Liposomal particles, methods for their production and their uses

Abstract

It is possible to synthesize liposomes known as "small unilamellar vesicles" (SUVs) in the 20–50 nm size range, but they face obstacles such as instability and aggregation leading to fusion between particles. This limits their use as a therapeutic delivery agent. Increasing the negative surface charge of SUVs by binding anionic entities such as DNA / RNA enhances the colloidal stability of these vesicles. Additionally, the dense spherical arrangement and radial orientation of nucleic acids exhibit unique biological and chemical properties, unlike their linear counterparts. These liposomal particles are non-toxic and, although anionic, can efficiently enter cells without the aid of cationic transfection agents in a non-immunogenic manner.These exceptional properties allow their use as delivery agents for gene regulation in different therapies and offer an alternative platform to metal-core spherical nucleic acids.

Description

This application claims the benefit of priority under Title 35 §119(e) of the United States Code of U.S. Provisional Application No. 61 / 911 334 filed on December 3, 2013, and U.S. Provisional Application No. 61 / 982 269 filed on April 21, 2014, the descriptions of which are incorporated herein in their entirety by reference. DECLARATION OF GOVERNMENTAL INTEREST The present invention was made with government support pursuant to HR0011-13-20018 granted by the Defense Advanced Research Projects Agency and CA151880 granted by the National Institutes of Health. The government has certain rights with respect to the invention. LIST OF SEQUENCES This application contains, as a separate part of the description, a computer-readable sequence listing (filename: 2013-201_SeqListing.txt. Created: December 3, 2014. 1893 bytes), which is incorporated in its entirety by this reference. FIELD OF INVENTION This description refers to liposomal particles, methods for their preparation, and their uses. Liposomal particles are useful in gene regulation and drug delivery. BACKGROUND OF THE INVENTION Chemistry has been explored to create liposomes and small unilamellar vesicles (SUVs). For example, Vogel et al., DNA Controlled Assembly of Lipid Membranes, U.S. Patent Publication No. 2010 / 0144848, describes how DNA modified with two lipophilic anchors can form liposomes or SUVs. This post-modification technique does not favor high surface density modification. Hook et al., Oligonucleotides Related to Lipid Membrane Attachment, U.S. Patent Publication No. 2013 / 0252852, describes liposomes or SUVs created with an oligonucleotide having a first and second nucleic acid strand and two or more hydrophobic anchoring residues located at its ends, wherein the hydrophobic anchoring residues are found in the bilayer. Since two cholesterol molecules are used to anchor a molecule to the lipid bilayer, this post-modification technique does not favor high surface density modification. Lu et al., Amphiphilic Substances and Functionalized Lipid Vesicles Including the Same, US patent publication 2010 / 0166842, describes liposomes or SUVs that comprise at least two nucleotide segments hybridized to each other. This vesicle, based on the non-post modification technique, is less effective in vesicle stabilization because it incorporates stabilizing residues on both sides of the lipid bilayer. Non-patent literature also reveals chemical methods for creating liposomes and SUVs, but each of these chemical methods has its own drawbacks. For example, "Liposome-Anchored Vascular Endothelial Growth Factor Aptamers," Bioconjugate Chem., 1998, 9, 573-582, describes the synthesis of liposomes functionalized with aptamer DNA and their application in selective targeting of cancer cells. The liposomes created using this method had an average size of 80 nanometers, contained aptamer DNA molecules on both sides of the bilipid layer, and did not demonstrate gene regulation. "Reversible Cell-Specific Drug Delivery with Aptamer-Functionalized Liposomes," Angew. Chem. Int. Ed. 2009, 48, 6494-6498, describes the synthesis of liposomes functionalized with aptamer DNA and their application in selectively targeting cancer cells and delivering drugs. The liposomes created using this method have an average size of between 140 and 200 nanometers, use a cholesterol unit to anchor the DNA in the lipid bilayer, comprise aptamer DNA molecules on both sides of the bilipid layer, and did not exhibit gene regulation. “Selective delivery of an anticancer drug with aptamer-functionalized liposomes to breast cancer cells in vitro and in vivo,” J. Mater. Chem. B, 2013, 1, 5288, describes the synthesis of aptamer DNA-functionalized liposomes and their application in selectively targeting cancer cells and delivering the drug. This work is an extension of the research described in “Reversible Cell-Specific Drug Delivery with Aptamer-Functionalized Liposomes,” mentioned above. As before, these liposomes use a cholesterol unit to anchor the DNA in the lipid bilayer, comprise aptamer DNA molecules on both sides of the bilipid layer, and did not exhibit gene regulation. The research in "Phospholipid Membranes Decorated by Cholesterol-Based Oligonucleotides as Soft Hybrid Nanostructures," J. Phys. Chem. B, 2008, 112, 10942-10952, characterizes liposomes functionalized with cholesterol DNA. In this report, liposomes measuring 33 to 35 nm were prepared from the lipid 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and subsequently functionalized with the cholesterol-modified DNA molecule. This report does not demonstrate gene regulation, and these particles utilize a cholesterol unit to anchor the DNA to the lipid bilayer. "Bivalent Cholesterol-Based Coupling of Oligonucleotides to Lipid Membrane Assemblies," J. Am. Chem. Soc. 2004, 126, 10224-10225, describes the development of partially double-stranded DNA containing two cholesterol units for attachment to the lipid bilayer. The use of two cholesterol units to attach a DNA strand to the lipid bilayer results in a reduction of the surface density of liposome-associated oligonucleotides. In “Quantification of Oligonucleotide Modifications of Small Unilamellar Lipid Vesicles,” Anal. Chem. 2006, 78, 7493–7498, researchers describe the development of a technique for quantifying DNA strands in a functionalized liposomal nanoparticle. The described particle comprises a partially double-stranded DNA containing two cholesterol units for attachment to the lipid bilayer. The use of two cholesterol units to attach a DNA strand to the lipid bilayer results in a reduction of the surface density of oligonucleotides associated with the liposome. “Single-Molecule Detection and Mismatch Discrimination of Unlabeled DNA Targets,” Nano Lett. 2008, 8, 183–188, describes 100-nanometer liposomes functionalized with a partially double-stranded DNA molecule containing two cholesterol units. This work is an extension of the research described in “Bivalent Cholesterol-Based Coupling of Oligonucleotides to Lipid Membrane Assemblies” and “Quantification of Oligonucleotide Modifications of Small Unilamellar Lipid Vesicles,” mentioned above. As before, these particles comprise a partially double-stranded DNA molecule containing two cholesterol units for attachment to the lipid bilayer. The use of the two cholesterol units to attach a DNA molecule to the lipid bilayer results in a reduction of the surface density of oligonucleotides associated with the liposome. "DNA-Induced Programmable Fusion of Phospholipid Vesicles," J. Am. Chem. Soc. 2007, 129, 9584-9585, is an analytical work on the fusion of liposomal nanoparticles functionalized with cholesterol DNA. The vesicles used in this work were at least 100 nanometers in size. “Determinants for Membrane Fusion Induced by Cholesterol-Modified DNA Zippers,” J. Phys. Chem. B, 2008, 112, 8264–8274, is an analytical work on the fusion of liposomal nanoparticles functionalized with cholesterol DNA and is a continuation of the work on “DNA-Induced Programmable Fusion of Phospholipid Vesicles” described above. This work combines sequence-specific fusion with the use of a partially double-stranded DNA containing two cholesterol units to anchor the oligonucleotide to the lipid bilayer (e.g., the partially double-stranded DNA found in “Quantification of Oligonucleotide Modifications of Small Unilamellar Lipid Vesicles” previously). “Liposome-Based Chemical Barcodes for Single Molecule DNA Detection Using Imaging Mass Spectrometry,” Nano Lett., 2010, 10, 732–737, is an analytical work on the detection of specific DNA targets according to the DNA sequence. This is an extension of the work by the same group that reported “DNA-Induced Programmable Fusion of Phospholipid Vesicles,” which combines sequence-specific fusion with a different DNA anchor (with a bischolesteryl anchor, see: Anal. Chem. 2006, 78, 7493–7498). "Programmable Assembly of DNA-Functionalized Liposomes by DNA" is an analytical work that describes the assembly of cholesterol-functionalized DNA liposomes. In this report, liposomes with hydrodynamic diameters of 114 and 251 nm were synthesized and subsequently functionalized synthetically with cholesterol-modified DNA molecules. The particles in this report utilize cholesterol anchoring of the oligonucleotide molecule to the lipid bilayer. BRIEF DESCRIPTION OF THE INVENTION Liposomes are spherical, self-enclosed structures of varying sizes consisting of one or more hydrophobic lipid bilayers with a hydrophilic core. The diameter of these lipid-based carriers ranges from 0.15 to 1 micrometer, which is considerably larger than the effective therapeutic range of 20–100 nanometers. Liposomes known as small unilamellar vesicles (SUVs) can be synthesized in the 20–50 nanometer size range, but they face challenges such as instability and aggregation, leading to interparticle fusion. This interparticle fusion limits the use of SUVs in therapies. To combat this instability, SUVs can be functionalized with polymers, peptides, DNA, and other molecules of interest using two different techniques. In the first approach, a modified molecule of interest is added to the lipid mixture, lipid film, or hybridization buffer solution during liposome synthesis. This approach results in a liposome containing a functional molecule of interest in both the inner and outer layers of the liposomal membrane. Generally speaking, structures created by this method are not stable at sizes smaller than 80 nanometers (nm). In an alternative approach, an SUV can be fabricated by anchoring a substrate of interest to the lipid bilayer of the preformed vesicle (a "post-modification technique"). This alternative approach yields a liposomal nanoparticle containing a functional molecule of interest in the outer layer of the liposomal membrane.It is worth noting that this alternative post-modification approach makes it possible to create liposomes of any size, even less than 50 nanometers. Therefore, in one respect, the description provides an architecture comprising a lipophilic end and a non-lipophilic end. The lipophilic end, in some forms, comprises tocopherol. In additional forms, the tocopherol is selected from the group consisting of alpha-tocopherol, beta-tocopherol, gamma-tocopherol, and delta-tocopherol. The non-lipophilic end, in some embodiments, is a charged polymer. In some embodiments, the charged polymer is an oligonucleotide. In related embodiments, the oligonucleotide comprises either RNA or DNA, and in several embodiments, the RNA is an inhibitory RNA (RNAi) that performs a regulatory function. In still other embodiments, the RNAi is selected from the group consisting of a small inhibitory RNA (sRNAi), an RNA that forms a triple nRfrnnn / zznz / E / YiAi structure with double-stranded DNA, and a ribozyme. In additional embodiments, the RNA is a Piwi-associated RNA (Piwi-RNA), or the RNA is a microRNA that performs a regulatory function. In some embodiments, the DNA is antisense DNA. In another aspect, the description provides a method for producing a description architecture comprising the provision of an oligonucleotide, the provision of a phosphoramidite-modified tocopherol, and the exposure of said oligonucleotide to said phosphoramidite-modified tocopherol to produce a description architecture. In an additional aspect, the description provides a liposomal particle with a substantially spherical geometry and comprising a lipid bilayer comprising multiple lipid groups and an oligonucleotide. The description contemplates, in several modalities, that such multiple lipid groups comprise a lipid selected from the group consisting of the lipid family of phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. In several forms, this lipid is selected from the group consisting of 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC), 1,2-dimiristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoylsn-phosphatidylcholine (POPC), 1,2-distearol-sn-glycero-3-phospho-(r-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1 '-rac-glycerol) (DOPG), 1,2-distearol-sn-glycero-3-phosphocholine (DSPG), 1,2-dpalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE). In additional embodiments, the oligonucleotide is a lipid-oligonucleotide conjugate containing a bound lipophilic group, wherein said bound lipophilic group is adsorbed onto the lipid bilayer. In several embodiments, the bound lipophilic group comprises tocopherol or cholesterol. The description also states that tocopherol, in various forms, is selected from the group consisting of a tocopherol derivative, alpha-tocopherol, beta-tocopherol, gamma-tocopherol, and delta-tocopherol. In still other forms, the description also states that the attached lipophilic group (i.e., the anchor lipid) comprises, for example, and not exhaustively, palmitoyl, dipalmitoyl, stearyl, or distearyl. In other embodiments, the oligonucleotide comprises either RNA or DNA. In additional embodiments, the RNA is non-coding RNA, and in still others, the non-coding RNA is inhibitory RNA (RNAi). The description further states that, in some