WO2018152327A1 - Enhancing stability and immunomodulatory activity of liposomal spherical nucleic acids - Google Patents

Abstract

The present disclosure is directed to liposomal spherical nucleic acids synthesized by directly functionalizing DNA to lipids on the surface of a liposome. This leads to a higher DNA shell density, increased serum stability. These attributes provide markedly increased cellular uptake and enhanced sequence-specific immunostimulation.

Description

ENHANCING STABILITY AND IMMUNOMODULATORY ACTIVITY OF LIPOSOMAL

SPHERICAL NUCLEIC ACIDS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit under 35 U.S.C. § 1 19(e) of U.S.

Provisional Application No. 62/459,129, filed February 15, 2017, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under FA9550-12-1 -0141 awarded by the Air Force Office of Scientific Research and U54CA199091 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] This application contains, as a separate part of the disclosure, a Sequence Listing in computer readable form (Filename: 2017-015_Seqlisting.txt; Size: 3.865 bytes; Created: February 15, 2018), which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0004] The present disclosure relates to liposomal particles having enhanced stability, methods of making the same, and uses thereof. Liposomal particles are useful in gene regulation and drug delivery.

BACKGROUND

[0005] Lipid-functionalized oligonucleotides (LONs) are an attractive platform for many uses that include materials assembly [Thompson et al., Nano Lett. 2010, 10, 2690; Dave et al., ACS Nano 201 1 , 5, 1304], detection [Pfeiffer et al., J. Am. Chem. Soc. 2004, 126, 10224], and therapeutic design [Patwa et al., Chem. Soc. Rev. 201 1 , 40, 5844; Raouane et al., Bioconjugate Chem. 2012, 23, 1091 ; Soutschek et al., Nature 2004, 432, 173; Liu et al., Nature 2014, 507, 519] due to their attractive chemical and biological properties that include increased stability in serum and straightforward assembly into nanocarriers through hydrophobic interactions [Patwa et al., Chem. Soc. Rev. 201 1 , 40, 5844; Raouane et al., Bioconjugate Chem. 2012, 23, 1091 ; Soutschek et al., Nature 2004, 432, 173; Liu et al., Nature 2014, 507, 519; Banga et al., J. Am. Chem. Soc. 2014, 136, 9866; Liu et al., Chemistry-A European Journal 2010, 16, 3791 ; Wolfrum et al., Nat. Biotechnol. 2007, 25, 1 149; Yoshina-lshii et al., J. Am. Chem. Soc. 2003, 125, 3696; Wang et al., Angew. Chem. Int. Ed. Engl. 2016, 55, 12063]. LONs can be used to synthesize spherical nucleic acids (SNAs), a class of nanomaterial that consists of a small spherical core (<100 nm) functionalized with a dense and highly oriented oligonucleotide shell [Mirkin et al., Nature 1996, 382, 607]. The nucleic acid shell allows SNAs to readily enter cells without the need for transfection agents [Banga et al., J. Am. Chem. Soc. 2014, 136, 9866;Rosi et al., Science 2006, 312, 1027], enhances their binding affinity for protein receptors [Chinen et al., Angew. Chem. Int. Ed. Engl. 2015, 54, 527] and complementary oligonucleotides [Mirkin et al., Nature 1996, 382, 607;Cutler et al., J. Am. Chem. Soc. 2012, 134, 1376], and reduces their susceptibility to degradation by endonucleases [Rosi et al., Science 2006, 312, 1027].

Because of these enhanced properties, SNAs have emerged as attractive agents for biodetection [Seferos et al., J. Am. Chem. Soc. 2007, 129, 15477;Chen et al., Angew. Chem. Int. Ed. Engl. 2013, 125, 2066; Zheng et al., Nano Lett. 2009, 9, 3258;Qu et al., Angew. Chem. Int. Ed. Engl. 2017, 56, 1855; Liu et al., J. Am. Chem. Soc. 2017, 139, 9471 ], drug delivery [Zheng et al., ACS Nano 2013, 7, 6545; Tan et al., J. Am. Chem. Soc. 2016, 138, 10834; Li et al., Adv. Mater. 2013, 25, 4386], gene regulation [Rosi et al., Science 2006, 312, 1027; Giljohann et al., J. Am. Chem. Soc. 2009, 131 , 2072; Ryou et al., Biochem. Biophys. Res. Commun. 2010, 398, 542; Zheng et al., Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 1 1975; Jensen et al., Sci. Transl. Med. 2013, 5, 209ra152], and immunomodulation

[Radovic-Moreno et al., Proc. Natl. Acad. Sci. U. S. A. 2015, 1 12, 3892]. Since the biological properties of SNAs are independent of the core type, they have been synthesized with a variety of templates including gold particles [Mirkin et al., Nature 1996, 382, 607], micelles [Liu et al., Chemistry-A European Journal 2010, 16, 3791 ; Li et al., Nano Lett. 2004, 4, 1055; Banga et al., J. Am. Chem. Soc. 2017, 139, 4278-4281 ], infinite coordination polymer particles [Calabrese et al., Angew. Chem. Int. Ed. Engl. 2015, 54, 476], liposomes [Banga et al., J. Am. Chem. Soc. 2014, 136, 9866], and proteins [Brodin et al., J. Am. Chem. Soc. 2015, 137, 14838]. For in vivo biological applications, the liposomal variants are more appealing since the vesicle cores are highly biocompatible, able to encapsulate a diverse range of molecules, and readily functionalized in a modular fashion [Zelphati et al., J.

Control. Release 1996, 41 , 99].

SUMMARY OF THE INVENTION

[0006] In some aspects, the disclosure provides a liposomal particle comprising a lipid bilayer comprising a plurality of lipids and a plurality of oligonucleotides, wherein each oligonucleotide is covalently bound to a lipid, and the oligonucleotide and the lipid comprise complementary reactive moieties that together form a covalent bond. In some

embodiments, the plurality of lipids comprises (a) a fatty acid chain portion comprising (1 ) 10 to 22 carbons and (2) 0 to 5 carbon-carbon double bonds; and (b) a hydrophilic head portion, wherein the hydrophilic head portion can be neutral, cationic, or anionic. In further embodiments, the fatty acid chain portion comprises 15 to 22 carbons and 0 to 2 carbon- carbon double bonds.

[0007] In some embodiments, the plurality of lipids comprises a lipid selected from the group consisting of the phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine family of lipids. In further embodiments, the lipid is selected from the group consisting of dioleoylphosphatidylcholine; dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine; dilauroylphosphatidylcholine,

dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and diarachidonylphosphatidylcholine (DAPC), dioleoylphosphatidylethanolamine,

dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE) distearoylphosphatidylethanolamine (DSPE), and a modified analog thereof.

[0008] In some embodiments, the reactive moiety on the lipid comprises an azide, an alkyne, a maleimide, a thiol, an alcohol, an amine, a carboxylic acid, an olefin, an

isothiocyanate, a N-hydroxysuccinimide, a phosphine, a nitrone, a norbornene, an oxanorbornene, a transcycloctene, an s-tetrazene, an isocyanide, a tetrazole, a nitrile oxide, a quadricyclane, or a carbodiimide. In further embodiments, the reactive moiety on the oligonucleotide is on a terminus of the oligonucleotide. In still further embodiments, the reactive moiety on the oligonucleotide comprises an alkyne, an azide, a maleimide, a thiol, an alcohol, an amine, a carboxylic acid, an olefin, an isothiocyanate, a N- hydroxysuccinimide, a phosphine, a nitrone, a norbornene, an oxanorbornene, a

transcycloctene, an s-tetrazene, an isocyanide, a tetrazole, a nitrile oxide, a quadricyclane, or a carbodiimide. In some embodiments, the alkyne comprises dibenzocyclooctyl (DBCO) alkyne or a terminal alkyne. In further embodiments, the lipid comprises an azide reactive moiety and the oligonucleotide comprises an alkyne reactive moiety, or vice versa. In still further embodiments, the alkyne reactive moiety comprises a DBCO alkyne.

