leading to systemic side effects and reduced therapeutic efficacy. The development of targeted delivery systems, particularly those utilizing lipid-based nanoparticles, offers significant improvements in delivering drugs selectively to diseased cells while sparing healthy tissues, or by delivering certain effectors selectively to one particular type or tissue.

[0005] Targeted delivery is a method for drug delivery systems that aims to direct therapeutic agents specifically to the desired site of action within the body. Targeted delivery uses ligands that recognize and bind to specific receptors on the surface oftarget cells. In this example, the ligands can be antibodies, peptides, aptamers, or small molecules that have a high affinity for their corresponding receptors. By attaching these ligands to drug carriers such as nanoparticles or liposomes, the drug delivery system can navigate through the body and selectively bind to the target cell. Once bound, the drug carrier can release the therapeutic agent directly to the targeted cells to ensure high local concentration of the therapeutic agent on the targeted site.SUMMARY

[0006] The present invention discloses novel lipid nanoparticles (LNPs) as advanced delivery systems for the targeted transport of therapeutic agents that include nucleic acids, proteins, small molecules, or toxins. These therapeutic agents or their precursors are encapsulated within the LNPs. As a result, selective targeting of specific cells or tissues in vivo is achieved by functionalizing the LNPs with ligands that bind to specific receptors, which may be overexpressed on the surface of target cells. Upon receptor binding, the LNPs undergo endocytosis, allowing for the release of encapsulated therapeutic agents into the cytosol. This technology can be applied in a cancer gene therapy that transfects cancer cells in vivo with LNPs carrying plasmid DNA encoding a glycosyltransferase. In this illustrative example, glycosyltransferase modifies cancer cell surfaces to present antigenic glycans, marking the cancer cells for destraction by antibodies specific to these glycans.

[0007] An illustrative embodiment of the present disclosure is a composition of matter for targeted therapeutic delivery, comprising one or more ionizable cationic lipids; a mixture of one or more helper lipids; and one or more lipids with ligands that target receptors for cell.

[0008] Another illustrative embodiment of the present disclosure is a method of targeted therapeutic delivery for treating a disease, comprising administering to a mammal in need thereof a compound comprising one or more ionizable cationic lipids; a mixture of one or more helper lipids; one or more lipids with ligands that target receptors for cell; and one or more nucleic acids.

[0009] Another illustrative embodiment of the present disclosure is a method of targeted therapeutic delivery for a cancer therapy, comprising administering to a mammal in need thereof a compound comprising one or more ionizable cationic lipids; a mixture of one or more helper lipids; one or more lipids with ligands that target receptors for cell; and one or more nucleic acids. The one or more nucleic acids causes cells of the mammal to express glycan.

[0010] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0012] Figure 1A-1C illustrate synthesis of DT7 and folate functionalized lipid nanoparticles with encapsulated plasmid DNA in accordance with an illustrative embodiment.

[0013] Figures 2 illustrates post-assembly modification of lipid nanoparticles with DT7 peptide ligand in accordance with an illustrative embodiment.

[0014] Figures 3A-3F illustrate plots for characterization of lipid nanoparticles functionalized with maleimide and DT7 peptides in accordance with an illustrative embodiment.

[0015] Figures 4 illustrates a plot of transfection results for lipid nanoparticles functionalized with DT7 peptides in accordance with an illustrative embodiment.

[0016] Figures 5 illustrates a plot of transfection results from a cell competition experiment for lipid nanoparticles functionalized with DT7 peptides in accordance with an illustrative embodiment.

[0017] Figures 6 illustrates a flowchart illustrating a process for targeted therapeutic delivery for treating a disease in accordance with an illustrative embodiment.DETAILED DESCRIPTION

[0018] Embodiments of this disclosure primarily includes a novel gene -based therapy for diseases with dual selectivity using targeting lipid nanoparticles (LNPs) to deliver therapeutic agents. In this illustrative example, examples of diseases can include cancer, neurological diseases, muscular diseases, or any genetic diseases. Embodiments of this disclosure can be a composition that serves as a carrier to deliver therapeutic agents.

[0019] In this illustrative example, different fundamental properties critically influence lipid nanoparticles’ performance, and therefore directly affect their efficacy and behavior in various applications. For example, properties impacting the performance of LNPs can include particle size, surface charge, polydispersity index (PDI), and surface modification. In this illustrative example, the size of lipid nanoparticles is a critical factor that affects both in vitro and in vivo performance. LNPs typically exhibit average diameters between 100 and 400 nm. For systemic drug delivery via intravenous (IV) injection, diameters for LNPs in the 10 to 200 nm range are considered optimal. In this illustrative example, LNPs that are smaller than 200 nm are preferred due to their ability to pass through liver sinusoidal endothelial fenestrae, and to be sterilized by filtration. In addition, a surface charge on lipid nanoparticles significantly influences interactions between LNPs and cellular membranes. In this illustrative example, surface charge can be experimentally determined by measuring the particles’ zeta (Q potential.

[0020] The ^-potential values also indicate colloidal dispersion stability by quantifying the degree of repulsion force. Typically, an LNP with a ^-potential exceeding +30 mV or falling below -30 mV exhibits robust electrostatic stability, effectively preventing LNP aggregation. Moreover, the surface charge on LNPs is crucial in promoting interactions with cell membranes and facilitating endosomal escape. In this illustrative example, anionic cell membranes generally repel anionic LNPs, cationic LNPs may induce cytotoxicity by directly disrupting cellular membranes. Preferably, neutral LNPs (-10 mV < ^-potential < 10 mV) can be employed to mitigate these issues. As a result, incorporating ionizable lipids in LNPs becomes essential as the overall surface charge of LNPs is dependent on the environmental pH. In this illustrative example, using ionizable lipids helps avoid undesirable electrostatic interactions with the cell membrane.In addition, a PDI < 0.2 is generally acceptable and indicates a relatively uniform LNP size distribution, which contributes to the homogeneity and reproducibility of the formulation. For LNPs with nucleic acid encapsulation, a desirable PDI can generally be achieved through the lipid composition. Tn this illustrative example, the protocol for mixing the aqueous nucleic acid phase with the organic lipid phase can be done manually by pipetting method. In other words, the two phases are mixed together in a container and pipetted up and down multiple times. However, the flow rate is difficult to control using this method. In an alternative example, the mixing can also be done in a microfluidic machine under controlled conditions. In this illustrative example, the flow rate by which the two phases flow through microtubes can be controlled by mixing the two phases in a specialized chamber. The microfluidic method is accurate and reproducible because all parameters are controlled by a computer. In this illustrative example, the lipid nanoparticles produced with a microfluidic machine typically have a lower PDI and the lipid nanoparticles may have small diameters.