embodiments, the RNAi is selected from the group consisting of a small inhibitory RNA (sRNA), a single-stranded RNA (sRNA) that forms a triplet structure with double-stranded DNA, and a ribozyme. In additional embodiments, the RNA is microRNA. In some embodiments, the DNA is antisense DNA. In several embodiments, the diameter of said liposomal particle is less than or equal to 50 nanometers. With respect to surface density, the description provides compositions and methods in which a liposomal particle comprises from about 10 to about 100 oligonucleotides or from about 10 to about 80 oligonucleotides. In some embodiments, the particle comprises 70 oligonucleotides. In some forms, the antisense oligonucleotide is a modified oligonucleotide. In another aspect of the description, a method is provided for producing a liposomal particle comprising adding a phospholipid to a solvent to form a first mixture comprising multiple liposomes, perturbing said multiple liposomes to create a second mixture comprising a liposome and a small unilamellar vesicle (SUV), isolating said SUVs with a particle size of between about 20 nanometers and 50 nanometers from said second mixture, and adding an oligonucleotide to the isolated SUVs to produce the liposomal particle. In some embodiments, the particle size of multiple liposomes in the first mixture is between approximately 100 nanometers and 150 nanometers. In additional embodiments, the particle size of the liposome and SUV in the second mixture is between approximately 20 nanometers and approximately 150 nanometers. In still other embodiments, the liposomal particle has a particle size less than or equal to approximately 50 nanometers. In some embodiments, the oligonucleotide is a lipid-oligonucleotide conjugate containing a bound lipophilic group, wherein said bound lipophilic group is adsorbed onto the lipid bilayer. In related embodiments, the bound lipophilic group comprises tocopherol and cholesterol. In other embodiments, the tocopherol is selected from the group consisting of a tocopherol derivative, alpha-tocopherol, beta-tocopherol, gamma-tocopherol, and delta-tocopherol. In additional embodiments, the oligonucleotide comprises either RNA or DNA. In some embodiments, the RNA is non-coding RNA. In additional embodiments, the non-coding RNA is inhibitory RNA (RNAi). The description further stipulates that, in additional embodiments, the RNAi is selected from the group consisting of a small inhibitory RNA (sRNA), a single-stranded RNA (sRNA) that forms a triple structure with double-stranded DNA, and a ribozyme. In some forms, the RNA is microRNA. In several forms, the DNA is antisense DNA. In some forms, the antisense oligonucleotide is a modified oligonucleotide. In another aspect of the description, a method is provided for inhibiting the expression of a gene comprising the hybridization step of a polynucleotide encoding said gene product with one or more oligonucleotides complementary to all or part of said polynucleotide and attached to the liposomal particle of the description, wherein said polynucleotide and said oligonucleotide hybridize into an extension of said polynucleotide with a degree of complementarity sufficient to inhibit the expression of said gene product. In some modalities, the expression of this gene product is inhibited in vivo. In additional modalities, the expression of this gene product is inhibited in vitro. In additional embodiments, the liposomal particle has a diameter of approximately 50 nanometers or less. In some embodiments, the oligonucleotide comprises either RNA or DNA. In some embodiments, the RNA is non-coding RNA. In related embodiments, the non-coding RNA is inhibitory RNA (RNAi). In several embodiments, the description also specifies that the RNAi is selected from the group consisting of a small inhibitory RNA (sRNA), a single-stranded RNA (sRNA) that forms a triplet structure with double-stranded DNA, and a ribozyme. In some embodiments, the RNA is a microRNA. In additional embodiments, the DNA is antisense DNA. In another aspect of the description, a method is provided for positively regulating the activity of a Toll-like receptor (TLR) comprising placing a cell having a Toll-like receptor in contact with a liposomal particle of the description. In some embodiments, the oligonucleotide is a TLR agonist. In other embodiments, the Toll-like receptor is selected from the group consisting of Toll-like receptor 1, Toll-like receptor 2, Toll-like receptor 3, Toll-like receptor 4, Toll-like receptor 5, Toll-like receptor 6, Toll-like receptor 7, Toll-like receptor 8, Toll-like receptor 9, Toll-like receptor 10, Toll-like receptor 11, Toll-like receptor 12, and Toll-like receptor 13. In an additional aspect, the description provides a method for negatively regulating the activity of a Toll-like receptor by bringing a cell with a Toll-like receptor into contact with a liposomal particle of the description. In some embodiments, the oligonucleotide is a TLR antagonist. In other embodiments, the Toll-like receptor is selected from the group consisting of Toll-like receptor 1, Toll-like receptor 2, Toll-like receptor 3, Toll-like receptor 4, Toll-like receptor 5, Toll-like receptor 6, Toll-like receptor 7, Toll-like receptor 8, Toll-like receptor 9, Toll-like receptor 10, Toll-like receptor 11, Toll-like receptor 12, and Toll-like receptor 13. In several instances, the description also includes the possibility of implementing a method, as described herein, in vitro. In other instances, the description includes the possibility of implementing a method, as described herein, in vivo. BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates the synthesis of small unilamellar vesicles (SUVs) functionalized with DNA or RNA on the surface of the lipid vesicle. Larger liposomes are sonicated into SUVs using a probe sonicator and separated from heavy impurities by ultracentrifugation. Figure 2 demonstrates the characterization of liposomal particles from small unilamellar vesicles (SUVs). Particle size data were obtained by dynamic light scattering (DLS) and images were obtained by transmission electron microscopy (TEM) before and after functionalization. Figures 3A-3C demonstrate the stability of liposomal particles stabilized with oligonucleotides having different lipophilic ends for anchoring the oligonucleotide to the liposome. Liposomes stabilized with tocopherol-modified oligonucleotides show better stability than liposomes alone, liposomes stabilized with cholesterol-modified oligonucleotides, and liposomes stabilized with stearyl-modified oligonucleotides. a) Gel electrophoresis image of FITC-encapsulated SUVs functionalized with oligonucleotides having different lipophilic ends; b) and c) Gel electrophoresis images of FITC-encapsulated SUVs functionalized with Cy5-labeled DNA. Figures 4A-4B demonstrate that liposomal particles stabilized with oligonucleotides exhibit good temperature stability and display the range of tocopherol-modified DNA concentrations used to stabilize the SUVs. a) Stability of liposomal ANS (LSNA) after storage at 37°C for 24 hours compared to LSNA stored at 4°C. b) Gel electrophoresis showing the range of α-tocopherol-modified DNA concentrations used to stabilize the SUVs. Figure 5 comprises confocal images demonstrating that the liposomal particles described herein are capable of entering cells. HeLa cells were treated with DNA (dT3o-Cy5 or dTso) at a concentration of 100 nM in serum-free media and analyzed after 16 hours. Figure 6 illustrates cell viability assay data demonstrating that liposomes stabilized with tocopherol-modified oligonucleotides do not exhibit a substantial cytotoxic effect on cells compared to liposomes that remain unmodified. Figure 7 illustrates the assembly of liposomal spherical nucleic acids (ANS) from a DOPC SUV and tocopherol-modified DNA. Figures 8A-8D illustrate stability studies of SUVs and LSNAs. (A) Dynamic light scattering profile of SUVs after heating in the buffer solution. (B) Dynamic light scattering profile of LSNAs after heating in the buffer solution. (C) Schematic representation of liposome decomposition in the presence of bovine serum albumin, a major component of fetal bovine serum. (D) Degradation of SUVs (upper trace) and LSNAs (lower trace) in the presence of 10% fetal bovine serum, as controlled by the release of encapsulated rhodamine dye, which causes increased fluorescence of the solution. Figures 9A-9B show (A) absorbance-controlled melting transition of liposomal ANS aggregates nAfrnnn / zznz / E / YiAi at 260 nm. (B) Absorbance spectrum of liposomal ASNs before aggregation (lower trace) and after aggregation in the presence of the linker DNA strand (upper trace). Figures 10A-10D show (A) confocal micrographs of SKOV3 cells incubated with 100 nM Cy5-labeled liposomal ANS for 24 hours. Cell nuclei are stained with Hoechst 33342. (B) Cytotoxicity measurements of liposomal ANS and the DharmaFECT-DNA complex in SKOV3 cells using the MTT assay. (C) Cellular uptake of the 5'-Cy5-labeled DNA strand and the 5'-Cy5-labeled liposomal ANS in SKOV3 cells quantified by flow cytometry after 1 hour (left bar in each group) and 36 hours (right bar in each group) of incubation. (D) HER2 gene inactivation in SKOV3 cells with anti-HER2 liposomal ANS constructs at a DNA concentration of 1 pM. Figure 11 illustrates a TEM micrograph of the SUVs after isolation and purification. Figures 12A-12B illustrate A) the equation used to calculate the total number of liposomes in a given solution. The lipid concentration can be determined by ICP. For most of the studies described herein, a working lipid concentration of 1.3 mM yields 1.361 × 10¹⁷ liposomes / L and a DNA loading of 71 strands of DNA per particle (4 pmol cm⁻²). B) Mobility of liposomal ANS particles according to the estimated oligonucleotide loading. Figures 13A-13C show the movement of FITC-encapsulated LSNAs functionalized with the 5'-Cy5-labeled DNA strand in a 1% agarose gel electrophoresis image. B) FITC channel showing the movement of the liposomal core in the gel due to the presence of the negatively charged DNA crown. C) Cy5 channel showing the difference in mobility due to size differences between a free strand and the functionalized strands in the liposomal construct. Both channels are located together in the same band. Figure 14 illustrates the Ramos-Blue™ NF-kB / AP-1 indicator system. Figure 15 shows the activation of Ramos-Blue cells when exposed to CpG-containing oligonucleotides. Figure 16 is a graphical representation of the synthesis of a liposomal ANS. Figure 17 shows an electrophoresis image of liposomal ANS genes functionalized on the surface with different amounts of oligonucleotides. The SUV concentration at 30 nm was 0.22 μM (determined by elemental analysis of phospholipid content and approximation of 2.2 × 10³ phospholipids per 30 nm SUV). Figure 18 shows the results of experiments in which liposomal particles were used to inactivate HIF1-α expression. Figure 19 shows the results of experiments in which liposomal nAbnnn / zznz / B / YiAi particles were used to inactivate BAX expression. DETAILED DESCRIPTION OF THE INVENTION Spherical nucleic acid nanoparticle conjugates (SNBs) are structures typically synthesized from inorganic nanoparticle templates and shells of highly oriented nucleic acid ligands immobilized on the surface of such particles [Mirkin et al., Nature 382: 607 (1996)]. SNBs have been prepared in a variety of different forms [Cutler et al., J. Am. Chem. Soc. 134: 1376 (2012); Will et al., In Nanomaterials for Biomedicine; American Chemical Society: Vol. 1119, pp. 1–20 (2012)]. Core compositions include gold, silica [Young et al., Nano Lett. 12:3867 (2012)], iron oxide [Cutler et al., Nano Lett. 10:1477 (2010); Zhang et al., Nat. Mater. 12:741 (2013)] and Ag [Lee et al., Nano Lett. 7: 2112 (2007)] with shell compositions consisting of DNA, RNA, ANB [Seferos et al., ChemBioChem 8: 1230 (2007)] and PNA [Lytton-Jean et al., Advanced Materials 21: 706 (2009)] have been prepared and explored in full.Hollow ANS structures consisting of crosslinked oligonucleotides [Cutler et al., J. Am. Chem. Soc. 133: 9254 (2011)] have been synthesized in conjunction with micelle-blocked copolymer structures [Li et al., Nano Lett. 4: 1055 (2004); Alemdaroglu et al., Advanced Materials 20: 899 (2008); Liu et al., Chemistry - A European Journal 16: 3791 (2010); Chien et al., Chem. Commun. 