[0009] In some embodiments, the oligonucleotide comprises RNA or DNA. In further embodiments, the RNA is a non-coding RNA, and in still further embodiments the non- coding RNA is an inhibitory RNA. In some embodiments, the inhibitory RNA is selected from the group consisting of a small inhibitory RNA (siRNA), a single-stranded RNA (ssRNA) that forms a triplex with double stranded DNA, and a ribozyme. In further embodiments, the RNA is a microRNA. In some embodiments, the DNA is antisense-DNA.

[0010] In some embodiments, diameter of the liposomal particle is less than or equal to about 50 nanometers.

[0011] In some embodiments, the particle comprises 50 to 500 oligonucleotides. In further embodiments, the particle comprises 150 to 350 oligonucleotides. In still further embodiments, the particle comprises 200 to 300 oligonucleotides. In some embodiments, the oligonucleotide is a modified oligonucleotide. In further embodiments, the

oligonucleotide further comprises a fluorescent tag.

[0012] The disclosure also provides, in some aspects, a method of inhibiting expression of a gene comprising hybridizing a polynucleotide encoding a gene product expressed from the gene with one or more oligonucleotides complementary to all or a portion of the polynucleotide, the complementary oligonucleotide comprising the oligonucleotide of a liposomal particle of the disclosure, wherein hybridizing between the polynucleotide and the complementary oligonucleotide occurs over a length of the polynucleotide with a degree of complementarity sufficient to inhibit expression of the gene product. In some embodiments, expression of the gene product is inhibited in vivo. In further embodiments, expression of the gene product is inhibited in vitro.

[0013] In some aspects, the disclosure provides a method for up-regulating activity of a toll-like receptor (TLR), comprising contacting a cell having the TLR with a liposomal particle of the disclosure. In some embodiments, the oligonucleotide is a TLR agonist. In further embodiments, the TLR is 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, tolllike receptor 9, toll-like receptor 10, toll-like receptor 1 1 , toll-like receptor 12, or toll-like receptor 13.

BRIEF DESCRIPTION OF FIGURES

[0014] Figure 1 shows the dissociation of LSNAs in the presence of other liposomal templates. A) A schematic representation of the FRET reporter system with two different types of LSNAs. The transfer of DNA from Particle B to Particle A results in an increase in fluorescence from FRET pair 1 (fluoroscein:Cy5) and decrease in fluorescence from FRET pair 2 (rhodamine:Cy5). B) The fret ratio for both FRET pairs are monitored over time at different ratios of particle A:particle B (filled). The FRET ratio for pair 1 (left) increases in intensity while the FRET ratio for pair 2 (right) shows a simultaneous decrease in

fluorescence due to the release of Cy5-labeled DNA from rhodamine-labeled liposomal core for cholesterol-tail (circle) and lipid-tail LSNAs (square). A slight increase in the FRET measurements for the lipid-tail SNAs due to the higher loading density that results in self- quenching of Cy5.

[0015] Figure 2 shows the dissociation of LSNAs in the presence of other liposomal templates. A) A schematic representation of the dissolution process for LSNAs in the presence of other liposomes. B) The FRET ratio of the cholesterol-tail and lipid-tail LSNAs in the presence of excess DOPC liposomes. C) The FRET ratio of both LSNAs incubated in buffer.

[0016] Figure 3 shows disassembly of LSNAs in serum. A) A schematic representation of the disassembly of FRET reporting LSNAs synthesized with Cy5 labeled DNA and rhodamine labeled lipids upon incubation in 10 vol% serum solution. B) Rhodamine fluorescence measured over time for cholesterol-tail and lipid tail LSNAs. C) The FRET ratio between rhodamine and Cy5 for both LSNAs. [0017] Figure 4 shows confocal images of U87-MG cells that were incubated with LSNAs (FRET particle B) synthesized with Cy5-labeled DNA (red) and rhodamine-labeled liposomes (green) for 1 and 24 hours. Increased uptake is observed after 1 hour in cells that were exposed to the lipid-tail LSNAs. After 24 hours, similar uptake is seen for both the types of LSNAs. The nuclei are stained with Hoechst (blue). Scale Bar: 50 μηι

[0018] Figure 5 shows acceptor photobleaching FRET imaging of LSNAs (A-B) Confocal images of a typical photobleaching experiment before (A) and after (B) photobleaching of the Cy5 labelled DNA (red) with rhodamine labelled liposomes (green) and hoescht stained nuclei (blue). The region of interest where the Cy5 is photobleaching is boxed with zoomed in images of the photobleaching regions for Cy5 DNA and rhodamine labelled liposomes shown below their respective image. Scale bars = 10 μηι. (C) The FRET efficiency for lipid- tail LSNAs with DNA shell densities of 300 and 150 strands/liposome and cholesterol-tail LSNA (150 strands/liposome) were calculated by measuring the fluorescence of rhodamine before and after photobleaching of Cy5. (n = 7-10 cells, ^*P < 0.05, ^**P < 0.01 , ^***P < 0.001 determined by ANOVA with a Tukey HSD post-hoc test)

[0019] Figure 6 shows activation of TLR9 by LSNAs in HEK-Blue cells. (A) Plot of the amounts of secreted alkaline phosphatase (SEAP) by HEK-Blue cells that have been exposed to cholesterol-tail (red) and lipid-tail (blue) LSNAs, as visualized by a colorimetric assay. Enhanced immunostimulatory activity is observed for the latter in comparison to the former. (B) A plot of a pulse-chase experiment where the particles were incubated for different periods of time with the cells. Lipid-tail LSNAs displayed higher activity for all time point (p < 0.01 ). T₂₀(SEQ ID NO:1 ) sequence functionalized LSNAs were used as a negative control.

[0020] Figure 7 shows the disassembly rate of liposomal SNAs assembled with different DNA densities were measured over time. The apparent rate of DNA shell disassembly decreases for lower DNA densities.

[0021] Figure 8 shows the distribution of liposomal template sizes measured with DLS.

[0022] Figure 9 depicts the fluorescence spectra of assembled FRET particles and SDS (0.1 vol%) treated particles.

[0023] Figure 10 depicts the fluorescence spectra of LSNAs containing rhodamine- labeled lipids mixed with LSNAs containing fluorescein labeled lipid when excited with a 480 nm wavelength laser, the peak excitation for fluoroscein. All particles were assembled with unlabeled DNA strands, such that FRET is only observed upon exchange of the labeled lipids. [0024] Figure 11 depicts FRET between labeled lipids within cholesterol-tail LSNAs was measured over time in 10 vol% FBS at 37 ^eC. The data were fit to a non-linear decay equation.

[0025] Figure 12 depicts the FRET signal monitored over time for lipid-tail LSNAs assembled with different DNA densities incubated in 10 vol% FBS.

[0026] Figure 13 shows A) The FRET ratio between Cy5-DNA and rhodamine-labeled liposomes at different concentrations of FBS. B) The FRET ratio was measured for two different concentrations of LSNA in a 10 vol% serum solution.

[0027] Figure 14 shows the activation of RAW Blue Macrophages by LSNAs. A) TLR9 activation resulting from overnight incubation with CpG and control (T25) LSNAs along with the linear CpG DNA. B) TLR9 activation from pulse-chase treatment with CpG LSNAs. The activation is normalized to that measured after overnight incubation at the same

concentration. (^*p < 0.01 , ANOVA with Bonferroni post-hoc test).