[0021] Further, surface modification such as PEGylation includes incorporating PEG imparts an external polymeric layer to the outer shell of LNPs, therefore effectively inhibiting the adsorption of serum proteins and components of the phagocytic system. This phenomenon extends the in vivo circulation time of LNPs, prevents particle aggregation, and enhances stability for LNPs even when the LNP is neutral in charge. In this illustrative example, PEG can shield LNPs from serum proteins such as Apo-E and albumins. However, excessive PEG in the LNP formulation can lead to excessive stability, hindering cellular internalization and intracellular release of the nucleic acids, ultimately reducing intracellular delivery. It is crucial to understand and optimize these properties to tailor LNPs for specific therapeutic or diagnostic applications, thereby improving LNPs’ efficacy in various biomedical settings.

[0022] In this illustrative example, ethanol dilution method is a well-established technique for assembling LNPs. For example, one equivalent volume of an ethanol solution containing all lipids is promptly combined with three equivalent volumes of an aqueous buffer solution containing the nucleic acid component. V arious methods can be employed to swiftly mix these two phases. For example, the methods can include pipette mixing, vortex mixing, and microfluidic mixing. In this illustrative example, the pipette mixing method includes manually pipetting the mixture up and down for producing small-scale LNP batches. Simplicity makes pipette mixing method a favoredchoice for optimization and characterization in in vitro studies, and for low-dose in vivo experiments.

[0023] As a result, a novel composition of lipid nanoparticles is proposed for targeted therapeutic delivery for diseases. In this illustrative example, composition can include various lipidic components. The composition can include one or more ionizable cationic lipids and permanently cationic lipids. In this illustrative example, ionizable cationic lipids permanently cationic lipids can constitute 45 mol% to 55 mol% of the lipid nanoparticles. In this illustrative example, the permanently cationic lipids are lipids that have quaternary ammonium salt, which is always positively charged. On the other hand, the ionizable lipids are cationic only at low pH and are neutral at higher pH. In this illustrative example, the one or more ionizable cationic lipids and permanently cationic lipids in the composition are used at a molar ratio of 4-9 (N / P ratio=4-9) equivalent to the phosphate groups in nucleic acids that are encapsulated by the composition. In this illustrative example, N / P ratios are nitrogen / phosphorus or a ratio of positive charge to negative charge.

[0024] In this illustrative example, ionizable cationic lipids and permanently cationic lipids can be at least one of heptadecan-9 -yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)octanoate or SM-102, (6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate or Dlin-MC3-DMA, 2-(2,2- di((9Z, 12Z)-octadeca-9, 12-dien- 1-yl)- 1 ,3-dioxolan-4-yl)-N,N-dimethylethan- 1 -amine or DLin-K2C-DMA, l,2-dioleoyl-3-dimethylammonium-propane or DOTAP, [(4- Hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) or ALC-0315, Didodecyldimethylammonium bromide or DDAB, or a combination thereof.

[0025] As used herein, the phrase “at least one of,” when used with a list of items, means different combinations of one or more of the listed items can be used, and only one of each item in the list may be needed. In other words, “at least one of” means any combination of items and number of items may be used from the list, but not all of the items in the list are required. The item can be a particular object, a thing, or a category.

[0026] For example, without limitation, “at least one of item A, item B, or item C,” may include item A, item A and item B, or item B. This example also may include item A, item B, and item C, or item B and item C. Of course, any combination of these items can be present. In some illustrative examples, “at least one of’ can be, for example, without limitation, two of item A; one of item B; and ten of item C; four of item B and seven of item C; or other suitable combinations.

[0027] In this illustrative example, the composition can also include a mixture of one or more helper lipids. In this illustrative example, the mixture of one or more helper lipids can include at least one of cholesterol, at least one of PEGylated lipids or polysarcosinylated lipids used for preventing aggregation and protein-hinding, and one or more non-PEGylated and non-polysarcosinylated phospholipids, or a combiantion thereof. In this illustrative example, the cholesterol can constitute 20 mol% to 50 mol% of the lipid nanoparticles, and preferably, 30 mol% to 45 mol% of the lipid nanoparticles.

[0028] The at least one of PEGylated lipids or polysarcosinylated lipids can include at least one of l,2-dimyristoyl-sn-glycero-3-succinyl-N-polysarcosine-25 or DMG- pSar25, 1 ,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol-2000 or DMG- PEG2000, 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethyleneglycol)-2000] or DSPE-PEG2000, or a combination thereof. In this illustrative example, the at least one of PEGylated lipids or polysarcosinylated lipids can constitute 2 mol% to 5 mol% of the lipid nanoparticles. In this illustrative example, the at least one of PEGylated lipids or polysarcosinylated lipids can increase circulation time and reduce immunogenicity for the composition of lipid nanoparticles.

[0029] The one or more non-PEGylated and non-polysarcosinylated phospholipids can include at least one of l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or POPC, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine or DOPE, l,2-distearoyl-sn-glycero-3- phosphocholine or DSPC, or a combination thereof. In this illustrative example, the at least one of PEGylated lipids or polysarcosinylated lipids can constitute 5 mol% to 12 mol% of the lipid nanoparticles.

[0030] Further, the composition can also include one or more lipids with ligands that target receptors for cells. In this illustrative example, ligands that target receptors for the cells can include ligands that target transferrin receptor l(TfRl / CD71) and folate receptor alpha (FRalpha). For example, the ligands can include DT7 peptide, holo- transferrin, or Ab(or Ab derivative) specific ligands for TfRl such as hul28.1, an aptamer, moieties, or any suitable ligand that target specific receptors on the surface of cells. In this illustrative example, the ligands that target receptors for the cells can constitute 0.2 mol% to 2 mol% of the lipid nanoparticles.