47:167 (2011)]. Although there is currently a great deal of structural and compositional diversity among known ANS, they all share some common properties and characteristics. Their polyvalent architectures allow for the cooperative joining of oligonucleotides and the formation of double structures that exhibit very tight fusion transitions. These properties have been exploited in the development of genomic detection systems with high sensitivity and high selectivity [Rosi et al., Chem. Rev. 105: 1547 (2005)].Although linear nucleic acids do not enter cells properly without polymers, peptides, or viral transfection agents, the three-dimensional structure of ANS is recognized by class A scavenger receptors [Patel et al., Bioconjugate Chem. 21: 2250 (2010); Choi et al., Proc. Nati. Acad. Sci. USA 110: 7625 (2013)] and is rapidly carried into more than 60 different cell types without the need for an auxiliary transfection agent [McAllister et al., J. Am. Chem. Soc. 124: 15198 (2002); Whitehead et al., Nat Rev Drug Discov8: 129 (2009); Zhang et al., Biomaterials 31: 1805 (2010)]. This property has made such structures important elements in both intracellular detection strategies [Zheng et al., Nano Lett. 9: 3258 (2009); Prigodich et al., ACS Nano 3: 2147 (2009)] and gene regulation via siRNA or antisense pathways [Rosi et al., Science 312: 1027 (2006); Agbasi-Porter et al., Bioconjugate Chem. 17: 1178 (2006); Giljohann et al., J. Am. Chem. Soc.131: 2072 (2009); Jensen et al., Science Translational Medicine 5: 209ra152 (2013)]. However, the barrier to therapeutic use is high, especially when such structures are made of materials with known clearance problems or unknown biodistribution characteristics. Ideally, an ANS structure produced from readily available starting materials that can be synthesized at scale and consists of components that have been part of FDA-approved pharmaceutical products would be preferable [Cutler et al., J. Am. Chem. Soc. 134: 1376 (2012); Farokhzad et al., Drug Delivery Rev. 58:1456 (2006)]. A strategy for producing such structures is provided herein, consisting of small liposomal cores stabilized with a dense shell of a loaded polymer with a hydrophobic tail that can be intercalated between the phospholipids that define the liposome structure. One of these charged polymers being considered for use is a nucleic acid.As with conventional ANS, these liposomal structures rapidly enter multiple cell lines and in some modalities are used to effectively inactivate gene expression through antisense pathways. Conventional ANS have been shown to enter cells derived from many organs and tissues including breast (SKBR3, MDA-MB-231, AU-565), brain (U87, LN229, U118), bladder (HT-1376, 5637, T24), colon (LS513), cervix (HeLa, SIHa), skin (C166, KB, MCF 10A), kidney (MDCK), brain (rat hippocampal neurons, astrocytes, glial cells), bladder, blood (PBMC, T lymphocytes), pancreas (human β-islets), skin (human), blood (Sup T1, Jurkat), leukemia (K562), liver (HepG2), kidney (293T), ovaries (CHO), fibroblast (NIH3T3), macrophage (RAW264.7). The architecture of spherical nucleic acids facilitates the entry of these constructs into cells by binding to the scavenger receptor A of a cell membrane receptor.Some non-exhaustive examples of cell lines that express this receptor are HeLa, SKOV-3, U87, Neuro 2A, RAW cells, HepG2, Hep3B, MDA-MB-468, MCF-7, C8S, C166 Bend3, A549, Rab9, HeyA8, Jurkat cells. The major drawback of using SUVs is their inherent instability in solution due to their high propensity to fuse into larger liposomal structures. This paper describes how functionalizing these structures with a dense layer of negatively charged DNA increases their stability, for example, by reducing particle-particle interaction due to the repulsion of the negatively charged particle surfaces. In the studies described herein, tocopherol-functionalized DNA was found to provide a higher density of DNA strands in the particle compared to other hydrophobic DNA analogs, significantly increasing particle stability. In addition to overall colloidal stability, the high DNA density will enhance the uptake of this nanoparticle via a refined receptor B pathway, enabling efficient delivery of genetic material to a cell.Finally, the dense DNA layer is expected to increase particle stability in a body, its circulation speed, and thus improve the biodistribution of this nanomedicine. This statement indicates that by increasing the negative charge of the SUV surface through the joining of anionic entities including, but not limited to, DNA and nAfrnnn / zznz / E / YiAi RNA enhances the colloidal stability of these vesicles. Additionally, the dense spherical arrangement and radial orientation of the nucleic acids exhibit unique biological and chemical properties, unlike their linear counterparts. These spherical nucleic acids (ANS) are non-toxic and, although anionic, can efficiently enter cells without the aid of cationic transfection agents in a non-immunogenic manner. Such exceptional properties make them suitable as delivery agents for gene regulation in various therapies. Liposome template-mediated ANS synthesis provides an alternative platform to metal-core ANS, which limits the therapeutic diversity of ANS with metal core bioaccumulation and the inability to encapsulate therapeutic entities. Further descriptions herein are provided of tocopherol-modified oligonucleotides, liposomal particles and methods for their preparation, and the uses of liposomal particles. In fact, the description can be incorporated in many different forms and should not be considered as limiting the embodiments stated herein. These embodiments are provided in sufficient detail in writing to describe and enable a person skilled in the art to prepare and use the invention, together with a description of the best way to implement the invention, as defined by the claims and their equivalents. Furthermore, many modifications and other variations of the methods described herein will occur to a person skilled in the art to which the invention belongs, with the benefit of the indications presented in the preceding descriptions and associated drawings. It is therefore understood that the invention is not to be limited to the specific variations described and that modifications and other variations are intended to be included within the scope of the appended claims. Although specific terms are used herein, they are used in a generic and descriptive sense only and are not intended to be exhaustive. Terminology Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the invention pertains. While in practice or in evaluating the invention any method and material similar or equivalent to those described herein may be used, the preferred methods and materials are described herein. Certain terms are defined first. Additional terms are defined throughout the descriptive report. For the sake of brevity, a description of a modality in terms of a small unilamellar vesicle (SUV), a liposomal ANS (LSNA), a liposomal particle, or a spherical nucleic acid (ANS) may also be applied to a modality that uses any of the preceding terms. By way of example, a method of gene expression regulation using a liposomal ANS may also be described herein as a method of gene expression regulation using a liposomal particle. Small unilamellar vesicles (SUVs) are liposomal particles less than 100 nanometers in size and are used as precursors to LSNAs. As such, SUVs and LSNAs may be considered subclasses of liposomal particles. The expressions used herein are intended to be "open" terms (for example, the expression "which includes" shall be interpreted as "which includes in a non-exhaustive way", the term "has" shall be interpreted as "has at least", the term "includes" shall be interpreted as "includes in a non-exhaustive way", etc.). Furthermore, in cases where a convention analogous to "at least one of A, B, and C, etc." is used, such a construction is generally used in the same sense that a person skilled in the art would understand the convention (for example, "a system with at least one of A, B, and C" would include, but not be limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together). Additionally, those skilled in the art will understand that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or in the figures, encompasses the possibility of including one of the terms, any of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A or B" or "A and B." All vocabulary such as "from", "to", "until", "at least", "greater than", "less than" and the like includes the expressed quantity and refers to intervals that can later be broken down into subintervals, as previously discussed. A range includes each individual member. Therefore, for example, a group that has 1-3 members refers to groups that have 1, 2, or 3 members. Similarly, a group that has 6 members refers to groups that have 1, 2, 3, 4, or 6 members, and so on. The verb "can" refers to the use or preferred selection of one or more options or choices among the various described modalities or characteristics. When no options or choices are described with respect to a particular modality or characteristic, the verb "can" refers to an affirmative action regarding how to do or use an aspect of a described modality or characteristic, or a definite decision to employ a specific skill with respect to a described modality or characteristic. In this latter context, the verb "can" has the same meaning and connotation as "has the ability to." As used herein, the articles "a" and "an" refer to one or more of one (for example, at least one) of the grammatical objects. Generally, "around" and "approximately" will mean an acceptable degree of error for the measured quantity, given the nature or precision of the measurements. Example error levels are between 20-25 percent (%), typically at 10%, and more commonly at 5% of a given value or range of values. The chemical structures described herein are named according to IUPAC nomenclature rules and include common names and abbreviations, where appropriate, accepted in the art. IUPAC nomenclature can be derived using chemical structure drawing software programs such as ChemDraw® (PerkinElmer, Inc.), ChemDoodle® (ChemLabs, LLC), and Marvin (ChemAxon Ltd.). The chemical structure governs the description to the extent that an IUPAC name is misused or otherwise conflicts with the chemical structure described herein. The headings, for example, (A), (B), (i), etc., are provided simply to facilitate reading of the specification and the claims. The use of the headings in the specification or claims does not require that the steps or elements be carried out in alphabetical or numerical order or in the order in which they are presented. This description outlines novel particles, termed liposomal particles, methods for their preparation, and their uses. These liposomal particles are more beneficial than other known liposomal-based materials because they are stable at a smaller particle size than other known liposomal particles, and the dense DNA coating enhances particle stability in the body and, consequently, increases the circulation rate of liposomal vesicles, thus improving the biodistribution of these particles in the body. A. Tocopherol-modified oligonucleotides In a first embodiment, an architecture comprising a tocopherol-modified oligonucleotide is described. A tocopherol-modified oligonucleotide comprises a lipophilic end and a non-lipophilic end. The lipophilic end comprises tocopherol and can be selected from the group consisting of a tocopherol derivative, alpha-tocopherol, beta-tocopherol, gamma-tocopherol, and delta-tocopherol. The lipophilic end, in other embodiments, comprises palmitoyl, dipalmitoyl, stearyl, or distearyl. The non-lipophilic end of the tocopherol-modified oligonucleotide is an oligonucleotide. The oligonucleotide is either RNA or DNA. The RNA can be an inhibitory RNA (RNAi) that performs a regulatory function and is selected from the group consisting of a small inhibitory RNA (pRNA), an RNA that forms a triple structure with double-stranded DNA, and a ribozyme. Alternatively, the RNA is a microRNA that performs a regulatory function. In even more complex forms, the RNA is a Piwi-associated RNA (Piwi-RNA). In some complex forms, the DNA is antisense DNA. The oligonucleotides contemplated for use according to the description are from nAfrnnn / zznz / E / γΐΛΐ approximately 5 to approximately 100 nucleotides in length. Methods and compositions are also considered in which the oligonucleotides are from approximately 5 to approximately 90 nucleotides in length, from approximately 5 to approximately 80 nucleotides in length, from approximately 5 to approximately 70 nucleotides in length, from approximately 5 to approximately 60 nucleotides in length, from approximately 5 to approximately 50 nucleotides in length, from approximately 5 to approximately 45 nucleotides in length, from approximately 5 to approximately 40 nucleotides in length, from approximately 5 to approximately 35 nucleotides in length, from approximately 5 to approximately 30 nucleotides in length, from approximately 5 to approximately 25 nucleotides in length, from approximately 5 to approximately 20 nucleotides in length, from approximately 5 to approximately 15 nucleotides in length.Oligonucleotides of approximately 5 to approximately 10 nucleotides in length, and all intermediate-length oligonucleotides of the sizes specifically described, are considered, provided the oligonucleotide is capable of achieving the desired result. Therefore, oligonucleotides of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36 nucleotides are considered. 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100 nucleotides in length. Modified oligonucleotides Specific examples of oligonucleotides include those containing modified backbones or internucleoside linkages of non-natural origin. Oligonucleotides with modified backbones include those that retain a phosphorus atom in the backbone and those that do not. Oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are considered to fall under the definition of "oligonucleotide." The main structures of modified oligonucleotides containing a phosphorus atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, festinates, phosphoramidates including 3'-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogues of these and those with reversed polarity in which one or more internucleotide linkages are a 3' to 3', 5' to 5' or 2' to 2' linkage.Also included are oligonucleotides with reversed polarity, comprising a single 3' to 3' linkage in the 3'-end internucleotide bond; that is, a single inverted nucleoside residue that may be abasic (lacking the nucleotide or having a hydroxyl group in its place). Salts, mixed salts, and free acid forms are also included. Representative U.S. patents indicating the preparation of the aforementioned phosphorus-containing bonds include U.S. patents Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5,536,821, and 5,541,306. 