[0028] Figure 15 depicts cellular uptake of LSNAs. A-B) Histograms of cellular

fluorescence intensity and the median fluorescence intensity for U87-MG cells (A) and Raw- Blue macrophages (B) incubated with structures incorporating Cy5 labeled DNA (^**P < 0.01 , ^***P < 0.001 ; ANOVA with a Bonferroni post-hoc test).

[0029] Figure 16 depicts cell viability following treatment with LSNAs at different concentrations. Cells treated with 10 vol% DMSO were utilized as a positive control. No cytoxicity was observed for any of the structures.

[0030] Figure 17 shows median fluorescence intensity of cells that were incubated with 500 nM of SNAs when treated with Fucoidan, an inhibitor of scavenger receptors on cells and also when not treated with any inhibitors (untreated). (^**P<0.01 ; ANOVA with Bonferonni post-hoc test).

[0031] Figure 18 shows examples of PE lipids with amine head groups used in synthesis of LSNAs herein.

[0032] Figure 19 depicts that the 1826 PS DNA conjugated to lipids on LSNAs outperform 1826 PS Linear oligonucleotides.

[0033] Figure 20 shows that the SNA architecture improves immune activation of 1826 PS oligonucleotide compared to the lipid-oligonucleotide conjugate.

DETAILED DESCRIPTION

[0034] Liposomal spherical nucleic acids (LSNAs) are an attractive therapeutic platform for gene regulation and immunomodulation due to their biocompatibility, chemically tunable structures, and ability to enter cells rapidly without the need for ancillary transfection agents. Such structures comprise small (<100 nm) liposomal cores functionalized with a dense, highly oriented nucleic acid shell, both of which are facilitate the biological activity of the LSNA. Here, the properties of LSNAs synthesized using conventional methods (i.e., anchoring cholesterol terminated oligonucleotides into a liposomal core) are compared to the LSNAs of the present disclosure, which are made by directly modifying the surface of a liposomal core having azide-functionalized lipids with alkyne {e.g., DBCO)-terminated oligonucleotides. The surface densities of the oligonucleotides are measured for both types of LSNAs, with the lipid-modified structures having approximately twice the oligonucleotide surface coverage compared to the conventional LSNAs. The stabilities and cellular uptake properties of these structures are also evaluated. The higher density, lipid-functionalized structures are more stable than conventional cholesterol-based structures in the presence of other unmodified liposomes and serum proteins as evidenced by fluorescence assays. Significantly, this new form of LSNA exhibits more rapid cellular uptake and increased sequence-specific toll-like receptor activation in immune reporter cell lines, making it a promising candidate for immunotherapy.

[0035] Herein, structures were synthesized with greater nucleic acid loading and less dynamic behavior in an effort to establish structure-function relationships between LSNAs and cellular environments. To accomplish this goal, LSNAs were synthesized using different hydrophobic anchors, lipid or cholesterol, chemically interfaced with DNA (Scheme 1 ) and various properties were measured - stability in buffer and serum, dissociation kinetics, cellular uptake properties, and ability to engage with toll-like receptors (TLRs) critical for immune-modulation. From these studies, it was determined that LSNAs synthesized with lipid-modified DNA lead to twice the oligonucleotide loading, resulting in substantively enhanced stability, more rapid cellular internalization, and greater sequence-specific TLR- mediated immune activation.

[0036] Spherical nucleic acids (SNAs) are a class of nanomaterials having a spherical core and a densely packed and highly oriented nucleic acid shell [Mirkin, et al. Nature 1996, 382 (6592), 607-9], and have emerged as attractive immunomodulatory [Radovic-Moreno, et al. Proc. Natl. Acad. Sci. U. S. A. 2015, 1 12 (13), 3892-3897] and gene regulatory

[Giljohann, et al. J. Am. Chem. Soc. 2009, 131 (6), 2072-3; Zheng, et al. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (30), 1 1975-1 1980] agents. The dense and highly oriented nucleic acid shell allows SNAs to readily enter cells without the need for transfection agents [Banga, et al. J. Am. Chem. Soc. 2014, 136 (28), 9866-9; Sprangers, et al. Small 2016], enhances their binding affinity for protein receptors [Chinen, et al. Angew. Chem., Int. Ed. 2015, 54 (2), 527-31 ] and complementary oligonucleotides [Mirkin, et al. Nature 1996, 382 (6592), 607-9; Cutler, et al. J. Am. Chem. Soc. 2012, 134 (3), 1376-91 ], and reduces their susceptibility to degradation by endonucleases [Banga, et al. J. Am. Chem. Soc. 2014, 136 (28), 9866-9]. SNAs have been synthesized with multiple different core types, including gold [Mirkin, et al. Nature 1996, 382 (6592), 607-9], infinite coordination polymers [Calabrese, et al. Angew. Chem., Int. Ed. 2015, 54 (2), 476-80], micelles [Li, et al. Nano Lett. 2004, 4 (6), 1055-1058], and liposomes [Banga, et al. J. Am. Chem. Soc. 2014, 136 (28), 9866-9]. Of these structures, liposomal spherical nucleic acids (LSNAs) are particularly attractive since the liposomal vesicle cores are biocompatible, able to encapsulate a diverse range of molecules, and readily functionalized in modular fashion [Zelphati, et al J. Controlled Release 1996, 41 (1 -2), 99-1 19]. Thus, LSNAs are attractive gene regulatory [Banga, et al. J. Am. Chem. Soc. 2014, 136 (28), 9866-9] and immunomodulatory [Radovic-Moreno, et al. Proc. Natl. Acad. Sci. U. S. A. 2015, 1 12 (13), 3892-3897] therapeutic candidates.

[0037] LSNAs are typically synthesized by intercalating nucleic acids that have been modified with hydrophobic moieties, such as cholesterol or tocopherol, into the lipid bilayer of the liposomal template [Banga et al., J. Am. Chem. Soc. 2014, 136, 9866; Radovic-Moreno et al., Proc. Natl. Acad. Sci. U. S. A. 2015, 1 12, 3892;Sprangers et al., Small 2016, 13]. However, the fluidic nature of the liposomal core [Reddy et al., Biophys. Acta 2012, 1818, 2271 ], and the hydrophilicity of the nucleic acid shell [Dayani et al., Biomacromolecules 2013, 14, 3380] make this structure intrinsically dynamic. Indeed, conventional liposome- based structures comprised of lipids and hydrophobic intercalators, such as tocopherol and cholesterol, are known to undergo inter-particle exchange [Mclean, et al. Biochemistry 1981 , 20 (10), 2893-2900]. For LSNAs, inter-particle component exchange could reduce the stability of the nucleic shell and ultimately the entire SNA structure. Studies on planar lipid bilayers and cationic vesicles with electrostatically adsorbed nucleic acids suggest such exchange may be possible [Frykholm, et al. Langmuir 2009, 25 (3), 1606-161 1 ; Frykholm, et al. Febs J. 2010, 277, 234-235]. The hydrophobic groups covalently anchored to the nucleic acids can modulate the dynamics of interparticle exchange; cholesterol-modified nucleic acids have weaker and more dynamic interactions with lipid bilayers, while nucleic acids modified with diacyl chains are significantly more stable [Pfeiffer et al., J. Am. Chem. Soc. 2004, 126, 10224; van der Meulen et al., Langmuir 2014, 30, 6525; Gambinossi et al., J. Phys. Chem. B 2010, 1 14, 7338]. Furthermore, in biological environments, the presence of serum proteins and natural lipid-based structures, which are known to readily interact with liposome-based conjugates [Kim et al., Arch. Pharm. Res. 1991 , 14, 336], in principle, could further destabilize LSNAs by interacting with the dissociated nucleic acid strands.