[0031] As depicted, the composition of the lipid nanoparticles mentioned above can be used for delivering therapeutic agents such as nucleic acids. In this illustrative example, the nucleic acids can include at least one of a mRNA, a siRNA, a plasmidDNA, a small activating RNA(saRNAs) or a combination thereof. The therapeutic agents in this disclosure primarily focus on nucleic acids, however, it should be understood that the therapeutic agents that can be carried by the composition of the lipid nanoparticles described in this disclosure can also include proteins, small molecules, or toxins. In this illustrative example, the nucleic acids are dissolved in an aqueous buffer at a slightly acidic pH such that optimal ionization of the ionizable cationic lipid can be achieved.

[0032] In this illustrative example, the composition of lipid nanoparticles can be mixed with nucleic acids in a number of ways. For example, the composition of lipid nanoparticles can be mixed with nucleic acids by pipetting. In this illustrative example, the composition of lipid nanoparticles is dissolved in ethanol and the nucleic acids are dissolved in an aqueous buffer. Solution for the composition of lipid nanoparticles can constitute 20%-50% of solution for the nucleic acids by volume. In this illustrative example, the aqueous buffer can be acetate, Phosphate-buffered saline (PBS), citrate, 4- (2-hydroxyethyl)-l -piperazineethanesulfonic acid or HEPES, or any buffer with slightly acidic pH to ensure the total ionization of the ionizable cationic lipid.

[0033] In this illustrative example, solution for the composition of lipid nanoparticles and solution for the nucleic acids are mixed in a microcentrifuge tube and homogenized by pipetting back and forth. After mixing, solution for the composition of lipid nanoparticles can be further diluted in a neutral buffer, typically IX PBS at pH 7.4. In this illustrative example, the mixture of lipid nanoparticles and nucleic acids can be adjusted with neutral buffer and purified via dialysis or Amicon filtration to remove organic solvent content and stabilize pH.

[0034] In an alternative embodiment, the composition of lipid nanoparticles can be mixed with nucleic acids by microfluidic mixing using a microfluidic device, followed by purification steps similar to direct mixing by pipetting, as described above.

[0035] In yet another example, the composition of lipid nanoparticles can also be modified post-assembly with the target ligands. In this example, the composition of lipid particles can include l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethyleneglycol)-2000, NHS ester] or DSPE-PEG2000-NHS, or 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethyleneglycol)-2000, maleimide] or DSPE-PEG2000-Mal at the same molar ratio as the desired ligand concentration. As a result, the ligand with an amino or thiol group can be covalently attached to the assembled lipid nanoparticles.

[0036] In this illustrative example, the mixture of lipid nanoparticles and nucleic acids can be sterilized by filtration through a 0.2 mm sterile PES filter and stored at 4°C avoided to maintain structural integrity and efficacy from freezing.

[0037] In this illustrative example, the mixture of lipid nanoparticles and nucleic acids is designed with optimized size, zeta potential, encapsulation efficiency, and PEG or polysarcosine shielding to avoid aggregation, protein corona formation, and sequestering by the liver, and minimize non-specific uptake. As a result, the mixture of lipid nanoparticles and nucleic acids is suitable for intravenous administration, intratumoral administration, and in vitro transfections.

[0038] Further, the mixture of lipid nanoparticles and nucleic acids has high selectivity from the ligand equipped by the lipid nanoparticles such that the mixture of lipid nanoparticles and nucleic acids can be used for targeting a specific cellular receptor of the target cells. In some illustrative examples, the mixture of lipid nanoparticles and nucleic acids can also be equipped with more than one type of ligand such that two or more receptors can targeted to further increase selectivity. In yet another illustrative example, the mixture of lipid nanoparticles and nucleic acids can include tissue-specific promoters in plasmid DNA to ensure gene expression only occurs in cells that are targeted. For example, tyrosinase promoter ensures that gene expression occurs only in melanocytes.

[0039] As depicted, the mixture of lipid nanoparticles and nucleic acids can be adapted to deliver a wide range of therapeutic agents for various diseases such as cancer, genetic disorders, neurological conditions, or any suitable disorders. For example, the mixture of lipid nanoparticles and nucleic acids can be used to deliver therapeutic agents to cancer cells for expressing alpha-Gal. In this illustrative example, the expression of alpha-Gal on cancer cells makes the cancer cells highly visible to immune system, thereby making the cancer cells targets for elimination by the immune system.

[0040] With reference now to Figure 1A-1C, an illustration for synthesis of DT7 and folate functionalized lipid nanoparticles with encapsulated plasmid DNA is shown in accordance with an illustrative embodiment.

[0041] In Figure 1A-1C, lipid nanoparticles 100 include various lipidic components. For example, the lipidic components has helper lipids that include cholesterol, DSPC as non-PEGylated and non-polysarcosinylated and permanently cationic phospholipid, and DMG-PEG(2000) as PEGylated lipid, and Dlin-MC3-DMAas ionizable cationic lipid. In this illustrative example, the lipidic components also include DSPE-PEG(2000)-DT7 (made from DSPE-PEG(2000)-Mal and Cys-DT7), and DSPE-PEG(2000)-Folate as lipids with ligand. In this illustrative example, DSPE- PEG(2000) is modified with DT7 peptide and folate as ligands for targeting TfRl and FRa receptors.

[0042] In this illustrative example, lipid nanoparticles 100 is designed to encapsulate therapeutic agent 102. Therapeutic agent 102 can be a plasmid DNA that includes melanocyte-specific promoter and gene codes for alpha- 1 ,3-Gal transferase. In this illustrative example, mixture 104 shows a composition with therapeutic agent 102 encapsulated by lipid nanoparticles 100 such that lipid nanoparticles 100 can delivery therapeutic agent 102 to target sites. In this illustrative example, mixture 104 targets overexpressed TfRl and FRa receptors simultaneously on cancerous melanocytes and enables cancerous melanocytes to express alpha- 1 ,3-Gal transferase after therapeutic agent 102 is delivered to cancerous melanocytes. The expression of alpha- 1,3-Gal transferase makes those cancerous melanocytes good candidates that are visible to immune system for eliminations.