5 550 111,5 563 253, 5 571 799, 5 587 361,5 194 599, 5 565 555, 5 527 899, 5 721 218, 5 672 697 and 5 625 050, whose descriptions are incorporated herein by means of this reference. The main structures of modified oligonucleotides that do not include a phosphorus atom have main structures formed by cycloalkyl or short-chain alkyl internucleoside linkages, cycloalkyl or alkyl and mixed heteroatom internucleoside linkages, or one or more short-chain heterocyclic or heteroaromatic internucleoside linkages. These include those with morpholine linkages, siloxane structures, sulfide, sulfoxide, and sulfone structures, formacetyl and thioformacetyl structures, methyleneformacetyl and thioformacetyl structures, riboacetyl structures, alkene-containing structures, sulfamate structures, methyleneimino and methylenehydrazine structures, sulfonate and sulfonamide structures, amide structures, and others that have mixed N, O, S, and CH2 component parts. For example, see U.S. Patent Nos.s5 034 506, 5 166 315, 5 185 444, 5 214 134, 5 216 141.5 235 033, 5 264 562, 5 264 564, 5 405 938, 5 434 257, 5 466 677, 5 470 967, 5 489 677, 5 541 307, 5 561 225, 5 596 086, 5 602 240, 5 610 289, 5 602 240, 5 608 046, 5 610 289, 5 618 704.5 623 070, 5 663 312, 5 633 360, 5 677 437, 5 792 608, 5 646 269 and 5 677 439, whose descriptions are incorporated herein in their entirety by means of this reference. In still other embodiments, oligonucleotide mimetics are those in which one or more sugars and / or one or more internucleotide bonds of the nucleotide units are replaced with groups of "non-natural origin." In one aspect, this embodiment includes a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an oligonucleotide is replaced with a backbone containing an amide. For example, see U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, and Nielsen et al., 1991, Science, 254: 1497-1500, descriptions of which are incorporated herein by reference. In still other embodiments, oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones are provided, and include —CH2—NH—O—CH2—, —CH2—N(CH3)—O—CH^, —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —O—N(CH3)—CH2—CH2— described in U.S. Patent Nos. 5,489,677 and 5,602,240. Oligonucleotides with morpholino backbones described in U.S. Patent No. 5,034,506 are also contemplated. In various forms, the linkage between two successive monomers in the oligonucleotide consists of 2 to 4, ideally 3, groups / atoms selected from —CH2—, —O—, —S—, —NRH—, >C=O, >C=NRH, >C=S, —Si(R)2—, —SO—, —S(O)2—, —P(O)2—, —PO(BH3) —, —P(O,S) —, —P(S)2—, —PO(R'j—, —PO(OCH3) —, and —PO(NHRH)—, wherein RH is selected from hydrogen and Cl-4 alkyl, and R is selected from Cl-6 alkyl and phenyl. Illustrative examples of such linkages are —CH2—CH2—CH2—, —CH2—CO—CH2—, —CH2—CHOH—CH2—, —O—CH2—O—, —O—CH2—CH2—, —O—CH2—CH= (which includes R5 when used as a link with a subsequent monomer), —CH2—CH2—O—, —NRH—CH2—CH2—, —CH2—CH2—NRh—, —CH2—NRh—CH2--, —O—CH2—CH2—NRh—, —NRH— CO—O—, —NRH—CO—NRH—, —NRH—CS—NRH—, —NRH—C(=NRH)—NRH—, —NRH— CO—CH2—NRH—O—CO—O—, —O—CO—CH2—O—, —O—CH2—CO—O—, — CH2—CO— NRH—, —O—CO—NRH—, —NRH—CO—CH2 —, —O—CH2—CO—NRH—, — O—CH2—CH2—NRH—, —CH=N—O—, —CH2—NRH—O—, —CH2—O—N= (which includes R5 when used as a link with a subsequent monomer), —CH2—O—NRH—, —CO—NRH— CH2—, — CH2—NRH—O—, — CH2—NRH—CO—, —O—NRH— CH^, —O—NRH, —O— CH2—S—, — S— CH2—O—, — CH2— CH2—S—, —O— CH2— CH2—S—, —S— CH2—CH= (which includes R5 when used as a link with a subsequent monomer), —S— CH2— CH2—, —S— CH2— CH2— O—, —S— CH2— CH2—S—, — CH2—S— CH2—, — CH2—SO— CH2—, — CH2—SO2— CH2—, —O—SO—O—, —O—S(O)2—O—, —O—S(O)2— CH2—, — O—S(O)2— NRH—, —NRH—S(O)2— CH2—;—O—S(O)2— CH2—, —O—P(O)2—O—, — O—P(O,S)—O—, —O—P(S)2—O—, —S—P(O)2—O—, — S—P(O,S)—O—, —S—P(S)2—O—, — O—P(O)2—s— , — O—P(O,S)—S—, —O—P(S)2—S—, —S—P(O)2—S—, — S—P(O,S)—S—, — S—P(S)2— S—, — O—PO(R'j—O—, — O—PO(OCH3)—O—, —O—PO(O CH2CH3)—O—, —O—PO(O CH2CH2S—R)—O—, —O—PO(BH3)—O—, —O—PO(NHRN)—O—, —O—P(O)2—NRHH—, — NRH—P(O)2—O—, — O—P(O,NRH)—O—, — CH2—P(O)2—O—, —O—P(O)2— CH2— y — O— Si(R)2—O—; among those to contemplate — CH2—CO—NRH—, — CH2—NRH—O—, —S— CH2—O—, — O— P(O)2—O—O—P(- O,S)—O—, —O—P(S)2—O—, —NRHP(O)2—O—, — O— P(O,NRH)—O—, — O—PO(R'j—O—, —O—PO(CH3)—O— y —O—PO(NHRN)—O—, in which RH selects hydrogen and Alquillo-Ci-4 and R are selected from alquillo-Ci-6 and phenyl. Additional illustrative examples are presented in Mesmaeker et. al., 1995, Current Opinion in Structural Biology, 5: 343-355 y Susan M. Freier y Karl-Heinz Altmann, 1997, Nucleic Acids Research, tomo 25: pages 4429-4443.; Further modified oligonucleotide forms are described in detail in U.S. patent application no. 20040219565, the description of which is incorporated herein in its entirety by reference. The modified oligonucleotides may also contain one or more substituted sugar residues. In certain respects, the preferred oligonucleotides comprise one of the following at the 2' position: OH, F, O-alkyl, S-alkyl or N-alkyl, O-alkenyl, S-alkenyl or nAfrnnn / zznz / E / YiAi N-alkenyl, O-alkynyl, S-alkynyl, or N-alkynyl or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups can be C1 to C2 alkyl or C2 to C10 substituted or unsubstituted alkenyl and alkynyl groups. Other embodiments include O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3]2, wherein n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following in the 2' position: lower alkyl Oi to Cw, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, Oalkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavage group, a marker group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. In one aspect, a modification includes 2'-methoxyethoxy (2'-O-CH2 CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., 1995, Helv. Chim. Acta, 78: 486-504) i.e., an alkoxy group.Other modifications include 2'-dimethylaminooxyethoxy, i.e., an O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the examples hereafter, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'O—CH2—O—CH2—N(CH3)2, also described in the examples hereafter. Still other modifications include 2'-methoxy (2'-O-CH3), 2'-aminopropoxy (2'OCH2CH2CH2NH2), 2'-allyl (2'-CH2-CH=CH2), 2'-O-allyl (2'-O-CH2-CH=CH2), and 2'-fluoro (2'F). The 2' modification can be at the arabino position (above) or the ribo position (below). In one respect, a 2' arabino modification is 2'-F. Similar modifications can also occur at other positions in the oligonucleotide, for example, at the 3' position of the sugar in the 3'-terminus nucleotide or in 2'-5' linked oligonucleotides and at the 5' position of the 5'-terminus nucleotide. Oligonucleotides can also have sugar mimetics, such as cyclobutyl groups instead of the pentofuranosyl sugar. See, for example, US patents no.° 4 981 957, 5 118 800, 5 319 080, 5 359 044, 5 393 878, 5 446 137, 5 466 786, 5 514 785, 5 519 134, 5 567 811, 5 576 427, 5 591 722, 5 597 909, 5 610 300, 5 627 053, 5 639 873, 5 646 265, 5 658 873, 5 670 633, 5 792 747 and 5 700 920, whose descriptions are incorporated herein in their entirety by this reference. In one aspect, a modification of the sugar includes blocked nucleic acids (NBAs) in which the 2'-hydroxyl group is attached to the carbon atom at the 3' or 4' position of the sugar ring, thus forming a bicyclic sugar moiety. In certain aspects, the linkage is a methylene group (—CH2—)n bridging the oxygen atom at the 2' position and the carbon atom at the 4' position, where n is 1 or 2. NBAs and their preparation are described in WO 98 / 39352 and WO 99 / 14226. nAbnnn / zznz / B / YiAi Oligonucleotides may also include base modifications or substitutions. As used herein, "unmodified" or "naturally occurring" bases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).Modified bases include other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracyl and cytosine, 5-propynyl, uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other adenines and guanines substituted in the position 8, 5-halo particularly 5-bromo, 5-trifluoromethyl and other uracils and cytosines substituted at position 5, 7-methylguanine and 7-methyladenine, 2-Fadenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Other modified bases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), G clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-pyrimido[5,4-b][1,4]benzoxazin2(3H)-one), carbazol cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Additional bases include those described in U.S. Patent No. 3,687,808, those described in The Concise Encyclopedia of Polymer Science and Engineering, pages 858–859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, and those described by Englisch et al., 1991, Angewandte Chemie, International Edition, 30:613, and those described by Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993. Certain bases of these are useful for increasing binding affinity and include 5-substituted pyrimidines, 6-azapyrimidines, and substituted N-2, N-6, and O-6 purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase the stability of the double nucleic acid at 0.6-1.2 °C and, in certain respects, combine with sugar modifications at 2'-O-methoxyethyl. See US patent no. 3,687,808, US patents no.s4 845 205, 5 130 302, 5 134 066, 5 175 273, 5 367 066, 5 432 272, 5 457 187, 5 459 255, 5 484 908, 5 502 177, 5 525 711, 5 552 540, 5 587 469, 5 594 121, 5 596 091, 5 614 617, 5 645 985, 5 830 653, 5 763 588, 6 005 096, 5 750 692 and 5 681 941, whose descriptions are incorporated herein by means of this reference. A "modified base," or similar term, refers to a compound that can be paired with a naturally occurring base (e.g., adenine, guanine, cytosine, uracil, and / or thymine) and / or can be paired with a base of non-natural origin. In certain respects, the modified base provides a Tm differential of 15, 12, 10, 8, 6, 4, or 2 °C or less. Examples of modified bases are described in EP 1 072 679 and WO 97 / 12896. "Nucleobase" refers to the naturally occurring nucleobases of adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), as well as non-naturally occurring nucleobases such as xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthin, 7-deazaguanine, N4,N4-ethanecytosine, N',N'-ethane-2,6-diaminopurine, 5-methylcytosine (mC), 5(C3—C6)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, and the "non-naturally occurring" nucleobases described in Benner et al., U.S. Patent No. 5. 432 272 and Susan M. Freier and Karl-Heinz Altmann, 1997, Nucleic Acids Research, Volume 25: pages 4429-4443. Therefore, the term "nucleobase" includes not only the known purine and pyrimidine heterocycles, but also their heterocyclic analogues and tautomers. Additional naturally occurring and non-naturally occurring nucleobases include those described in U.S. Patent No.° 3 687 808 (Merigan, et al.), in chapter 15 by Sanghvi, in Antisense Research and Application, Ed. ST Crooke and B. Lebleu, CRC Press, 1993, in Englisch et al., 1991, Angewandte Chemie, international edition, 30: 613-722 (especially see pages 622 and 623, and in Concise Encyclopedia of Polymer Science and Engineering, JI Kroschwitz Ed., John Wiley & Sons, 1990, pages 858-859, Cook, Anti-Cancer Drug Design 1991,6, 585-607, each of which is incorporated herein in full by this reference). It is also intended that the expressions "nucleoside base" or "base unit" include compounds such as heterocyclic compounds that can act as nucleobases that include certain "universal bases" that are not nucleoside bases in the more classical sense, but that act as nucleoside bases.Specifically, the universal bases mentioned are 3-nitropyrrole, optionally substituted indoles (e.g., 5-nitroindole), and optionally substituted hypoxanthine. Other desirable universal bases include pyrrole, diazole, or triazole derivatives, which are among those universal bases known in the art. B. Production methods for tocopherol-modified oligonucleotides In a second embodiment, methods for producing tocopherol oligonucleotides are described. First, a phosphoramidite-modified tocopherol and oligonucleotide are provided. Then, the oligonucleotide is exposed to the phosphoramidite-modified tocopherol to create the modified tocopherol oligonucleotide. While not intended to be limited, the practitioner may use any chemical method to link the tocopherol to the oligonucleotide, including amide bonding or click chemistry. C. Liposomal particles In a third embodiment, liposomal particles are described. The liposomal particle has at least a substantially spherical geometry, an inner and an outer side, and comprises a lipid bilayer. The lipid bilayer is composed of a first lipid and a second lipid. In some embodiments, the first and second lipids are identical. In other embodiments, the first and second lipids are different. The lipid primer is selected from the phosphocholine lipid family or the phosphoethanolamine lipid family. Non-exhaustively, the first lipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-diestearoyl-s / i-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-diestearoyl-sn-glycero-3-phosphocholine (DSPG), 1,2-dipalmitoyls / i-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadecenoyl)sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-d¡hexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE). The second lipid is selected from the phosphocholine lipid family or the phosphoethanolamine lipid family. Non-limitingly, the second lipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1 rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmito¡l-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Zoctadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE). Furthermore, the liposomal particle comprises a tocopherol-modified oligonucleotide, in which the lipid bilayer adsorbs the lipophilic end of the tocopherol-modified oligonucleotide. The tocopherol is selected from the group consisting of alpha-tocopherol, beta-tocopherol, gamma-tocopherol, and delta-tocopherol. The non-lipophilic end of the tocopherol-modified oligonucleotide is an oligonucleotide. In several embodiments, this oligonucleotide is either RNA or DNA. The RNA may be an inhibitory RNA (RNAi) that performs a regulatory function, and in several embodiments, it is selected from the group consisting of a small inhibitory RNA (pRNA), an RNA that forms a triplet structure with double-stranded DNA and a ribozyme. Alternatively, and in additional embodiments, the RNA is a microRNA that performs a regulatory function. Optionally, the DNA is antisense DNA. In still additional embodiments, the RNA is a Piwi-associated RNA (Piwi-RNA). In other words, the description provides a liposomal particle with a substantially spherical geometry comprising a lipid bilayer consisting of multiple lipid groups and an oligonucleotide. In several embodiments, the antisense oligonucleotide is a modified oligonucleotide. In some embodiments, the