[0038] Any loss of the nucleic acid shell is likely to alter the interactions between LSNAs and cells. Indeed, prior SNA research has shown that greater oligonucleotide density leads to increased cellular uptake [Giljohann et al., Nano Lett. 2007, 7, 3818]. Along with the dynamic behavior of LSNAs, the conventional approaches to synthesizing LSNAs result in lower oligonucleotide densities in comparison to SNAs with inorganic cores [Hurst et al., Anal. Chem. 2006, 78, 8313]. These observations point toward the importance of increasing oligonucleotide density and maintaining stability of LSNA architectures in physiological environments.

[0039] Prior research has shown that increased nucleic acid density leads to increased cellular uptake, yet the approaches for making conventional LSNAs often lead to structures with lower oligonucleotide densities when compared to SNAs with inorganic cores [Hurst, et al. Anal. Chem. 2006, 78 (24), 8313-8]. This observation points towards the need for developing methods for increasing oligonucleotide loading in LSNA architectures. Provided herein is a new strategy for synthesizing LSNAs that involves the generation of a liposomal vesicle comprised of lipids with azide functional groups (Scheme 1 ). Such structures can then be readily and covalently modified with oligonucleotides with tails that contain dibenzocyclooctyl (DBCO) groups. This strategy essentially trades the cholesterol or tocopherol anchoring groups associated with conventional LSNAs for lipid anchoring groups. However, the ability to preform the vesicle with the appropriate number of azide functional groups, allows one to modulate and maximize oligonucleotide surface coverage. The two classes of LSNAs are compared, henceforth referred to as cholesterol-tail and lipid-tail LSNAs (Scheme 1 ), with regard to their structures, stabilities in buffer and serum, dissociation kinetics, cellular uptake properties, and ability to engage with TLRs critical for immune-modulation. With lipid-tail SNAs, oligonucleotide loading can be increased by a factor of 2, which results in substantively enhanced stability, increased cellular uptake, and increased sequence specific TLR-9 immune activation as measured with a Quanti-Blue assay.

Scheme 1

Lipid-az de

[0040] Synthesis of LSNAs: LSNAs were synthesized by modifying the surface of small unilamellar vesicle (SUV) templates (50 nm size; Figure 8) using two different

functionalization strategies (Scheme 1 ). In the first strategy, cholesterol-tail LSNAs were synthesized via literature methods by adding cholesterol-modified DNA to 1 ,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC)-based SUVs in HEPES-buffered saline (HBS; Scheme 1 A) [Banga et al., J. Am. Chem. Soc. 2014, 136, 9866]. In the second strategy, an azide- functionalized 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) derivative, 1 ,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6-azidohexanoyl) (DPPE-Az), was used as the minor lipid component (0.5 - 1 0 mol%) in the formation of DOPC-based SUVs with azide groups presented on the surface of the vesicle (Scheme 1 B). While DPPE-azide was used in the specific example, other lipids modified to include an azide (N₃) moiety at one terminus can be used to form a SUV.

[0041 ] The lipid comprises a fatty acid chain portion and a hydrophilic head portion. In some cases, the fatty acid chain portion comprises 1 0-22 (e.g., 12-22, 1 5-22, 1 5-20, 10, 1 1 , 12, 1 3, 14, 1 5, 16, 1 7, 1 8, 19, 20, 21 , or 22) carbons with 0-5 (e.g., 0, 1 , 2, 3, 4, 5, 1 -2, 0-2, 2-3, 0-3, 1 -4, 1 -3, 1 -5) carbon-carbon double bonds (also referred to as "unsaturation" of the fatty acid chain) throughout the fatty acid chain. The hydrophilic head portion can be modified to comprise the reactive moiety. The hydrophilic head portion can be neutral, cationic (positively charged), or anionic (negatively charged). Contemplated lipids include phosphatidylcholine with both saturated and unsaturated fatty acid chains, including dioleoylphosphatidylcholine; dimyristoylphosphatidylcholine (DMPC);

dipentadecanoylphosphatidylcholine; dilauroylphosphatidylcholine;

dipalmitoylphosphatidylcholine (DPPC); distearoylphosphatidylcholine (DSPC); and diarachidonylphosphatidylcholine (DAPC); phosphatidylethanolamines, such as

dioleoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE) and distearoylphosphatidylethanolamine (DSPE). The choline or ethanolamine moiety can be modified derivatives that provide an reactive {e.g., azide) moiety. The synthesis method can also be generalized to other PE lipids. The amines on these lipids can be modified to have any of a plurality of functional groups. Examples of these functional groups include maleimide, azide, and alkyne (Figure 18).

[0042] DBCO-modified DNA strands were then covalently conjugated to the azide groups through Cu-free click chemistry [Baskin, et al. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (43), 16793-16797]. While DBCO-modified DNA was used in this example, other alkyne moieties can be used instead, including a terminal alkyne (HC≡C-) or an internal alkyne (RC≡C-, where R comprises an alkyl). The alkyne moiety can be attached to the oligonucleotide via a linker as shown in the example, e.g., a linker modified to include a phosphoramidite that can used with an oligo synthesizer to attach to one terminus of the oligonucleotide of interest for inclusion in the LSNA.

[0043] While the specific examples shown use DNA, oligonucleotides in general are contemplated. Oligonucleotides can comprise DNA, RNA, modified DNA and/or RNA, or a combination thereof. The oligonucleotide can comprise any sequence, and include both natural and unnatural nucleotides. The sequence of the oligonucleotide can be any desired sequence, and in some cases can be selected in view of the desired end use {e.g., for use in gene targeting, or to stimulate or inhibit a selected target).

[0044] Oligonucleotides contemplated for use according to the disclosure are from about 5 to about 100 nucleotides in length. Methods and compositions are also contemplated wherein the oligonucleotide is about 5 to about 90 nucleotides in length, about 5 to about 80 nucleotides in length, about 5 to about 70 nucleotides in length, about 5 to about 60 nucleotides in length, about 5 to about 50 nucleotides in length about 5 to about 45 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 35 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to about 15 nucleotides in length, about 5 to about 10 nucleotides in length, and all oligonucleotides intermediate in length of the sizes specifically disclosed to the extent that the oligonucleotide is able to achieve the desired result. Accordingly, oligonucleotides of 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24,25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 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 are contemplated.

[0045] Modified Oligonucleotides. Specific examples of oligonucleotides include those containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are considered to be within the meaning of "oligonucleotide."

[0046] Modified oligonucleotide backbones 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, phosphinates, phosphoramidates including 3'-amino phosphoramidate and

aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5', or 2' to 2' linkage. Also contemplated are oligonucleotides having inverted polarity comprising a single 3' to 3' linkage at the 3'-most internucleotide linkage, i.e. a single inverted nucleoside residue which may be abasic (the nucleotide is missing or has a hydroxyl group in place thereof). Salts, mixed salts and free acid forms are also contemplated. Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, U.S. Pat. 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 ; 5,541 ,306; 5,550,1 1 1 ; 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, the disclosures of which are incorporated by reference herein.

[0047] Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts. See, for example, U.S. Patent Nos. 5,034,506; 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, the disclosures of which are incorporated herein by reference in their entireties.

[0048] In still other embodiments, oligonucleotide mimetics wherein both one or more sugar and/or one or more internucleotide linkage of the nucleotide units are replaced with "non-naturally occurring" groups. In one aspect, this embodiment contemplates a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone. See, for example US Patent Nos. 5,539,082; 5,714,331 ; and 5,719,262, and Nielsen et al., 1991 , Science, , 254: 1497-1500, the disclosures of which are herein incorporated by reference.