[0043] With reference now to Figure 2, an illustration of post-assembly modification of lipid nanoparticles with DT7 peptide ligand is shown in accordance with an illustrative embodiment.

[0044] In Figure 2, mixture 200 shows pDNA encapsulated by lipid nanoparticles that are functionalized with maleimide (Mai). In some illustrative examples, synthesis for mixture 200 can start with a molar ratio of DLin-MC3-DMA: cholesterol: DSPC: DMG-PEG2000 and DSPE-PEG2000-Mal of 50: 38.5: 10: 1: 0.5, respectively. However, to identify the most favorable lipid composition yielding optimal physicochemical characteristics for lipid nanoparticles, various lipid nanoparticles can be synthesized by varying the DMG-PEG2000 mol% (1, 2, 2.5, 3, and 5 mol%) of the total lipid composition. Throughout these experiments, the ionizable cationic lipid to negative charges in the pDNA (N / P) ratio was maintained at 6, and the percentages of DLin-MC3-DMA, DSPC, and DSPE-PEG2000-Mal were held constant. The incremental 8 adjustments in the DMG-PEG2000 mol% are compensated by proportionately reducing the amount of cholesterol. Subsequently, after determining the optimal composition based on the DMG-PEG2000 mol%, different N / P ratios are tested while keeping the lipid formulation constant to enhance the characteristics of lipid nanoparticles further. In this illustrative example, mixture 200 is conjugated with DT7peptide 204 to yield mixture 204 that includes DT7 peptide as ligand that targets hTfRl- expressing cells.

[0045] With reference now to Figure 3A-3F, plots for characterization of lipid nanoparticles functionalized with maleimide and DT7 peptides are shown in accordance with an illustrative embodiment. In this illustrative example, lipid nanoparticles functionalized with maleimide and DT7 peptides can be examples of mixture 200 and mixture 202 in Figure 2.

[0046] In this illustrative example, plots in Figure 3A-3F show characterization for both lipid nanoparticles functionalized with maleimide (LNP-Mal) and DT7 peptides (LNP-DT7). For example, dynamic light scattering (DLS), ^-potential analysis, and pDNA encapsulation efficiency (EE) are determined, hi plot 300, plot 302, and plot 304, the N / P ratio of 6 is constantly maintained such that an optimal amount of DMG- PEG2000 for lipid nanoparticle formation and pDNA encapsulation can be investigated.

[0047] For example, plot 300 shows sizes of lipid nanoparticles for different concentration of DMG-PEG2000. In this example, at a low concentration of 1 mol% of DMG-PEG2000, LNP-Mal is obtained with a size of approximately 115 nm and a PDI of 0.08. However, after the DT7 conjugation, noticeable particle aggregation occurred, showing that only 1 mol% of DMGPEG2000 compromised the LNP-DT7's stability.

[0048] The issue of aggregation can be resolved by incrementally raising the DMG- PEG2000 concentration in the overall formulation. A progressive reduction in size was observed in all lipid nanoparticles as the DMG-PEG2000 concentration increased from 2 to 5 mol%. In all cases, LNP-DT7 particles exhibited a consistent increase of around 59 nm in hydrodynamic size compared to their LNP-Mal precursors, with PDIs remaining below 0.2 in all formulations.

[0049] In plot 302, both LNP-Mal and LNP-DT7 are considered neutral in charge before and after DT7 conjugation. In this illustrative example, plot 302 displays t,- potential values ranging from -10 to 10 mV. ^-potential measurements are conducted at physiological pH, where the amine moiety in the ionizable lipid DLin-MC3-DMA (pKa = 6.44) was mostly deprotonated, which places the ^-potential for lipid nanoparticles in the neutral range. In this illustrative example, the ^-potential of all LNP-DT7 particles shifted from positive to negative values compared to their LNP-Mal precursors.

[0050] Plot 304 shows dependence of the pDNA EE on the DMG-PEG2000 content by varying the DMG-PEG2000 concentration between 2 mol% and 5 mol%. In this illustrative example, DMG-PEG2000 concentration of 2 mol%, 2.5 mol%, and 3 mol%demonstrates similar EEs of approximately 90%, while lipid nanoparticles with 5 mol% DMG-PEG2000 exhibited a significantly lower EE. In this illustrative example, the higher concentration of DMG-PEG2000 appears to impact assembly mechanism for lipid nanoparticles. In addition, the overall yield remained consistent across all formulations, ranging from 82% to 96%.

[0051] In this illustrative example, plot 304 indicates that lipid nanoparticles featuring DMG-PEG2000 concentrations of 2 mol%, 2.5 mol%, and 3 mol% exhibit similar physicochemical properties, with the lipid nanoparticles at 3 mol% of DMG- PEG2000 showing marginally superior overall characteristics. Preliminary transfection assays revealed that the lipid nanoparticles with 3 mol% of DMG-PEG2000 yield the highest percentage of transfection. Additionally, the decision to proceed with the higher DMG-PEG2000 concentration of 3 mol% is influenced by its potential to mitigate protein corona formation and reduce the likelihood of nonspecific uptake in a potential in vivo scenario. In other words, the DMG-PEG2000 concentration at 3 mol% is determined as optimal concentration for DMG-PEG2000 in lipid nanoparticles.

[0052] On the other hand, plot 306, plot 308, and plot 310 shows results for experiments for identifying optimal amount of the ionizable DLin-MC3-DMA for particle formation and pDNA encapsulation. In this illustrative example, four different N / P ratios (4, 6, 8, and 10) are tested.