multiple lipid groups comprise a lipid selected from the group consisting of the phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine lipid family. In additional embodiments, the oligonucleotide is a lipid-oligonucleotide conjugate containing a bound lipophilic group, wherein said bound lipophilic group is adsorbed onto the lipid bilayer. In various embodiments, the bound lipophilic group comprises tocopherol, palmitoyl, dipalmitoyl, stearyl, distearyl, or cholesterol. Alternatively, the liposomal particle also comprises a therapeutic agent encapsulated on its inner side. In further embodiments, a liposomal particle of the described nature also comprises a therapeutic agent directly or indirectly bound to the liposomal particle. For example, and without limitation, indirect binding includes the attachment of an oligonucleotide that, in turn, binds to the liposomal particle. In some formulations, the liposomal particle additionally comprises a diagnostic agent encapsulated on its inner side. In some formulations, this diagnostic agent is gadolinium. Regarding the surface density of oligonucleotides on the surface of a liposomal particle described herein, it is envisaged that a liposomal particle described herein comprises from approximately 1 to approximately 100 oligonucleotides on its surface. In various embodiments, a liposomal particle comprises from approximately 10 to approximately 100, or from approximately 10 to approximately 90, or from approximately 10 to approximately 80, or from approximately 10 to approximately 70, or from approximately 10 to approximately 60, or from approximately 10 to approximately 50, or from approximately 10 to approximately 40, or from approximately 10 to approximately 30, or from approximately 10 to approximately 20 oligonucleotides on its surface. In additional embodiments, a liposomal particle comprises at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 oligonucleotides on its surface. D. Methods for producing liposomal particles In a fourth modality, methods for producing liposomal particles are described. First, a phospholipid, solvent, and a tocopherol-modified oligonucleotide are provided. Then, the phospholipid is added to the solvent to form a first mixture comprising liposomes. The liposomes in the first mixture are between approximately 100 nanometers and approximately 150 nanometers in size. Next, the liposomes are perturbed to create a second mixture comprising liposomes and small unilamellar vesicles (SUVs). The liposomes and SUVs in the second mixture range in size from approximately 20 nanometers to approximately 150 nanometers. Next, SUVs with a particle size between approximately 20 nanometers and approximately 50 nanometers are isolated from the second mixture. Finally, the tocopherol-modified oligonucleotide is added to the isolated SUVs to produce a liposomal particle. The particle size of the liposomal particles created by a method described in nAfrnnn / zznz / E / YiAi is less than or equal to about 50 nanometers. In some forms, multiple liposomal particles are produced and these have an average diameter less than or equal to about 50 nanometers (e.g., about 5 nanometers to about 50 nanometers, or about 5 nanometers to about 40 nanometers, or about 5 nanometers to about 30 nanometers, or about 5 nanometers to about 20 nanometers, or about 10 nanometers to about 50 nanometers, or about 10 nanometers to about 40 nanometers, or about 10 nanometers to about 30 nanometers, or about 10 nanometers to about 20 nanometers).In additional modalities, the particles of the multiple liposomal particles created by a method of the description have an average diameter less than or equal to about 20 nanometers, or less than or equal to about 25 nanometers, or less than or equal to about 30 nanometers, or less than or equal to about 35 nanometers, or less than or equal to about 40 nanometers, or less than or equal to about 45 nanometers. In other words, in some respects, the description provides a method for producing a liposomal particle comprising adding a phospholipid to a solvent to form a first mixture comprising multiple liposomes, perturbing said multiple liposomes to create a second mixture comprising a liposome and a small unilamellar vesicle (SUV), isolating said SUVs with a particle size of between about 20 nanometers and 50 nanometers from said second mixture, and adding an oligonucleotide to the isolated SUVs to produce the liposomal particle. E. Uses of liposomal particles in gene therapy / regulation The methods for inhibiting gene product expression provided herein include those in which gene product expression is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% compared to gene product expression. in the absence of a liposome spherical nucleic acid.In other words, the methods provided comprise those that result in essentially any degree of inhibition of expression of a target gene product. The degree of inhibition in vivo is determined from a sample of body fluid or a biopsy sample, or using established imaging techniques. Alternatively, the degree of inhibition is determined in a cell culture assay, generally as a predictable measure of the degree of inhibition that can be expected in vivo from the use of a specific type of liposomal spherical nucleic acid and a specific oligonucleotide. In some aspects of the description, a liposomal particle is envisioned as fulfilling both a gene inhibition function and a therapeutic agent delivery function. In these aspects, a therapeutic agent is encapsulated within a liposomal particle of the description and further functionalized with one or more oligonucleotides designed to inhibit the target gene expression. In additional modalities, a therapeutic agent is attached to a liposomal particle of the description. In several respects, the methods include the use of an oligonucleotide that is 100% complementary to the target polynucleotide, i.e., totally coincident, although in other respects, the oligonucleotide is at least (meaning greater than or equal to) about 95% complementary to the polynucleotide with respect to the oligonucleotide in its entirety, at least about 90%, at least about 85%, about 75%, at least about 70%, about 60%, at least about 55%, about 45%, at least about 40%, at least about 80%, at least less about 65%, at least less about 50%, at least less about 35%, at least about 30%, at least about 25%, at least about 20% complementary to the polynucleotide with respect to the oligonucleotide in its entirety, to the extent that the oligonucleotide is able to achieve the desired degree of inhibition of a target gene product. The technique understands that the sequence of an antisense compound does not need to be 100% complementary to that of its target nucleic acid to hybridize specifically. Furthermore, an oligonucleotide can hybridize in one or more segments such that adjacent or intervening segments are involved in the hybridization event (for example, a loop or hairpin structure). The percentage of complementarity is determined with respect to the oligonucleotide along its entire length. For example, given an antisense compound in which 18 of the 20 nucleotides are complementary to a 20-nucleotide region in a 100-nucleotide target polynucleotide, the oligonucleotide would have 90 percent complementarity.In this example, the remaining non-complementary nucleotides may be grouped or interspersed with complementary nucleobases and do not need to be contiguous to each other or to complementary nucleotides. It is possible to routinely determine the percentage of complementarity of an antisense compound with a target nucleic acid region using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the technique (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). Accordingly, a fifth modality provides methods for using liposomal particles in gene regulation therapy. This method comprises the step of hybridizing a polynucleotide encoding the gene product with one or more oligonucleotides complementary to all or part of the polynucleotide and attached to a liposomal particle, wherein the polynucleotide and the oligonucleotide hybridize along a portion of the polynucleotide with a degree of complementarity sufficient to inhibit the expression of the gene product. The liposomal particle has a diameter of approximately 50 nanometers or less. Inhibition of gene expression can occur in vivo or in vitro. The oligonucleotide used in this method is either RNA or DNA. The RNA can be inhibitory RNA (RNAi), which performs a regulatory function, and in several forms, it is selected from the group consisting of small inhibitory RNA (sRNAi), an RNA that forms a triplet structure with double-stranded DNA and an hbozyme. Alternatively, the RNA is microRNA, which performs a regulatory function. In some forms, the DNA is antisense DNA. In another aspect of the description, a liposomal particle is used in a method for treating traumatic brain injury (TBI). In the United States, more than 244,000 cases of TBI have occurred in the military since 2000, and it is the leading cause of death and disability in individuals under 45 years of age. Furthermore, it is difficult to predict the neurological consequences of minor incidents and very challenging to treat the secondary phase of the trauma (e.g., inflammation, ischemia, and apoptosis). Therefore, in some embodiments, the methods of the invention relate to the use of a liposomal particle designed to select and regulate the expression of a gene product involved in TBI. For example, and not limited to, the target gene product is selected from the group consisting of histone deacetylase (HDAC), BCL2-associated protein X (BAX), a metallopeptidase / metalloproteinase array (MMP, including, but not limited to, metallopeptidase array 9 (MMP-9)), a hypoxia-induced factor (HIF, including, but not limited to, hypoxia-induced factor 1 alpha (HIF1-a)), and calpain. F. Uses of liposomal particles in immune regulation Toll-like receptors (TLRs) are a class of proteins expressed on sentinel cells that play a fundamental role in regulating the innate immune system. The mammalian immune system employs two general strategies to combat infectious diseases. Exposure to pathogens rapidly triggers an immune response characterized by the production of immunostimulatory cytokines, chemokines, and polyreactive IgM antibodies. Exposure to pathogen-associated molecular patterns (PAMPs) expressed by a diverse group of infectious microorganisms activates the innate immune system. Members of the Toll-like receptor family mediate PAMP recognition. TLR receptors, such as TLR4, TLR8, and TLR9, which respond to specific oligonucleotides, are located within special intracellular compartments called endosomes.The modulation mechanism of TLR 4, TLR 8 and TLR 9 receptors is based on protein and DNA interactions. Synthetic immunostimulatory oligonucleotides containing CpG motifs similar to those found in bacterial DNA stimulate a TLR-like response. Consequently, immunomodulatory ODNs have several potential therapeutic uses, including the treatment of cancer and immunodeficiency. The use of liposomal nanoparticles functionalized with immunomodulatory ODNs allows for greater absorption preference and, therefore, greater therapeutic efficacy. Notably, smaller particles (25 to 40 nm), such as those provided herein, penetrate tissue barriers more effectively and thus provide more efficient activation of innate immune responses. Therefore, small liposomal nanoparticles of 30 nm in size, functionalized and stabilized with DNA containing a functional CpG motif, would provide an even better therapeutic effect. Negative regulation of the immune system would involve inactivating the gene responsible for Toll-like receptor expression. This antisense approach involves using liposomal nanoparticles functionalized with antisense oligonucleotide sequences to inactivate the expression of any Toll-like protein. Therefore, in a sixth modality, methods for using particles to modulate Toll-like receptors are described. The method positively or negatively regulates the Toll-like receptor through the use of a TLR agonist or a TLR antagonist, respectively. The method comprises placing a cell with a Toll-like receptor in contact with a liposomal particle. The modulated Toll-like receptors include Toll-like receptor 1, Toll-like receptor 2, Toll-like receptor 3, Toll-like receptor 4, Toll-like receptor 5, Toll-like receptor 6, Toll-like receptor 7, Toll-like receptor 8, Toll-like receptor 9, Toll-like receptor 10, Toll-like receptor 11, Toll-like receptor 12, and Toll-like receptor 13. G. Uses of liposomal particles in Nanoflare technology In further aspects of the description, a liposomal particle is used to detect an intracellular target. Such methods are described in U.S. Patent No. 8,507,200, which is incorporated herein by reference in its entirety. In short, an oligonucleotide containing a recognition sequence specific to a target molecule is attached to a liposomal particle as described herein. Therefore, as used herein, "recognition sequence" refers to a sequence that is partially or fully complementary to a target molecule of interest. Initially, the liposomal particle is associated with a linked oligonucleotide containing a recognition sequence for a marker sequence. As used herein, a "marker sequence" is understood to refer to a partially or fully complementary sequence, and thus capable of hybridization, to the recognition sequence. The marker sequence is labeled with a detectable tag (such as, but not limited to, a fluorophore) and is also referred to as Nanoflare. In various respects, the marker sequence comprises a smaller, equal, or larger number of bases than the recognition sequence, such that binding of the recognition sequence to its target molecule causes the release of the hybridized marker sequence, resulting in a detectable and measurable change in the tag attached to the marker sequence. The examples below illustrate the invention and are not intended to be exhaustive. EXAMPLES Example 1 - General All reagents were