[0049] In still other embodiments, oligonucleotides are provided with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and including— CH₂— NH— O— CH₂— ,— CH₂— N(CH₃)— O— CH₂—„— CH₂— O— N(CH₃)— CH₂— ,— CH₂— N(CH₃)— N(CH₃)— CH₂— and—0—N(CH₃)—CH₂—CH₂— described in US Patent Nos. 5,489,677, and 5,602,240. Also contemplated are oligonucleotides with morpholino backbone structures described in US Patent No. 5,034,506.

[0050] In various forms, the linkage between two successive monomers in the

oligonucleotide consists of 2 to 4, desirably 3, groups/atoms selected from— CH₂— ,— O— ,

— S— ,— NR^H— , >C=0, >C=NR^H, >C=S,— Si(R")₂— ,—SO—,— S(0)₂— ,— P(0)₂— ,—

PO(BH₃)— ,— P(0,S)— ,— P(S)₂— ,— PO(R")— ,— PO(OCH₃)— , and— PO(NHR^H)— , where RH is selected from hydrogen and Ci_₄-alkyl, and R" is selected from d-e-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=(including R⁵ when used as a linkage to a succeeding monomer),— CH₂— CH₂— O— ,—

NR^H— CH₂— CH₂— ,— CH₂— CH₂— NR^H— ,— CH₂— NR^H— CH₂— -,— O— CH₂— CH₂—

NR^H— ,— NR^H— CO— O— ,— NR^H— CO— NR^H— ,— NR^H— CS— NR^H— ,— NR^H— C(=NR^H)—

NR^H— ,— NR^H— CO— CH₂— NR^H— O— CO— O— ,— O— CO— CH₂— O— ,— O— CH₂— CO—

O— ,— CH₂— CO— NR^H— ,— O— CO— NR^H— ,— NR^H— CO— CH₂— ,— O— CH₂— CO—

NR^H— ,— O— CH₂— CH₂— NR^H— ,— CH=N— O— ,— CH₂— NR^H— O— ,— CH₂— O—

N=(including R⁵ when used as a linkage to a succeeding monomer),— CH₂— O— NR^H— ,—

CO— NR^H— CH₂— ,— CH₂— NR^H— O— ,— CH₂— NR^H— CO— ,— O— NR^H— CH₂— ,— O— NR ,— O— CH₂— S— ,— S— CH₂— O— ,— CH₂— CH₂— S— ,— O— CH₂— CH₂— S— ,— S— CH₂— CH=(including R⁵ when used as a linkage to a succeeding monomer),— S— CH₂— CH₂— ,— S— CH₂— CH₂— O— ,— S— CH₂— CH₂— S— ,— CH₂— S— CH₂— ,— CH₂— SO— CH₂— ,— CH₂— S0₂— CH₂— ,— O— SO— O— ,— O— S(0)₂— O— ,— O—

S(0)₂— CH₂— ,— O— S(0)₂— NR^H— ,— NR^H— S(0)₂— CH₂— ;— O— S(0)₂— CH₂— ,— O— P(0)₂— O— ,— O— P(0,S)— O— ,— O— P(S)₂— O— ,— S— P(0)₂— O— ,— S— P(0,S)— O— , — S— P(S)₂— O— ,— O— P(0)₂— S— ,— O— P(0,S)— S— ,— O— P(S)₂— S— ,— S— P(0)₂— S— ,— S— P(0,S)— S— ,— S— P(S)₂— S— ,— O— PO(R")— O— ,— O— PO(OCH₃)— o— ,— O— PO(0 CH₂CH₃)— O— ,— O— PO(0 CH₂CH₂S— R)— O— ,— O— PO(BH₃)— O— ,— O— PO(NHR^N)— O— ,— O— P(0)₂— NR^H H— ,— NR^H— P(0)₂— O— ,— O— P(0,NR^H)— O— ,— CH₂— P(0)₂— O— ,— O— P(0)₂— CH₂— , and— O— Si(R")₂— O— ; among which— CH₂— CO— NR^H— ,— CH₂— NR^H— O—— S— CH₂— O— ,— O— P(0)₂— O— O— P(- 0,S)— O— , — O— P(S)₂— O— ,— NR^H P(0)₂— O— ,— O— P(0,NR^H)— O— ,— O— PO(R")— O— ,— O— PO(CH₃)— O— , and— O— PO(NHR^N)— O— , where RH is selected form hydrogen and C₁-4- alkyl, and R" is selected from d.₆-alkyl and phenyl, are contemplated. Further illustrative examples are given in Mesmaeker et. al., 1995, Current Opinion in Structural Biology, 5: 343-355 and Susan M. Freier and Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol 25: pp 4429-4443.

[0051] Still other modified forms of oligonucleotides are described in detail in U.S. Patent Application No. 20040219565, the disclosure of which is incorporated by reference herein in its entirety.

[0052] Modified oligonucleotides may also contain one or more substituted sugar moieties. In certain aspects, oligonucleotides comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-0- alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Other embodiments include 0[(CH₂)_nO]_mCH₃,

0(CH₂)_nOCH₃, 0(CH₂)_nNH₂, 0(CH₂)_nCH₃, 0(CH₂)_nONH₂, and 0(CH₂)_nON[(CH₂)_nCH₃]₂, where n and m are from 1 to about 10. Other oligonucleotides comprise one of the following at the 2' position: Ci to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, CI, Br, CN, CF₃, OCF₃, SOCH₃, S0₂CH₃, ON0₂, N0₂, N₃, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter 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'-0-CH₂CH₂OCH₃, also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al., 1995, Helv. Chim. Acta, 78: 486-504) i.e., an alkoxyalkoxy group. Other modifications include 2'- dimethylaminooxyethoxy, i.e., a 0(CH₂)₂0N(CH₃)₂ group, also known as 2'-DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-0— CH₂— O— CH₂—

N(CH₃)₂, also described in examples herein below.

[0053] Still other modifications include 2'-methoxy (2'-0— CH₃), 2'-aminopropoxy (2'- OCH₂CH₂CH₂NH₂), 2' allyl (2'-CH₂— CH=CH₂), 2'-0-allyl (2'-0— CH₂— CH=CH₂) and 2'-fluoro (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position. In one aspect, a 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, for example, at the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. See, for example, U.S. Pat. Nos. 4,981 ,957; 5,1 18,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,81 1 ; 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, the disclosures of which are incorporated herein by reference in their entireties.

[0054] In some cases, a modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is in certain aspects is a methylene (— CH₂— )_n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0055] Oligonucleotides may also include base modifications or substitutions. As used herein, "unmodified" or "natural" 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- hydroxymethyl cytosine, 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-halouracil 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 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7- methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified bases include tricyclic pyrimidines such as phenoxazine cytidine(1 H-pyrimido[5 ,4- b][1 ,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1 H-pyrimido[5 ,4-b][1 ,4]benzothiazin- 2(3H)-one), G-clamps such as a substituted phenoxazine cytidine {e.g. 9-(2-aminoethoxy)-H- pyrimido[5,4-b][1 ,4]benzox- azin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2- one), pyridoindole cytidine (H-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. Further bases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et ai, 1991 , Angewandte Chemie, International Edition, 30: 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these bases are useful for increasing the binding affinity and include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O- 6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1 .2°C. and are, in certain aspects combined with 2'-0-methoxyethyl sugar modifications. See, U.S. Pat. Nos. 3,687,808, U.S. Pat. Nos. 4,845,205; 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; 5,525,71 1 ; 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 , the disclosures of which are incorporated herein by reference.

[0056] A "modified base" or other similar term refers to a composition which can pair with a natural base {e.g., adenine, guanine, cytosine, uracil, and/or thymine) and/or can pair with a non-naturally occurring base. In certain aspects, the modified base provides a T_m differential of 15, 12, 10, 8, 6, 4, or 2°C. or less. Exemplary modified bases are described in EP 1 072 679 and WO 97/12896.