[0053] In plot 306, the transition from an N / P ratio of 4 to 6 resulted in a significant lipid nanoparticle size reduction and a notable enhancement in PDI. Since the majority of amine groups in DLin-MC3-DMA carry a positive charge in acetate buffer (pH 4), the strong electrostatic interaction between positively charged ionizable lipids and negatively charged pDNA at a higher N / P ratio prompted the formation of compacted lipid nanoparticles with smaller diameters. This observation indicates that at N / P = 4, there are insufficient positively charged lipids to complex with all negatively charged pDNA molecules, which allows a portion of the pDNA to remain unbound in the solution. Further increases in the N / P ratio from 6 to 8 and 10 maintained the size relatively constant while the PDI reached unacceptable levels, particularly after the conjugation of DT7 peptide. Additionally, higher N / P ratios of 8 and 10 are believed to diminish the pDNA release capacity from endosomes, resulting in reduced transfection efficiency of the lipid nanoparticles.

[0054] Plot 308 shows data associated with ^-potential. In this illustrative example, all lipid nanoparticles can be considered neutral in charge, with ^-potential valuesranging between -10 and 10 mV. Further, a consistent shift from positive 10 to negative ^-potential values is observed when comparing the LNP-Mal group with the LNP-DT7 group. In plot 308, the pDNA EE% reveals an improvement from approximately 60% at N / P = 4 to around 90% at N / P = 6.

[0055] As a result, LNP-Mal particles exhibit more favorable physicochemical properties than the corresponding LNP-DT7 particles. The latter displayed an increase in size and PDI, along with a decrease in EE% and overall yield. This pattern can be attributed to the additional manipulation and purification treatments the LNP-DT7 particles had to undergo during their synthesis. In other words, LNP-DT7 with 3 mol% of DMG-PEG2000 and an N / P ratio of 6 is determined to be the optimal formulation because lipid nanoparticles formed under these conditions exhibit particle sizes suitable for in vivo gene delivery (~ 150 nm), a low desirable PDI after conjugation (~0. 145), a high encapsulation efficiency (>80%) and a high yield of >90%.

[0056] With reference now to Figure 4, a plot of transfection results for lipid nanoparticles functionalized with DT7 peptides is shown in accordance with an illustrative embodiment. In this illustrative example, lipid nanoparticles functionalized DT7 peptides can be examples of LNP-DT7 in Figure 3.

[0057] In this illustrative example, two genetically modified cell lines are used to validate the selective transfection of LNP-DT7 particles toward hTfRl-expressing cells: CHO-TRVb-neo and CHO-TRVb-hTfRl. Both cell lines originated from the CHO- TRVb cell line, characterized by the absence of detectable cell surface TfRl receptor. In this illustrative example, the CHO-TRVb-hTfRl cell line exclusively expresses hTfRl, whereas the CHO-TRVb-neo cells were transfected with an empty vector. Both CHO-TRVb cell lines are maintained in F-12 (HAM) medium supplemented with 10% fetal bovine serum and 1 mg / mL G418 as a selection marker. In this illustrative example, both cell lines provide robust positive control (CHO-TRVb-hTfRl ) and negative control (CHO-TRVb-neo) regarding transferrin receptor expression.

[0058] In this illustrative example, cells were seeded at a concentration of 8,000 cells / well for all transfection experiments. Subsequently, the cells were treated with five or six different concentrations of LNP-DT7, corresponding to encapsulated pDNA amounts of 100, 200, 300, 400, 500, 600, 800, or 1000 ng / well. In this illustrative example, the lipid nanoparticles are prepared with a composition that includes 50 mol% of DLin-MC3-DMA, 10 mol% of DSPC, 36.5 mol% of cholesterol, 3 mol% of DMG- PEG2000, and 0.5 mol% of DSPE-PEG2000-Mal.

[0059] In Figure 4, individual transfection results with LNP-DT7 using CHO- TRVb-neo cell line and CHO-TRVb-hTfRl cell line are shown. The percentages of mGL-positive cells of both sets of experiments were compared at various pDNA concentrations. In this illustrative example, the results consistently show low mGL expression between approximately 1% and 4% in all transfection experiments using CHO-TRVb-neo cells with LNP-DT7. This result confirms the limited affinity of LNP- DT7 particles toward hTfRl -negative CHO-TRVb-neo cells. In contrast, the percentages of mGL-positive CHO-TRVb-hTfRl cells demonstrated a pronounced and dose-dependent increase, which reaches approximately 20% transfection efficiency at a pDNA dose of 1000 ng / well. At higher pDNA doses of 1500 and 2000 ng / well, the percentage of mGL positive cells increased only slightly and appeared to reach a plateau at 22%. Notably, achieving a 20% transfection efficiency at this dose using pDNA encapsulated in a lipid nanoparticle surpasses the reported outcomes in existing technologies, where comparable percentages were only attained with higher pDNA doses (5000 ng pDNA), and only via the ApoE / LDL-R pathway.

[0060] In this illustrative example, the transfection selectivity of the LNP-DT7 can be quantified at a specific pDNA concentration as the ratio between mGL-positive CHO-TRVb-hTfRl and mGL-positive CHO-TRVb-neo cells. For example, at a pDNA concentration of 600 ng / well a fivefold higher transfection of the CHO-TRVb-hTfRl is achieved when compared to CHO-TRVb-neo cells under the same conditions. The ratio of transfected CHOTRVb- neo cells to transfected CHO-TRVb-hTfRl cells of 1 : 5 corresponds to a ratio of 17% : 83% when considering the sum of all transfected cells as 100% at a pDNA concentration of 600 ng / well. In other words, 17% of the transfections occurred nonspecifically.

[0061] With reference now to Figure 5, a plot of transfection results from a cell competition experiment for lipid nanoparticles functionalized with DT7 peptides is shown in accordance with an illustrative embodiment. In this illustrative example, lipid nanoparticles functionalized DT7 peptides can be examples of LNP-DT7 in Figure 3.

[0062] Figure 5 shows transfection results for lipid nanoparticles functionalized with DT7 peptides from a cell competition experiment. In this illustrative example, equal amounts of CHO-TRVbhTfRl and CHO-TRVb-neo cells are seeded together into the same wells prior to being subjected to transfection with different amounts of LNP- DT7. In this illustrative example, seeding equal amounts of CHO-TRVbhTfRl andCHO-TRVb-neo cells together reflects a more realistic scenario since cancer cells and healthy cells are mixed together in human bodies.