obtained from suppliers in their highest purity form and used without further purification. HPLC was performed on a Varian Prostar system. UV / Vis spectrophotometry was recorded on a Varian Cary 300 spectrophotometer. Fluorescence spectra were obtained on a SPEX FluoroLog fluorometer. Examples 2 - Oligonucleotide synthesis Oligonucleotides were synthesized on a micromolar scale (1.0) using an automated DNA synthesizer (ABI 3400, Applied Biosystems, Inc.). After cleavage and deprotection with aqueous ammonium hydroxide (55 °C, 14 hours), the DNA was purified by reversed-phase HPLC and quantified using a UV spectrometer. Example 3 - Symptoms of liposomal particles The lipid monomer (40 pmol of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) dissolved in chloroform) was added to a 20 mL container and evaporated before being subjected to overnight lyophilization to remove the solvent, resulting in a thin lipid film. The film was then rehydrated with HBS buffer (5.0 mL, 20 mM Hepes buffer, 150 mM NaCl at pH 7.4), followed by vigorous mixing to form a liposomal suspension, and then sonicated in an ice bath for 30 minutes without pulsation. The resulting suspension was ultracentrifuged at 104,986 g and 4 °C for 90 minutes. The phospholipid concentration was determined by elemental analysis. The modified DNA / RNA strands were then synthesized using standard solid-phase phosphoramidite chemistry on an Expedite nucleotide synthesis system. The strands were cleaved from the solid support and purified by reversed-phase high-performance liquid chromatography. Finally, the appropriate DNA / RNA (16 μM) was added to the 1.3 mM SUV solution and allowed to be stirred overnight. The particles were purified the following day using spin filters with a 100 kDa cutoff. The particles were then analyzed by TEM and dynamic light scattering. Figure 13 illustrates gel electrophoresis of liposomal particles encapsulated with FITC and surface functionalized with CY5-labeled DNA. Example 4 - Visualization of cellular absorption of liposomal particles To visualize LSNA uptake, HeLa cells were cultured overnight in a Lab-Tek® II Chamber#1.5 German Coverglass system (Nalge Nunc International) and incubated with Cy5-labeled LSNA (DNA concentration 0.1 pM). After 16 hours of incubation, the medium was replaced with fresh medium, and live cells were stained with Hoechst 33342 (Invitrogen) according to the manufacturer's instructions. All images were acquired using a Zeiss 510 LSM instrument with 40x magnification via a Mai Tai 3308 laser (Spectra-Physics). Fluorescence emission was recorded at 390–465 nm and 650–710 nm with excitation at 729 nm and 633 nm, respectively (Figure 5). The left panel of Figure 5 illustrates the entry of liposomal fluorescein into HeLa cells, while the right panel shows the colocalization of fluorescein and Cy5, suggesting delivery of the entire liposome into the cell. Example 4 - Cell Viability The cytotoxicity of liposomal particles was evaluated using the Alamar Blue® assay (Invitrogen). Briefly, HeLa cells were placed in a 96-well plate in 200 pL of medium and incubated for 24 hours. The cells were then treated with FITC-encapsulated SUVs and DNA-functionalized LSNA at varying phospholipid concentrations (0, 32.5, 65, and 162.5 pM). After 16 hours, the medium was removed, the cells were washed three times with PBS, and incubated with 90 pL of fresh culture medium plus 10 pL of Alamar Blue reagent (Resazuhna) for 4 hours. The cells were analyzed by testing for excitation at 560 nm and emission at 590 nm. Example 5 - SUV Preparation Materials The lipid monomer 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was purchased from Avanti Polar Lipids, Inc., either as a dry powder or in a chloroform solution, and was used without further purification. Phosphoramides and other DNA synthesis reagents were purchased from Glen Research, Inc. in their purest form and were used as received from the manufacturer. Instrumentation The freeze-drying process was carried out using a Freezone freeze dryer (Labconco, Kansas City, MO). Sonication was performed using a titanium alloy solid probe sonicator (Vibra-Cell™ VC 505, 500 Watt, Sonics & Materials, Inc., Newtown, CT) at 40% intensity and 20 kHz pulseless. Ultracentrifugation was performed using a Beckman-Coulter Avanti J30I (Beckmann-Coulter, Inc., Indianapolis, IN). Transmission electron microscopy (TEM) was performed using a Hitachi-2300 STEM electron microscope. Dynamic light scattering (DLS) was determined using a Malvern Zetasizer Nano-ZS instrument (Malvern Instruments, UK). MALDI-ToF analysis was performed using a Bruker Autoflex III SmartBean mass spectrometer (Bruker Daltonics Inc., MA, USA). Fluorescence measurements were taken using a Fluorlog-3 system (HORIBA Jobin Yvon Inc., NJ, USA). UV-Vis spectrometry was determined using a Cary 5000 UV-Vis spectrophotometer (Varan Inc., CA, USA). Oligonucleotide synthesis Oligonucleotides were synthesized by phosphoramidite synthesis on solid support Strand Name Application Sequence (5'-3) Cy5-tagged T25 strand Size analysis, DNA density determination, and stability assays 5'-Cy5-T25-tocopherol-3' (SEQ ID NO: 1) Fusion strand 1 Fusion analysis 5'-tocopherol-Aio-TCT CTT GGA-3' (SEQ ID NO: 2) Fusion strand 2 Fusion analysis 5'-TGC GTA GAC-A10 tocopherol-3' (SEQ ID NO: 3) Linker strand Fusion analysis 5'-ACG CAT CTG TCC AAG AGA-3' (SEQ ID NO: 4) HER2 antisense Gene regulation 5'-CTC CAT GGT GCT CAC- Thio-tocopherol-3' (SEQ ID NO: 5) HER2 antisense Cy5-tagged Cell imaging and uptake 5'- Cy5-CTC CAT GGT GCT CAC- T10tocopherol-3' (SEQ ID NO: 6) Disordered antisense Gene regulation 5'-GAG CTG CAC GCT GCC GTC A-T10tocopherol-3' (SEQ ID NO: 7) Synthesis of small unilamellar vesicles The volume of lipid monomer stock solution (25–50 mg) was added to a 20 mL container, which was then placed in a 25 mL glass beaker, and the solvent was carefully evaporated under a stream of nitrogen. The resulting lipid monomer was subsequently dried overnight under vacuum to remove any residual chloroform. The lipid film was then hydrated with 20 mM HBS (5.0 mL), followed by vortex mixing to form a liposomal suspension. This suspension was sonicated for 30 minutes while maintaining the temperature of the lipid mixture below 10 °C (cooling with an ice-water bath). After sonication, the suspension was ultracentrifuged at 100,000 x g for 90 minutes at 122 °C. After centrifugation, the clear supernatant containing the desired small unilamellar vesicles (SUVs) was collected and the pellet was discarded (Figure 1).The SUV particles obtained were further extruded through a polycarbonate membrane (30 nm pore size) to obtain particles with a narrower size distribution. The SUVs obtained were analyzed using dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques (Figure 11). The final phospholipid concentration in a given sample was determined by induced plasma mass spectrometry (ICP-MS). The amount of liposomes in solution and the amount of oligonucleotides on the surface of a liposome can be calculated according to the equation illustrated in Figure 12. Preparation of DNA-functionalized liposomal ANS The desired 15 μM 3'-tocopherol-modified oligonucleotide was added to a 1.3 mM SL)V ([phospholipid]) solution and allowed to be shaken overnight to prepare liposomal ANS. The resulting solution was then purified by gel filtration chromatography on a crosslinked Sepharose column (Sepharose CL 4B, Aldrich). Particle size distribution was analyzed according to DLS. Samples were placed on plasma-cleaned carbon TEM grids and further stained with uranyl acetate solution (2% w / v) (staining for 2 minutes, followed by washing with water and drying) for TEM visualization of liposomal ANS. Grid images were then obtained using a Hitachi-2300 STEM electron microscope. Liposomal ANS gel electrophoresis All gel electrophoresis experiments were performed on a 1% agarose gel in 1xTBE buffer (trisborate, EDTA). Samples were loaded into the wells using glycerol (30% v / v, 5 pL) as a loading agent. The gel chamber was filled with 1xTBE buffer and pre-cooled with ice. Gels were electrophoresis-treated at 70 V for 1 hour at 10°C, and gel images were acquired using Fluorchem Q with a Cy5 filter. Quantification of DNA density on the liposomal surface Increasing concentrations of Cy5-labeled 3'-tocopherol modified DNA were incubated overnight in a fixed SUV concentration ([P] 1.3 mM) to determine the DNA loading on the liposome surface. The liposomal ANS were then analyzed by gel electrophoresis. The constructs were dissolved in 1% SDS solution, and the absorbance was measured at 260 nm. This absorbance was then calculated using the extinction coefficient of the respective DNA strand to quantify the density of functionalized DNA on the SUVs. The number of liposomes in the corresponding solution was calculated using the theoretical equation, assuming that the liposomal phospholipid concentration remained constant after functionalization. Fusion tests A two-component nanoparticle system with liposomal ANS was formed, containing strands complementary to the linker strand, as described in Table 1. The aggregates were formed by adding two DNA-functionalized liposomal ANS and hybridized to the linker strand in a 1:1 ratio (total DNA concentration: 1.5 μM, volume: 1 mL). The absorbance spectrum of the liposomal ANS with the linker was obtained using a Cary 5000 UV-Vis spectrometer and compared to the absorbance spectrum of liposomal ANS without the linker. The aggregates were then subjected to a gradual temperature increase at a rate of 0.25 °C / min from 20 to 65 °C, and the absorbance at 260 nm of the aggregates was observed. Rhodamine encapsulation Dry DOPC monomer (25 mg) was resuspended in 20 mM sulforhodamine B in HBS (5 mL). The resulting suspension was gradually extruded through a series of polycarbonate membranes at 100 nm, 80 nm, 50 nm, and 30 nm. Rhodamine-containing liposomes were separated from free rhodamine by gel filtration chromatography on crosslinked Sepharose (Sepharose CL-4B, Aldrich). The resulting particles were functionalized with tocopherol and DNA conjugates using the previously described procedure. The rhodamine-containing liposomes and liposomal ANS were suspended in 10% fetal bovine serum in HBS, and stain release was monitored in a Fluorlog-3 system by exciting the sample at 420 nm and measuring the intensity at 480 nm to assess the serum stability of the constructs. Cell culture studies SKOV-3 cells were acquired from the American Type Culture Collection (ATCC) and cultured in McCoy 5A medium with 10% heat-inactivated fetal bovine serum, 100 U of penicillin, and 50 pg of streptomycin, and maintained at 37 °C with 5% CO2 according to ATCC instructions. Cells were plated 24 hours prior to treatment at 50% confluence for cell studies. Confocal microscopy of liposomal ANS SKOV3 cells were placed in 35 mm FluoroDish™ chamber plates at 50% confluence to visualize cellular internalization of liposomal ANS. Cells were incubated with Cy5-labeled liposomal ANS (0.1 μM DNA concentration) in medium for 20 hours, followed by three washes with 1x PBS containing 0.01% (v / v) Tween 20, which was then replaced with fresh medium. Nuclei were stained with Hoechst 3342 (Invitrogen) according to the manufacturer's protocol. Live cells were then imaged using a Zeiss LSM 510 inverted laser scanning confocal microscope with a Mai Tai 3308 laser (Spectra-Physics) at 40x magnification. Hoechst was excited at 780 nm and recovered at 390-495 nm, and was excited at 640 nm and emission was produced at 650-710 nm. Flow cytometry experiments Cells were placed in a 96-well plate in 100 pL of medium and incubated with either 0.1 pM cell-free DNA or liposomal ANS for 24 hours to compare cell uptake of liposomal ANS to cell-free DNA. Untreated cells were used as a negative control. After incubation, cells were washed three times with 1 x PBS containing 0.01% (v / v) Tween 20 and then trypsinized to form a suspension. Flow cytometry was performed on the cell suspension using a Cy5 intensity channel on a Guava easyCyte 8H (Millipore, USA) with the signal from the untreated cells as the background intensity. Error values ​​were calculated using the standard error of the mean of the median signal from different wells representing the same sample. Cytotoxicity studies (MTT assay): SKOV-3 cells were placed in a 96-well plate 24 hours prior to the experiment to evaluate the cytotoxicity of liposomal ANS. Cells were treated with liposomal ANS at different DNA concentrations for 24 hours. The cytotoxicity of liposomal ANS was compared with that of DharmaFECT® 1 (Dharmacon), a commercially available transfection agent. Cells were transfected with different concentrations of DharmaFECT® 1 transfected DNA according to the manufacturer's protocol. Untreated cells were used as a negative control. After the 24-hour incubation period, cells were washed three times daily with 1x PBS, incubated with alamarBIue® solution (Thermo Fisher Scientific Inc.), and incubated at 37 °C in 5% CO2 for 4 hours. Fluorescence emission at 590 nm was recorded using a BioTek Synergy H4 Hybrid Reader. Western blot to quantify HER2 protein inactivationSKOV-3 cells were placed in a 6-well plate and incubated overnight at 37°C in 5% CO2. The cells were incubated with liposomal antisense anti-HER2 ANS and liposomal disordered ANS. After 24 hours of treatment, the medium was replaced with fresh medium, and the cells were allowed to grow for an additional 48 hours. The cells were harvested and resuspended in 100 pL of mammalian cell lysis buffer (Cell Signaling, MA, USA) containing phosphatase and protease inhibitors to analyze for HER2 protein inactivation (Thermo Scientific, IL, USA). The protein concentration in the cells was determined using a BCA protein assay kit (Pierce, IL, USA). Equal amounts (20 pg) of protein were fractionated on Precast 4-20% gradient gel (BioRad) and transferred to nitrocellulose membranes (Thermo Scientific, IL, USA).The membrane was blocked with a 5% (w / v) solution of dried skim milk in Tris-buffered saline (TBS). Proteins