[0057] By "nucleobase" is meant the naturally occurring nucleobases 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-deazaxanthine, 7- deazaguanine, N⁴,N⁴-ethanocytosin, N',N'-ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C³— C⁶)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5- methyl-4-tr- iazolopyridin, isocytosine, isoguanine, inosine and the "non-naturally occurring" nucleobases described in Benner et al., U.S. Pat. No. 5,432,272 and Susan M. Freier and Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp 4429-4443. The term

"nucleobase" thus includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Further naturally and non-naturally occurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan, et al.), in Chapter 15 by Sanghvi, in Antisense Research and Application, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, in Englisch et al., 1991 , Angewandte Chemie, International Edition, 30: 613-722 (see especially pages 622 and 623, and in the Concise Encyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed., John Wiley & Sons, 1990, pages 858-859, Cook, Anti-Cancer Drug Design 1991 , 6, 585-607, each of which are hereby incorporated by reference in their entirety). The term "nucleosidic base" or "base unit" is further intended to include compounds such as heterocyclic compounds that can serve like nucleobases including certain "universal bases" that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases. Especially mentioned as universal bases are 3-nitropyrrole, optionally substituted indoles {e.g., 5-nitroindole), and optionally substituted hypoxanthine. Other desirable universal bases include, pyrrole, diazole or triazole derivatives, including those universal bases known in the art.

[0058] While Scheme 1 and the specific examples are directed to an oligonucleotide having an alkyne reactive moiety and a lipid having an azide reactive moiety, the reverse is also contemplated. Further, the oligonucleotide can be modified to include any

complementary reactive moiety to a reactive moiety on the lipid so as to form a covalent bond between the oligo and the lipid. Complementary reactive moieties are moieties that react to form a covalent bond. Non-limiting examples of such complementary groups include an alcohol and a carboxyl group {e.g., carboxylic acid, carboxylic halide, carbodiimide, maleimide) to form an ester bond, a amine and a carboxyl group to form an amide bond, a thiol and a carboxyl group to form a thioester bond, and an alkyne and an azide to form a triazolyl ring. Other reactive moieties contemplated include an olefin, an isothiocyanate, a N- hydroxysuccinimide, a phosphine, a nitrone, a norbornene, an oxanorbornene, a

transcycloctene, an s-tetrazene, an isocyanide, a tetrazole, a nitrile oxide, and a

quadricyclane. Specific reactive moieties pairings contemplated for the oligonucleotide and lipid covalent bond formation include maleimide-thiol, and use of a carbodiimide {e.g., EDC, DCC) to form an amide bond between a carboxylic acid and amine.

[0059] The disclosed strategy relies on two potential advantages: 1 ) the number of potential anchoring sites {e.g., azides) can be controlled prior to surface modification through the stoichiometry used to create the initial vesicle, and 2) the saturated lipid-tail has lower diffusivity in a DOPC-based lipid bilayer than cholesterol, minimizing the dissociation of oligonucleotides already anchored to the liposomal vesicle [Filippov, et al. Biophys. J. 2003, 84 (5), 3079-3086; Machan, et al. Biochim. Biophys. Acta, Biomembr. 2010, 1798 (7), 1377- 1391 ; Gilbert et al., Biochemistry 1975, 14, 444;Smith et al., J. Mol. Biol. 1972, 67, 75].

Since the hydrophobic anchors for the two different strategies have different affinities for the liposomal template, the DNA loading for each was determined. To determine the

stoichiometry that led to maximum DNA loading, increasing amounts of either DBCO-DNA or cholesterol-DNA were incubated with their respective liposomal templates overnight at 25° C (Table 1 ). Following purification by size-exclusion chromatography, the number of strands per liposome was assessed. Lipid-tail LSNAs had significantly greater DNA loading compared to cholesterol-tail analogs (Table 1 ). This constitutes a substantive increase in DNA shell density, which should affect particle interactions with proteins and cells.

[0060] Cholesterol-tail LSNAs (approximately! 50 strands/liposome) prepared as- described have significantly reduced DNA loading compared to direct lipid-tail LSNAs (-300 strands/liposome). The number of strands per liposome can be controlled by the amount of modified lipid used in the making of the SUV. A higher concentration of oligonucleotide can be achieved by a higher concentration of modified lipid used. The concentration of the modified lipid can be, for example, 0.5 mol% to 10 mol%, 1 mol% to 9 mol%, or 2 mol% to 8 mol%, of the total amount of lipid used to form the SUV.

Table 1 . Number of strands per particle when incubated with different ratios of liposome to

DNA.

Lipid-tail LSNAs

Mole Fraction of Azide in Liposome

10% 5% 2.5%

Ratio of DNA to Total Azide Strands/Liposome

10:1 330 330 190

5:1 320 340 160

1 :1 200 190 1 10

Cholesterol-tail LSNA

Cholesterol DNA Added

Strands/Liposome

(μΜ DNA/mM Lipid)

15 70

20 90

30 140

50 150

[0061] Thus, the disclosure contemplates LSNAs comprising from about 50 to about 500 oligonucleotides. In some embodiments, a particle of the disclosure comprises from about 100 to about 450 oligonucleotides, or from about 100 to about 400 oligonucleotides, or from about 150 to about 350 oligonucleotides. In further embodiments, a particle of the disclosure comprises from about 200 to about 300 oligonucleotides. In some embodiments, a particle of the disclosure comprises from about 200 to about 500 oligonucleotides. In some embodiments, a particle of the disclosure comprises from about 200 to about 400 oligonucleotides. In some embodiments, a particle of the disclosure comprises from about 300 to about 500 oligonucleotides. In further embodiments, a particle of the disclosure comprises from about 300 to about 400 oligonucleotides. In still further embodiments, a particle of the disclosure comprises at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, or at least about 500 oligonucleotides. In some embodiments, a particle of the disclosure comprises about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 oligonucleotides. It will be understood that the aforementioned number of oligonucleotides refers to the number of covalent bonds between an oligonucleotide and a lipid. The oligonucleotide itself can be single-stranded or double stranded. In some embodiments, it is contemplated that a particle comprises single stranded oligonucleotides, double stranded oligonucleotides, or a combination thereof.

[0062] The disclosed lipid functionalization strategy differs from previous methods reported for preparing DNA liposome conjugates [Dave, et al. ACS Nano 201 1 , 5 (2), 1304- 12], which used maleimide-functionalized lipids that were subsequently reacted with DNA terminated with a thiol modification. A strain-promoted azide-alkyne cycloaddition is fully compatible with most nucleic acids. In addition, the azide-modified lipids are less prone to hydrolysis in comparison to maleimide-modified analogs [Baldwin, et al. Bioconjugate Chem. 201 1 , 22 (10), 1946-53]. Utilizing SUVs with varying amounts of azide-modified lipid allows one to directly tailor the amount of DNA on the surface of the resulting SNA structure (See Table 1 ). The studies presented herein indicate that the amount of DNA attached to the core increases with increasing DPPE-Az content, up to 10 mol% of the lipid.