[0063] In a similar fashion, cells were seeded at a concentration of 8,000 cells / well for all transfection experiments. Subsequently, the cells were treated with five or six different concentrations of LNP-DT7, corresponding to encapsulated pDNA amounts of 100, 200, 300, 400, 500, 600, 800, or 1000 ng / well. In this illustrative example, the lipid nanoparticles are prepared with a composition that includes 50 mol% of DLin- MC3-DMA, 10 mol% of DSPC, 36.5 mol% of Cho, 3 mol% of DMG-PEG2000, and 0.5 mol% of DSPE-PEG2000-Mal.

[0064] In this illustrative example, successfully transfected cells are detected by flow cytometry based on their mGL expression, and CHO-TRVb-hTfRl and CHO- TRVb-neo cells are distinguished from each other based on the detection of the hTRl expression using the fluorescent mouse anti-hTfRl (CD71) monoclonal antibody [(OKT9 (OKT-9)), PerCP-eFluorTM 710, eBiosciencesTM.

[0065] Plot 500 shows the quantitative, dose-dependent transfection results using mixtures of CHO-TRVb-hTfRl and CHO-TRVb-neo cells prepared as mentioned above. As depicted in plot 400 in Figure 4, the cells display a dose-dependent transfection efficiency with increasing quantities of pDNA. When compared to the transfection of CHO-TRVbhTfRl cells only, an overall lower percentage of mGL positive cells was observed. However, plot 500 shows that using a 1 : 1 mixture of CHO-TRVb-hTfRl and CHO-TRVb-neo cells also resulted in a clear preferential transfection of the CHO-TRVb-hTfRl, albeit with a lower selectivity when compared to the individual transfection experiments shown in plot 400 in Figure 4. For example, the selectivity for the CHO-TRVb-hTfRl was only fourfold when exposing 4000 CHO- TRVb-hTfRl and 4000 CHO-TRVb-neo cells with 300 ng pDNA. A ratio of transfected CHO-TRVb-neo / transfected CHO-TRVb-hTfRl of 1 : 4 corresponds to 20% : 80%. In other words, 20% of the transfections occurred non- specifically.

[0066] In addition, the selectivity for the CHO-TRVbhTfRl cells decreased to only threefold at 600 ng pDNA. Despite lower selectivity in the cell competition experiment, the observed preferential transfection of the CHO-TRVb-hTfRl cells has to be attributed to the interaction between hTfRl and DT7.

[0067] With reference now to Figure 6, a flowchart illustrating a process for targeted therapeutic delivery for treating a disease is shown in accordance with anillustrative embodiment. The process in this figure can be implemented using lipid nanoparticles described in Figure 1-5.

[0068] The process begins by administer to a mammal in need thereof a compound comprising: one or more ionizable cationic lipids; a mixture of one or more helper lipids; one or more lipids with ligands that target receptors for cells; and one or more nucleic acids (step 600). In this step, the compound can be used for cancer therapy such that the one or more nucleic acids causes cells of the mammal to express glycan. The process terminates thereafter.

[0069] As a result, the proposed lipid nanoparticles can be used to encapsulate and deliver therapeutic agents to targeted cells for treating diseases. For example, the proposed novel lipid nanoparticles with therapeutic agents can be used for treating cancers. In this illustrative example, cancer cells can be transfected with a gene (pDNA) coding for the enzyme alpha-1 ,3- galactosyl transferase (alpha- l ,3-GalT) in vivo. To accomplish this, the lipid nanoparticles can be modified with targeting ligands to deliver encapsulated DNA to cancer cells that overexpress transferrin receptor 1 (TfRl) and / or folate receptor alpha (FRalpha). In order for the lipid nanoparticles to perform a cancer-targeted delivery, the lipid nanoparticles can be designed to have TfRl- targeting peptides, antibodies (Abs such as hul28.1), aptamers, or transferrin, and / or FRalpha-targeting ligands such as folate. Upon binding, the lipid nanoparticles are internalized by receptor-mediated endocytosis and the targeted delivery of lipid nanoparticles via the TfRl and / or FRalpha receptors is the first level of selectivity.

[0070] The transfection of cancer cells results in cancer destruction and anti-tumor immunity. Many different types of cancer overexpress receptors TfRl and / or FRalpha. For example, cells for melanoma, lung cancer , breast cancer , and colorectal cancer all overexpress receptors TfRl and / or FRalpha. TfRl has been recognized as a universal cancer receptor, and both TfRl and FRalpha have been widely targeted with anti-cancer drugs.

[0071] In this illustrative example, the transfection of cancer cells is done with a gene that codes for alpha- 1,3-GalT, which is an enzyme responsible for capping the glycans of glycoproteins and lipids with alpha-Gal, or a combination of alpha- 1,3- GalT and tetanus toxoid (TT) fragment C. The TT fragment C provides T cell epitopes for eliciting specific anti-alpha-Gal Abs in addition to the already existing anti-alpha-Gal Abs present in normal human serum. The transcription is controlled by a cell typespecific promoter such that the transcription cannot occur in any other tissues that arenot targeted. For example, the transcription will be controlled with a melanocytespecific tyrosinase promoter in melanoma. This is the second level of selectivity for transfecting only cancer cells.

[0072] As depicted, transfection of such TfRl and / or FRalpha-expressing cancer cells in vivo with the DNA gene ggtal (or mRNA) coding for alpha- 1,3 -galactosyl transferase (alpha- l,3GalT or an additional gene coding for tetanus toxoid fragment C) can occur through selective delivery by lipid nanoparticles equipped with TfRl- targeting ligands and FRalpha-binding folate. In addition, different cell specific promoters can be used for different types of cancer. For example, in the case of targeting melanoma with a plasmid DNA-carrying lipid nanoparticles, the transcription is under the control of a melanocyte-specific promoter such as tyrosinase promoter to add another level of selectivity. Thus, dual selectivity will cause transfection almost exclusively in melanoma cells.

[0073] The successfully transfected cancer cells will express the enzyme alpha- l,3GalT, which leads to massive galactosylation on N-glycans and glycolipids. As a result, N-glycans and glycolipids are now endcapped with alpha-Gal displayed on the exterior of each transfected cancer cell. In some illustrative examples, T cell epitopes are provided to elicit specific anti-alpha-Gal Abs for additional expression of TT fragment C.