were detected using primary rabbit antibodies against HER2 (1:1000) and GADPH (1:500), followed by secondary anti-rabbit antibodies (1:10,000) (LI-COR Biosciences, NE, USA). The fluorescence signal was recorded using the Odyssey® infrared imaging system (LI-COR Biosciences, NE, USA). Synthesis A typical liposomal ANS was synthesized in two steps (Figure 7). The first step involves the preparation of 30 nm diameter unilamellar vesicles from lipid monomers. This particle size is ideal for ANS transfection and falls within the appropriate range to maximize blood flow and minimize renal clearance. Unfortunately, liposomes in this size range are often unstable and fuse to form larger structures. Therefore, one of the aims of this work was to determine a pathway for synthesizing such structures and avoiding particle growth pathways. DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), an unsaturated lipid with two oleic acid derivatives extending from a phosphate moiety and a quaternary ammonium group at one end, was selected for the preparation of small unilamellar vesicles (SUVs). In a standard experiment, a suspension of DOPC monomers in 20 mM HBS was sonicated to produce SUV particles averaging 30 nm in diameter. The particles were isolated by centrifugation (100,000 x g). Further extrusion of this material through a polycarbonate membrane with 30 nm pores yielded particles with a polydispersity index (PDI) of 0.11 in an overall yield of 70%. The particles were then redispersed in saline solution, and DLS was used to confirm their diameter of 30 ± 3 nm, which was subsequently confirmed by negative-staining TEM analysis. The second step of the synthesis involves surface functionalization of the liposome with a nucleic acid derivative containing a hydrophobic tocopherol moiety, which inserts into the lipid bilayer and defines the SUV. Although a variety of hydrophobic terminal groups may be suitable [Pfeiffer et al., J. Am. Chem. Soc. 126: 10224 (2004); Banchelli et al., J. Phys. Chem. B 112: 10942 (2008); Dave et al., ACS Nano 5: 1304 (2011); Jakobsen et al., Bioconjugate Chem. 24: 1485 (2013)], α-tocopherol (a form of vitamin E) was chosen due to its biocompatibility and low cost. α-Tocopherol was installed into nucleic acid (DNA) strands by conventional oligonucleotide synthesis with commercially available tocopherol phosphoramidite derivative (Glenn Research).Liposomal ANS were synthesized by incubating a suspension of SUV (1.3 mM lipids) with tocopherol and nucleic acid conjugates (16 mM) at a lipid-to-nucleic acid ratio of 8:1 for 12 hours at room temperature. The liposome-free nucleic acid and tocopherol were then removed from the sample using size-exclusion chromatography on a Sepharose column (Sepharose 4LB). In the case of DNA, a significant reduction in zeta potential from -1 to -23 was observed after this step, indicating surface functionalization of liposomes with the negatively charged nucleic acid. Furthermore, dynamic light scattering (DLS) analysis of the final nanoparticle samples showed an increase in particle size from 30 to 46 nm, consistent with the charge of the 8–9 nm long double structure.To quantitatively determine the average number of nucleic acid strands charged on the surface of a liposome, liposomal ANS was dissolved in the presence of Triton X for release. The final nucleic acid concentration was determined by measuring absorbance at 260 nm with respect to a calibrated oligonucleotide standard. The DNA-coated liposomal ANS showed an average of 70 strands per particle (Figure nAfrnnn / zznz / E / YiAi). 17) This density is lower than that of a typical gold-based ANS structure [Hurst et al., Anal. Chem. 78:8313 (2006)], but it is sufficient to exhibit many of the cooperative properties of such structures. A graphical representation of a liposomal ANS is provided in Figure 16. These liposomal ANS structures have several characteristic properties. First, they are remarkably stable compared to the native 30 nm liposomal constructs from which they are derived (Figure 2, Figure 8). For example, if SUVs are stored without a surface oligonucleotide layer for four days at 37 °C (physiological temperature), they fuse together and form larger polydisperse structures (structures averaging 100 nm with some micrometer-sized entities). In contrast, liposomal ANSs show no signs of fusion or particle degradation over the same time period under virtually identical conditions.This increased stability of the liposomal ANS system is likely due to repulsive forces between the negatively charged nucleic acid strands comprising the liposomal ANS surface, which stabilizes individual particles and inhibits particle-particle fusion interactions [Lí et al., Bioconjugate Chem. 24: 1790 (2013)]. Furthermore, the negatively charged DNA crown on the liposomal ANS serves as a protective layer for the liposomal core and prevents its degradation in the presence of serum proteins [Sénior et al., Life Sci. 30:2123 (1982); Kim et al., Arch. Pharmacal Res. 14: 336 (1991); Sulkowski et al., J. Mol. Struct. 744-747: 737 (2005)]. For example, the serum stability of the liposomal ANS system was evaluated by measuring the release of a sulforhodamine dye physically incorporated into the core of a liposomal ANS at a self-inactivating concentration of 20 mM (core concentration).In this experiment, disruption of the liposomal core leads to the release of sulforhodamine dye from within the particle and subsequent self-inactivation, resulting in increased fluorescence [Versluis et al., J. Am. Chem. Soc. 135: 8057 (2013)]. In a standard experiment, rhodamine-containing liposomal nanoparticles were incubated in 10% fetal bovine serum at 37 °C, and fluorescence spectra were recorded continuously for 3 hours. The same stability study was performed for non-functionalized particles. Similar to the thermal stability studies, the DNA-functionalized particles remained stable in serum throughout the experiment. No dye release was observed during the 3-hour incubation period. In contrast, incubation of clean DOPC liposomes led to a significant release of the rhodamine fluorophore, indicating rapid breakdown of the liposomal structure in serum (Figure 8).A second property of liposomal ANS is their ability to cooperatively bind to complementary nucleic acids. This is a feature specific to all ANS and derives from the highly oriented and compactly packed configuration of the surface nucleic acids. Two sets of liposomal ANS nanoparticles, each produced with different DNA sequences (particle A and particle B), were synthesized to explore the binding and subsequent fusion properties of the liposomal ANS constructs. A DNA linker sequence complementary to the oligonucleotide sequences of the liposomal ANS was used to facilitate polymerization by hybridization. Adding the linker sequence to an equimolar mixture of the two liposomal ANS particles resulted in aggregation, as observed by DLS, and eventually, a lamellar precipitate formed [Dave et al., ACS Nano 5: 1304 (2011)].These aggregates were resuspended in 20 mM HBS (150 mM NaCl) and a melting analysis was performed by absorbance control at 260 nm. Notably, a very narrow melting transition was observed at 47.5 °C (full width at half height of the first derivative is approximately 2 °C), which is a clear indication of an ANS structure with a high nucleic acid surface density (Figure 9). The ability to enter cells without the need for auxiliary transfection agents is an important property of anabolic-androgenic amino acids (ANAs) [Cutler et al., J. Am. Chem. Soc. 134:1376 (2012)]. To determine whether liposomal ANAs exhibit this behavior, ovarian cancer ascites (SKOV3, American Type Culture Collection) cells were incubated in the presence of liposomal ANAs synthesized with 5' Cy5-tagged DNA in the absence of any transfection agents at different DNA concentrations. The uptake of liposomal ANAs in SKOV3 cells was analyzed using confocal microscopy and flow cytometry. Notably, the liposomal ANAs readily entered the cells in large quantities after 1 hour of incubation, demonstrating their potential as intracellular probes and targeting agents.Furthermore, no significant uptake of free DNA strands (tagged at the 5' end with Cy5) was detected in SKOV3 cells even after 36 hours of incubation under identical conditions. Similar to ANS-Au, high uptake of liposomal ANS in SKOV3 cells did not result in toxicity, even at high concentrations (Figure 10). In contrast, the use of DharmaFECT to attempt to deliver DNA similar to that delivered by liposomal ANS resulted in significant cytotoxicity, reducing cell viability to 35% during a 24-hour incubation period. After establishing that liposomal antisense antisense antibodies (ANS) are not cytotoxic, liposomal ANS capable of inactivating the human epidermal growth factor receptor 2 (HER2), an oncogene overexpressed in SKOV3 cells, was synthesized [Zhang et al., J. Am. Chem. Soc. 134: 16488 (2012)]. SKOV3 cells were incubated in the presence of anti-HER2 liposomal ANS and control liposomal ANS (each at a total DNA concentration of 1 μM) to compare the efficacy of the liposomal ANS antisense activity to that of conventional transfection systems. After 72 hours of incubation, the cells were harvested and analyzed for protein content by Western blot. It is noteworthy that HER2 protein levels were reduced by 85% in the presence of anti-HER2 liposomal ANS compared to the internal reference gene for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Figure 10).Taken together, these results demonstrate the possibility of using liposomal ANS to produce both cell transfection and gene regulation. In summary, a scalable synthetic route for novel metal-free liposomal ANS has been developed. Such structures are readily produced from non-toxic and easily accessible starting materials. The nucleic acid architecture not only stabilizes these small liposomal structures but also facilitates their internalization by SKOV3 cells. Consequently, such structures prove useful as biocompatible gene regulatory constructs that exhibit many of the attractive properties of more conventional gold nanoparticle-based ANS. Example 6 - Evaluation of liposomal particles in Ramos-Blue™ cells Ramos-Blue™ cells are NF-κB / AP-1 indicator B lymphocytes. Ramos-Blue is a B cell line that stably expresses an NF-κB / AP-1 (secreted embryonic alkaline phosphatase)-induced SEAP indicator gene. When stimulated, they produce SEAP in the supernatant, which can be monitored using the QUANTI-Blue assay. QUANTI-Blue is a SEAP detection medium that turns blue in the presence of SEAP (Figure 14). When exposed to CpG-containing oligonucleotides, Ramos-Blue cell activation was detected (Figure 15). Representative compounds based on the CpG oligonucleotide 7909 TLR agonist 9 (5'-TCGTCGTTTTGTCGTTTTGTCGTT3' (SEQ ID NO: 8)).These include CpG 7909 with a densely functionalized phosphodiester structure on 13 nm gold nanoparticles (ANS CpG 7909-po (Au)), CpG 7909 with an all-phosphorothioate structure (CpG 7909-ps), a liposomal SNA ANS with a phosphodiester CpG 7909 (targeting 7909 (particle)), a liposomal ANS with the C and G of the all-phosphodiester oligonucleotide structure inverted to eliminate the TLR 9 binding site (control 7909 (particle)), CpG 7909 with an unformulated phosphodiester and 3'-tocopherol lipid structure in a liposomal ANS (targeting 7909 (tocopherol)), and a control sequence with inverted C and G also unformulated in a liposomal ANS (control of 7909 (tocopherol)).Serial dilutions of these compounds were prepared and then incubated with Ramos-Blue cells, a cell line expressing secreted alkaline phosphatase (SEAP) with activation of the pro-inflammatory transcription factor NF-κB overnight. SEAP levels in the cell culture medium were analyzed using a probe with the QuantiBlue kit (InVivogen). Activation was measured by light absorption at 650 nm. nAfrnnn / zznz / E / YiAi Example 7 - Use of liposomal particles to regulate HIF1-a Liposomal particles were designed to individually target HIF1-α and BAX to further demonstrate the efficacy of the described composition. Experiments used the Neuro-2a (N2A) cell line, a rapidly growing mouse neuroblastoma cell line. Exposure of N2A cells to liposomal particles targeting HIF1-α (Figure 18) and BAX (Figure 19) resulted in a significant reduction in the amount of the target gene product. In each experiment, the relative amount of mRNA expression was determined by quantitative PCR (qOCR) 72 hours after the start of treatment of N2A cells in 6-well plates. Cells were treated with the liposomal particles for 24 hours in OptiMEM before the liposomal particles were removed and the medium replaced with MEM and 10% fetal bovine serum (FBS). In the experiments where HIF1-α was selected as the target, N2A cells were first subjected to hypoxia with Cocl2 stimulation, which increased HIF1-α mRNA expression by approximately 50%. The N2A cells were then exposed to liposomal particles functionalized with siRNA directed against HIF1-α. This exposure resulted in approximately 50% HIF1-α inactivation (Figure 18). In the case of experiments in which BAX was selected as the target, the treatment of N2A cells resulted in approximately 65% ​​inactivation of BAX mRNA by liposomal particles and more than 50% inactivation of BAX mRNA by lipid micelles (as measured against control liposomal ANS) (Figure 19). These experiments demonstrated that the liposomal particles described herein very effectively inhibit target gene expression in mammalian cells. It will be evident to those skilled in the art that the present invention is not limited to the illustrative examples and can be manifested in other specific forms without departing from its essential attributes. Therefore, it is intended that the examples be regarded in all respects as illustrative and not restrictive, and that reference be made to the appended claims, rather than to the preceding examples, and that all changes falling within the meaning and scope of equivalence of the claims be included, which are therefore also intended to be encompassed.