[0063] Dynamics of interparticle exchange of DNA The dynamics of interparticle DNA exchange for both cholesterol-tail and lipid-tail LSNAs were assessed in buffer using a FRET reporter within the LSNAs (Figure 1 A). In the design provided herein, LSNAs were synthesized with a rhodamine-labeled lipid and Cy5-labeled DNA (see Table 2). These particles were mixed with 50 nm DOPC liposomes to measure the decay of the particles. An interparticle exchange of DNA results in a decrease in the FRET signal between rhodamine and Cy5, providing for a quantitative measure of DNA exchange (See Table 6 for particle compositions). Accordingly, addition of the FRET reporter LSNA to the DOPC liposomes at room temperature (21 ^eC) results in a decreased FRET signal for the rhodamine-labeled LSNAs (Figure 1 B, right panel). Controls experiments consisting of incubating the LSNAs without other liposomes resulted in no appreciable decay, indicating that DNA is only lost through interactions with other liposomes. The rate of DNA transfer between particles is significantly slower for lipid-tail LSNAs in comparison to the cholesterol-tail LSNAs (Table 2). To minimize the effect of DNA loading on these studies, particles with similar DNA loading (-150 strands/liposome) were utilized in the study. Table 2

Particle type Application Sequence SEQ ID

NO:

T20 " Quantification 5'-T₂₀ -Cholesterol-3' 2

Cholesterol

T20 - DBCO Quantification ^'5'-Τ₂₀ -DBCO^fc-3' 3

T20-BHQ.2- Stability study 5' -T₂₀-BHQ^c-Cholesterol-3' 4

Cholesterol

T20 -BHQ2- Stability study 5' -T₂₀-BHQ-DBCO-3' 5

DBCO

T₂₀-Cy5- Stability study 5' -T₂₀-Cy5-Cholesterol-3' 6

Cholesterol

T₂₀ -Cy5- Stability study 5' -T₂₀-Cy5-DBCO-3' 7

DBCO

IS-1826- Immunostimulation 5'-TCCATGACGTTCCTGACGTT-T₅- 8 cholesterol (Spacerl 8^a)₂-Cholesterol-3'

IS-1826- Immunostimulation 5'-TCCATGACGTTCCTGACGTT-T₅- 9

DBCO (Spacer18^a)₂-DBCO-3'

Scrambled- Immunostimulation 5'-T₂₀ -Cholesterol-3' 2

Cholesterol

Scrambled- Immunostimulation 5'-T₂₀-DBCO-3' 3

DBCO

aSpacer18 = 18-0-Dimethoxytritylhexaethyleneglycol,1 -[(2-cyanoethyl)-(A/,A/-diisopropyl)]- phosphoramidite. ^bDBCO = 5'-Dimethoxytrityl-5-[(6-oxo-6-(dibenzo[b,f]azacyclooct-4-yn-1 - yl)-capramido-A/-hex-6-yl)-3-acrylimido]-2'-deoxyuridine,3'-[(2-cyanoethyl)-(A/,A/-diisopropyl)]- phosphoramidite. ^cBHQ =5'-Dimethoxytrityloxy-5-[(A/-4"-carboxyethyl-4"-(A/-ethyl)-4'-(4-Nitro- phenyldiazo)-2'-methoxy-4'-methoxy-azobenzene)-aminohexyl-3-acrylimido]-2'-deoxyuridine- 3'-[(2-cyanoethyl)-(A/,A/-diisopropyl)]-phosphoramidite.

[0064] Although DNA density is important, LSNA constructs also must remain assembled in physiological environments for it to exhibit its architecture-dependent properties. To determine the stability of the LSNAs, the kinetics of interparticle DNA exchange for both cholesterol-tail and lipid-tail LSNAs were measured in liposome-containing buffer and serum protein environments. As a reporter of the assembly state of the structures, Forster resonance energy transfer (FRET) LSNAs were synthesized using a rhodamine-labeled lipid and Cy5-labeled DNA (detailed sequences are listed in Table 3, so that FRET can occur between the fluorophore-labeled lipids and DNA when the LSNA is fully assembled (Figure 9). To assess the rate at which the DNA shell dissociated from its original liposomal template and inserted into a different lipid bilayer, FRET reporter particles, synthesized with -150 strands/particle, were mixed with an excess (-100 fold by liposome) of 50 nm DOPC liposomes (without DNA). The addition of the cholesterol-tail FRET reporter LSNA to the DOPC liposomes at room temperature (21 ^eC) and physiologic temperature (37 ^eC) resulted in an exponential decay of FRET signal (k_obs = 2.1 ± 0.1 ^χ 10^"3 s^"1 and 1 .0 ± 0.08 ^χ 10^"2 s^"1 respectively Figure 2B) indicating rapid dissociation of the LSNA. In contrast, lipid-tail LSNAs showed minimal decay at room temperature and dissociation rates of 7.1 ± 0.2 χ 10^"" s^"1 at 37^e C. Consistent with the hypothesis and previous findings, the rate of exchange between particles is significantly slower for lipid-tail LSNAs in comparison to the cholesterol- tail LSNAs. As a control, the LSNAs were incubated in buffer over the same time and displayed no decay (Figure 2C), indicating that disassembly of the LSNAs only occurs in the presence of other liposomes.

Table 3. The oligonucleotides and oligonucleotide-modified materials used in this study.

Particle type Application Sequence SEQ ID NO:

T₂₅ - Quantification 5'-T₂₀ -Cholesterol-3' 2

Cholesterol

T₂₅ - DBCO Quantification 5'-T₂₀ -DBCO^¾-3' 3

T₂₀-Cy5- Stability study 5' -T₂₀-Cy5^c-Cholesterol-3' 6

Cholesterol

T₂₀ -Cy5- Stability study 5' -T₂o-Cy5^c-DBCO-3' 7

DBCO

IS-1826- Immunostimulation 5'-TCCATGACGTTCCTGACGTT-T₅- 8

cholesterol (SpaceM 8^a)₂-Cholesterol-3'

IS-1826- Immunostimulation 5'-TCCATGACGTTCCTGACGTT-T₅- 9

DBCO (Spacer18^a)₂-DBCO-3'

Scrambled- Immunostimulation 5'-T₂₀-( Spacerl 8)₂-Cholesterol-3' 10

Cholesterol

Scrambled- Immunostimulation 5'-T₂₀-(Spacer18)₂-DBCO-3' 1 1

DBCO

IS- 1826 Immunostimulation 5'-TCCATGACGTTCCTGACGTT-3' 12 ^aSpacer18 = 1 8-0-Dimethoxytritylhexaethyleneglycol,1 -[(2-cyanoethyl)-(/V,/V-diisopropyl)]- phosphoramidite. ^DBCO = 5'-Dimethoxytrityl-5-[(6-oxo-6-(dibenzo[b,f]azacyclooct-4-yn-1 -yl)- capramido-/V-hex-6-yl)-3-acrylimido]-2^,-deoxyuridine,3^,-[(2-cyanoethyl)-(/V,/\/-diisopropyl)]- phosphoramidite. ^cCy5 =1 -[3-(4-monomethoxytrityloxy)propyl]-1 '-[3-[(2-cyanoethyl)-(N,N- diisopropylphosphoramidityl)propyl]-3,3,3',3'-tetramethylindodicarbocyanine chloride.