[0074] In this illustrative example, cells with alpha-Gal are highly antigenic and become heavily opsonized by anti-alpha-Gal Abs. There are different possible sources of anti-alpha- Gal Abs, for example anti-alpha-Gal Abs present in all normal human sera, Chagas disease patients or elicited through immunization (e.g., through the expression of alpha-Gal-TT fragment C), and mono- or polyclonal anti-alpha-Gal Abs that are passively administered. All these anti-alpha-Gal Abs can opsonize the alpha- Gal-expressing cancer cells resulting in the rapid destruction of the cancer cells by complement-dependent cytotoxicity (CDC), Ab-dependent cell-mediated cytotoxicity (ADCC), and / or Ab-dependent cell-mediated phagocytosis (ADCP). In addition, ADCP leads to a secondary (adaptive) broad-spectrum anti-tumor immunity. In other words, the expression of alpha-Gal on cancer cells per se would make the tumor more visible to cells that are part of the innate immune cells resulting in tumor destruction and immunogenicity.

[0075] For example, Gala3LN can be expressed in very large quantities (-106 epitopes / cell) on melanoma cell surfaces through transduction with the al,3GalT gene(ggtal; UniProt accession # Q9DBU1). The fact that this epitope does not cause autoimmune conditions makes it an attractive antigen for tumor destruction. In mammals (except for humans and Old-World nonhuman primates), the biosynthesis of Gala3LN occurs enzymatically on N-glycans and glycolipids with terminal Galbl,4GlcNAc (LacNAc) residues.

[0076] In this illustrative example, lipid nanoparticles can be engineered with properties that control their biodistribution. For example, negatively charged lipid nanoparticles get trapped by macrophages while positively charged LNPs suffer from fast plasma clearance when intravenously delivered. It is therefore imperative that the lipid nanoparticles are neutral with a zeta potential near 0, which is controllable through the N / P ratio. In addition, controlling the size of the lipid nanoparticles is also critical. It is well established that the majority of lipid nanoparticles in blood circulation are removed by the reticuloendothelial system (RES), which is mainly composed of the liver and the spleen. Since the vessels of RES organs are fenestrated, lipid nanoparticles smaller than the fenestrae (~ 100 nm in diameter) can diffuse through to reach targets. Further, the plasma stability for the lipid nanoparticles can be increased by increasing polyethylene glycol (PEG) content in the lipid nanoparticles. In this illustrative example, a high PEG content protects lipid nanoparticles from non-specific protein binding, which would cause loss of their targeting abilities. Also, PEG prevents cellular uptake unless the lipid nanoparticles have targeting ligands for receptor- mediated endocytosis.

[0077] In this illustrative example, selective targeting of melanoma cells for the lipid nanoparticles can be achieved by equipping the lipid nanoparticles with the peptide hrpyiah (DT7) and folate on the surface to target the melanoma cells’ overexpressed transferrin and folate receptors, TfRl receptors and FRa receptors, respectively. TfRl receptors and FRa receptors have low expression levels in most healthy tissues, but are overexpressed by many cancers. TfRl receptors and FRa receptors have been targeted in many anti-cancer treatments because both receptors internalize their respective ligand and its attached cargo. In this illustrative example, equipping the lipid nanoparticles with DT7 and folate will direct the transfection predominantly to melanoma cells.

[0078] In addition, the transcription of the al,3GalT gene will be under the control of a melanocyte-specific promoter. As a result, uptake by tissues other than melanocytes will be ineffective. In this illustrative example, selectivity from peptidehrpyiah (DT7), folate, and melanocyte- specific promoter ensure that a-Gal expression will occur almost exclusively on melanoma cells.

[0079] In an alternative illustrative example, the proposed novel lipid nanoparticles with therapeutic agents can be used for treating neurological conditions. In this illustrative example, lipid nanoparticles targeting TfRl receptors can be used to deliver therapeutic agents across the blood-brain barrier (BBB) by binding TfRl receptors that are expressed on endothelial cells of the blood-brain barrier (BBB). TfRl-binding lipid nanoparticles of the disclosure can be used to treat neurological conditions in which expression of TfRl on the endothelial cells of BBB can facilitate delivery of a therapeutic to the brain, or other parts of the central nervous system by transcytosis. In this illustrative example, genetic disease mucopolysaccharidosis type II (Hunter syndrome) can be treated using the TfRl - binding LNPs to deliver the missing enzyme (or the gene encoding for the enzyme) iduronate- 2-sulfatase (IDS) across the BBB and into CNS cells as enzyme-replacement therapy. In some other illustrative examples, Pompe disease, Hurler-Scheie syndrome (MPS I), and Sanfilippo syndrome (MPS IIIA) can be treated with TfRl-binding lipid nanoparticles to deliver missing the enzymes (or the gene encoding for the enzymes) alglucosidase alfa, iaronidase, and heparansulfatase, respectively. In yet another illustrative example, TfRl-binding lipid nanoparticles can also be useful in the delivery of one or more therapeutic agents across the BBB for treating primary or secondary parkinsonism and Alzheimer’s disease.

[0080] In yet another alternative illustrative example, the proposed novel lipid nanoparticles with therapeutic agents can be used for treating muscular dystrophies. For example, TfRl-binding lipid nanoparticles can be used to deliver nucleic acids such as antisense oligonucleotides or siRNA to muscle cells to treat conditions such as Duchenne muscular dystrophy (DMD) and myotonic dystrophy.

[0081] In this illustrative example, TfRl-binding lipid nanoparticles can be used to treat muscular dystrophies by correcting defective proteins utilizing exon skipping through delivering nucleic acids such as antisense-oligonucleotide (AON) or siRNA into muscle cells. In this illustrative example, examples of muscular dystrophies can include Duchenne muscular dystrophy (DMD), myotonic dystrophy type 1 (DM1), and facioscapulohumeral muscular dystrophy (FSHD).