Claims

1. A liposomal particle with a substantially spherical geometry comprising: a lipid bilayer comprising a variety of lipid groups and an oligonucleotide.

2. The liposomal particle of claim 1, wherein said variety of lipid groups comprises a lipid selected from the group consisting of the phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine family of lipids.

3. The liposomal particle of claim 2, wherein said lipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimiristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadeceno¡l)-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dihexadecanoyl-snglycero-3-phosphoethanolamine (DPPE).

4. The liposomal particle of any of claims 1-3, wherein the oligonucleotide is a lipid and oligonucleotide conjugate containing a bound lipophilic group, wherein the lipid bilayer adsorbs said bound lipophilic group.

5. The liposomal particle of claim 4, wherein the bound lipophilic group comprises tocopherol or cholesterol.

6. The liposomal particle of claim 5, wherein the tocopherol is selected from the group consisting of a tocopherol derivative, alpha-tocopherol, beta-tocopherol, gamma-tocopherol, and delta-tocopherol.

7. The liposomal particle of any of claims 1-6, wherein said oligonucleotide comprises RNA or DNA.

8. The liposomal particle of claim 7, wherein said RNA is a non-coding RNA.

9. The liposomal particle of claim 8, wherein said non-coding RNA is an inhibitory RNA (RNAi).

10. The liposomal particle of claim 8 or claim 9, wherein the RNAi is selected from the group consisting of a small inhibitory RNA (pRNA), a single-stranded RNA (sRNA) forming a triple structure with double-stranded DNA, and a ribozyme.

11. The liposomal particle of claim 8 or claim 9, wherein the RNA is microRNA.

12. The liposomal particle of claim 7, wherein said DNA is antisense DNA.

13. The liposomal particle of any of claims 1-12, wherein the diameter of said liposomal particle is less than or equal to 50 nanometers.

14. The liposomal particle of any of claims 1-13, wherein the particle comprises from about 10 to about 80 oligonucleotides.

15. The liposomal particle of claim 14, wherein the particle comprises 70 oligonucleotides.

16. The liposomal particle of any of claims 1-15, wherein said oligonucleotide is a modified oligonucleotide.

17. A method for producing a liposomal particle comprising: a. adding a phospholipid to a solvent to form a first mixture comprising a variety of liposomes, b. disrupting said variety of liposomes to create a second mixture comprising a liposome and a small unilamellar vesicle (SUV), c. isolating said SUV with a particle size of between about 20 nanometers and 50 nanometers from said second mixture, d. adding an oligonucleotide to the isolated SUV to produce the liposomal particle.

18. The method of claim 17, wherein the particle size of the liposome variety in said first mixture is between about 100 nanometers and 150 nanometers.

19. The method of claim 17 or claim 18, wherein the particle size of the liposome and the SUV in said second mixture is between about 20 nanometers and about 150 nanometers.

20. The method of claim 17, wherein said liposomal particle has a particle size less than or equal to about 50 nanometers.

21. The liposomal particle of any of claims 17-20, wherein the oligonucleotide is a lipid and oligonucleotide conjugate containing a bound lipophilic group, wherein the lipid bilayer adsorbs said bound lipophilic group.

22. The method of claim 21, wherein the bound lipophilic group comprises tocopherol or cholesterol.

23. The method of claim 22, wherein the tocopherol is selected from the group consisting of a tocopherol derivative, alpha-tocopherol, beta-tocopherol, gamma-tocopherol and delta- nAfrnnn / zznz / E / YiAi 40 tocopherol.

24. The method of any of claims 17-23, wherein said oligonucleotide comprises RNA or DNA.

25. The method of claim 24, wherein said RNA is a non-coding RNA.

26. The method of claim 25, wherein said non-coding RNA is an inhibitory RNA (RNAi).

27. The method of claim 26, wherein the RNAi is selected from the group consisting of a small inhibitory RNA (sRNA), a single-stranded RNA (sRNA) forming a triple structure with double-stranded DNA, and a ribozyme.

28. The method of claim 24, wherein the RNA is a microRNA.

29. The method of claim 24, wherein said DNA is antisense DNA.

30. The method of any of claims 17-29, wherein the oligonucleotide is a modified oligonucleotide.

31. A method for inhibiting the expression of a gene comprising the step of hybridizing a polynucleotide encoding said gene product with one or more oligonucleotides complementary to all or part of said polynucleotide and attached to the liposomal particle of any of claims 1-16, wherein said polynucleotide and said oligonucleotide are hybridized to an extension of said polynucleotide with a degree of complementarity sufficient to inhibit the expression of said gene product.

32. The method of claim 29, wherein the expression of said gene product is inhibited in vivo.

33. The method of claim 29, wherein the expression of said gene product is inhibited in vitro.

34. The method of any of claims 31-33, wherein said liposomal particle has a diameter of about less than 50 nanometers or equal to it.

35. The method of any of claims 31-34, wherein said oligonucleotide comprises RNA or DNA.

36. The method of claim 35, wherein said RNA is a non-coding RNA.

37. The method of any of claim 36, wherein said non-coding RNA is an inhibitory RNA (RNAi).

38. The method of claim 37, wherein the RNAi is selected from the group consisting of a small inhibitory RNA (siRNA), a single-stranded RNA (sRNA) forming a triple structure with double-stranded DNA, and a ribozyme.

39. The method of claim 35, wherein said RNA is a microRNA.

40. The method of claim 35, wherein said DNA is antisense DNA.

41. A method for upregulating the activity of a Toll-like receptor (TLR) comprising contacting a cell having the Toll-like receptor with a liposomal particle 41 of any of claims 1-16.

42. The method of claim 41, wherein the oligonucleotide is a TLR agonist.

43. The method of claim 41 or claim 42, wherein said Toll-like receptor is selected from the group consisting of Toll-like receptor 1, Toll-like receptor 2, Toll-like receptor 3, Toll-like receptor 4, Toll-like receptor 5, Toll-like receptor 6, Toll-like receptor 7, Toll-like receptor 8, Toll-like receptor 9, Toll-like receptor 10, Toll-like receptor 11, Toll-like receptor 12 and Toll-like receptor 13.

44. A method for downregulating the activity of a Toll-like receptor (TLR) comprising contacting a cell having the Toll-like receptor with a liposomal particle of any of claims 1-16.

45. The method of claim 44, wherein the oligonucleotide is a TLR antagonist.

46. ​​The method of claim 44 or claim 45, wherein said Toll-type receptor is selected from the group consisting of Toll-type receptor 1, Toll-type receptor 2, Toll-type receptor 3, Toll-type receptor 4, Toll-type receptor 5, Toll-type receptor 6, Toll-type receptor 7, Toll-type receptor 8, Toll-type receptor 9, Toll-type receptor 10, Toll-type receptor 11, Toll-type receptor 12 and Toll-type receptor 13.

47. The method of any of claims 41-46 as implemented in vitro.

48. The method of any of claims 41-46 that is put into practice