[0082] It should be understood that the proposed novel lipid nanoparticles can also deliver other nucleic acids such as other DNA, mRNA, siRNA, saRNAs, proteins, and any suitable small-molecule drugs for cells that express TfRl and / or FRalpha.Therefore, this technique can also be used for treating cancer in other ways, and it can be used for treating diseases other than the diseases mentioned above. For example, the proposed novel lipid nanoparticles can be used as a delivery vehicle for gene therapy that results in the expression of a particular protein that was missing.

[0083] This nanotechnology transfects specifically cancer cells with dual selectivity. Unlike standard chemotherapy, it minimizes damage to healthy cells and tissues. Cancer cells that are usually poorly immunogenic are made highly visible to the immune system. This technology takes full advantage of the capabilities of both the innate and adaptive immune system, thus eliminating cancer at all immunological fronts.

[0084] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the present disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present disclosure as defined by the appended claims.

Claims

CLAIMS:What is claimed is:

1. A composition of matter for targeted therapeutic delivery, comprising: one or more ionizable cationic lipids and permanently cationic lipids; a mixture of one or more helper lipids; and one or more lipids with ligands that target receptors for cells.

2. The composition of claim 1 , wherein the composition is used for delivering one or more nucleic acids to a cell that contains the receptors targeted by the one or more lipids with the ligands.

3. The composition of claim 2, wherein the one or more nucleic acids comprise at least one of a mRNA, a siRNA, a plasmid DNA, a small activating RNA(saRNAs) or a combination thereof.

4. The composition of claim 1 , wherein the mixture of one or more helper lipids comprises at least one of cholesterol, at least one of PEGylated lipids or polysarcosinylated lipids used for preventing aggregation and protein-binding, and one or more non-PEGylated and non-polysarcosinylated phospholipids, or a combiantion thereof.

5. The composition of claim 4, wherein the at least one of PEGylated lipids or polysarcosinylated lipids comprise at least one of l,2-dimyristoyl-sn-glycero-3- succinyl-N-polysarcosine-25, 1 ,2-dimyristoyl-rac-glycero-3 -methoxypoly ethylene glycol-2000, 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethyleneglycol)-2000], or a combination thereof.

6. The composition of claim 4, wherein the one or more non-PEGylated and non- polysarcosinylated phospholipids comprise at least one of l-palmitoyl-2-oleoyl-sn- glycero-3 -phosphocholine, 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- distearoyl-sn-glycero-3-phosphocholine, or a combination thereof.

7. The composition of claim 4, wherein the one or more non-PEGylated and non- polysarcosinylated phospholipids is present in the composition with an amount that ranges from 5 mol% to 12 mol% of the composition.

8. The composition of claim 4, wherein the cholesterol is present in the composition with amount that ranges from 20 mol% to 50 mol% of the composition.

9. The composition of claim 4, wherein the at least one of PEGylated lipids or polysarcosinylated lipids are present in the composition with amount that ranges from 2 mol% to 5 mol% of the composition.

10. The composition of claim 1, wherein the one or more lipids with ligands are present in the composition with an amount that ranges from 0.2 mol% to 2 mol% of the composition.

11. The composition of claim 1 , wherein the one or more ionizable cationic lipids and permanently cationic lipids are present in the composition with amount that ranges from 45 mol% to 55 mol% of the composition.

12. The composition of claim 1, wherein the one or more ionizable cationic lipids and permanently cationic lipids comprise at least one of heptadecan-9-yl 8-((2- hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31 -tetraen- 19-yl 4-(dimethylamino)butanoate, 2-(2,2-di((9Z,12Z)- octadeca-9,12-dien-l-yl)-l,3-dioxolan-4-yl)-N,N-dimethylethan-l -amine, 1 ,2-dioleoyl- 3-dimethylammonium-propane, [(4-Hydroxybutyl)azanediyl]di(hexane-6, 1 -diyl) bis(2- hexyldecanoate), Didodecyldimethyl ammonium bromide, or a combiantion thereof.

13. The composition of claim 1, wherein the one or more lipids with ligands target transferrin receptor l(TfRl) of cells.

14. The composition of claim 1, wherein the one or more lipids with ligands target folate receptor alpha (FRalpha) of cells.

15. The composition of claim 1, wherein the one or more lipids with ligands target transferrin receptor l(TfRl) of cells and folate receptor alpha (FRalpha) of cells simultaneously.

16. A method of targeted therapeutic delivery for treating a disease, comprising: administering to a mammal in need thereof a compound comprising: one or more ionizable cationic lipids; a mixture of one or more helper lipids; one or more lipids with ligands that target receptors for cells; and one or more nucleic acids.

17. The method of claim 16, wherein administering comprises at least one of intravenous or intratumoral administration.

18. The method of claim 16, wherein the one or more lipids with ligands target transferrin receptor l(TfRl) of cells.

19. The method of claim 16, wherein the one or more lipids with ligands target folate receptor alpha (FRalpha) of cells.

20. The method of claim 16, wherein the one or more lipids with ligands target transferrin receptor l(TfRl) of cells and folate receptor alpha (FRalpha) of cells simultaneously.

21. The method of claim 16, wherein the mixture of one or more helper lipids comprises at least one of cholesterol, at least one of PEGylated lipids or polysarcosinylated lipids, and one or more non-PEGylated and non-polysarcosinylated phospholipids, or a combiantion thereof.

22. The method of claim 16, wherein the compound is used for in vitro transfections for cells with receptors that are targeted by the one or more lipids with ligands.

23. The method of claim 16, wherein the compound administered to the mammal is used to treat cancer.

24. The method of claim 16, wherein the compound administered to the mammal is used to treat neurologic disorders.

25. The method of claim 16, wherein the compound administered to the mammal is used to treat muscular disorders.

26. A method of targeted therapeutic delivery for a cancer therapy, comprising: administering to a mammal in need thereof a compound comprising: one or more ionizable cationic lipids; a mixture of one or more helper lipids; one or more lipids with ligands that target receptors for cells; and and one or more nucleic acids, wherein the one or more nucleic acids causes cells of the mammal to express glycan.

27. The method of claim 26, wherein the mixture of one or more helper lipids comprises at least one of cholesterol, at least one of PEGylated lipids or polysarcosinylated lipids, and one or more non-PEGylated and non-polysarcosinylated phospholipids, or a combiantion thereof.

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