[0065] Previous studies have reported that the rate of spontaneous dissociation of fluorophore- modified DOPE from liposomal bilayers is significantly slower (1 .16 χ 10^"5 s^"1 at 37^e C) than the dissociation rates reported here for the DNA modified with cholesterol or lipid [Silvius et al., Biochemistry 1993, 32, 13318]. Since the rate of dissociation for lipids is significantly slower than those observed in the disclosed particles, it was hypothesized that the DNA shell was dissociating much quicker than the lipid components. To confirm this hypothesis, control particles featuring rhodamine-labeled lipids and unlabeled DNA were mixed with particles containing carboxyfluorescein-labeled lipids and unlabeled DNA (see Table 6 for composition). The fluorescence spectrum was measured after 0, 0.25, and 24 hour incubation at 25 ^°C, revealing only minimal exchange of the dye-labeled lipids (Figure 10). [0066] Dynamics in a protein-rich environment The protein-rich environment in serum can destabilize LSNAs by interacting with the lipid components or the DNA, either on the particle or when dissociated [Wolfrum et al., Nat. Biotechnol. 2007, 25, 1 149; Zelphati et al., Biochim. Biophys. Acta, Lipids Lipid Metab. 1998, 1390, 1 19]. The stability of the LSNAs was measured in a 10 vol % serum protein environment using the FRET reporter LSNAs. Disruption of the LSNAs due to interactions with serum proteins would result in decreased FRET (Figure 3A). Indeed, the FRET signals decreased and the rhodamine-labeled lipids were turned on for both LSNA structures over time (Figures 3B & 3C), although at different rates. The lipid-tail LSNAs show a > 20-fold extended half-life in comparison to the cholesterol-tail analogs with observed dissociation rates of 2.8 ± 0.4 ^χ 10^"4 s^"1 and 7.9 ± 1 .1 x 10^"3 s^"1 , respectively. The increased stability of lipid-tail LSNAs should allow such structures to remain intact and enter cells via known endosomal pathways [Choi et al., Proc. Natl. Acad. Sci. U. S. A. 2013, 1 10, 7625], which, in the context of immunotherapy, should equate to a larger therapeutic payload. In addition, this result suggests that serum proteins actively disrupt LSNAs as evidenced by the four-fold increase in exchange rate for the lipid- tail LSNAs in the presence of serum proteins when compared to the system containing only LSNAs mixed with unmodified liposomes, but in the absence of proteins where only passive dissociative exchange is possible.

[0067] Note that although for both classes of LSNAs there is a significant decrease in the FRET signal over the course of the experiments, a baseline level of FRET remains, which indicates that some DNA remains attached to the lipid bilayer. To confirm that the decrease in FRET is due to the loss of the DNA shell and not the fluorophore-labeled lipids in the core, a LSNA composed of carboxyfluorescein- and rhodamine-labeled lipids (1 mol % each) with unlabeled cholesterol-tail DNA was incubated in a 10 vol % serum solution. The dissociation rate of the lipids from the core was significantly slower (k_obs = 5.7 ± 0.2 ^χ 10^"5 s^"1) than that measured between the DNA shell and the template (Figure 1 1 ). This supports the conclusion that the dissociation rate of the DNA shell is faster than the disassembly of the lipid core in protein-rich environments.

[0068] Since the density of the DNA shell can potentially alter interactions between LSNAs and serum proteins due to electrostatic considerations and oligonucleotide sequence- and density-specific interactions [Zwanikken et al., Phys. Chem. C 201 1 , 1 15, 16368], the stabilities of the lipid-tail LSNAs were evaluated as a function of shell density. The particles with higher DNA shell densities reached equilibrium, as determined by the FRET ratio, at later time points compared to those with lower shell densities (Figure 12), showing that increased DNA loading leads to longer structural retention.

[0069] Since the concentration of the particles and serum proteins could potentially impact the dissociation rates of the system, the dissociation rate as a function of different serum and particle concentrations was measured. To examine the serum protein concentration dependent dissociation, the LSNAs were incubated with increasing serum protein concentrations, consisting of 10, 20, and 30 vol % FBS. There was no observable increase in dissociation rate at higher serum concentrations (Figure 13A). In addition, the effect of particle concentration was evaluated by incubating two different particle concentrations, 100 and 500 nM by DNA, with 10 vol % FBS (Figure 13B). Again, no significant concentration dependence was observed. Taken together, these results suggest that LSNA dissociation is independent of serum protein and particle concentration for ranges typically used in cell experiments and is primarily dependent on the hydrophobic anchor attached to the DNA.

[0070] Cellular Uptake of LSNAs. A characteristic property of SNAs is that the nucleic acid shell facilitates their rapid cellular internalization by engaging scavenger class A receptors, among others, on the cell membrane [Choi et al., Proc. Natl. Acad. Sci. U. S. A. 2013, 1 10, 7625]. As such, increased surface loading of DNA on the LSNAs should lead to higher rates of cellular uptake. Structures that have slower dissociation rates should result in higher DNA densities facilitating cellular uptake. To study the effect of LSNA stability and DNA shell density on cellular uptake, two different cell lines were tested: U87-MG

glioblastoma cells and RAW-Blue macrophages. The cells were incubated with both types of LSNAs, cholesterol-modified DNA, and DBCO-modified DNA, which were all synthesized with Cy5-labeled phosphorothioate (PS) DNA, and then evaluated using flow-cytometry.

[0071] Notably the U87-MG cells showed increased uptake of the lipid-tail LSNAs after 1 hour compared to the cholesterol-tail LSNAs (Figure 15A). After 2 hours incubation, the lipid-tail LSNAs no longer displayed an advantage (Figure 15A). Confocal imaging of the cells corroborated this result with greater Cy5 fluorescence intensity after 1 hour of incubation for the lipid-tail LSNA (Figure 4). Significantly, the uptake of both types of LSNAs as well as cholesterol-tail DNA is greater than that observed for DBCO-modified PS DNA, which is not capable of assembling into a spherical architecture like the other DNA structures used.

[0072] The macrophages also displayed enhanced uptake of the lipid-tail LSNAs in comparison to cholesterol-tail LSNAs (Figure 15B). These cells showed rapid uptake of the lipid-tail LSNAs and greater total uptake even after 4 hours of incubation (Figure 15B).

Surprisingly, the cholesterol-tail LSNAs and cholesterol-modified DNA did not have any enhancement in uptake over the DBCO-modified DNA. This stands in contrast to what was observed for the glioblastoma derived U87-MG cells. The differences between the cell lines is likely due to differences in the levels of expression of cell membrane receptors.

[0073] It was hypothesized that the increased uptake of the lipid-tail LSNAs stems from its more stable and higher density DNA shell facilitating interactions with scavenger receptors on the cell surface, thus, enhancing cellular internalization [Giljohann et al., Nano Lett. 2007, 7, 3818; Choi et al., Proc. Natl. Acad. Sci. U. S. A. 2013, 1 10, 7625]. To test this hypothesis, U87-MG cells were treated with Fucoidan, an inhibitor of scavenger receptors, prior to incubation with LSNAs. Following this treatment, the overall uptake of the LSNAs

decreased, and no significant difference between the uptake of cholesterol-tail and lipid-tail particles was observed (Figure 17). This result is consistent with the conclusion that the enhanced uptake of the lipid-tail structures at early time points stems largely from scavenger receptor mediated endocytosis.

[0074] Since both cholesterol-tail and lipid-tail DNAs are capable of cellular internalization, due to their ability to independently form self-assembled micellar structures that mimic the SNA architecture [Li et al., Nano Lett. 2004, 4, 1055; Banga et al., J. Am. Chem. Soc. 2017, 10.1021 /jacs.6b13359] or bind non-specifically to cell membranes [Radovic-Moreno et al., Proc. Natl. Acad. Sci. U. S. A. 2015, 1 12, 3892; Luo et al., Nat. Biotechnol. 2000, 18, 33], confocal microscopy and flow cytometry data alone are not enough to determine if the constructs are being internalized as fully or partially intact structures. To answer this question, the FRET efficiency of the particles inside cells was measured by imaging the rhodamine fluorescence before/after photobleaching of the Cy5 dye (Figure 5). Intact particles have increased rhodamine fluorescence after photobleaching of the Cy5-labeled DNA (Figure 4A). In contrast, disassembled particles, where the Cy5-labeled DNA has detached from the initial liposomal structure, have minimal increases in rhodamine fluorescence. After 1 hour of incubation, cells treated with the lipid-tail LSNAs exhibit increased FRET compared to those treated with cholesterol-tail LSNAs (Figure 5C), consistent with the former being more stable. Significantly, lipid-tail LSNAs loaded with higher DNA densities (300 strands/LSNA) continued to retain more DNA strand per liposome after 1 and 2 hours of incubation compared to those assembled with lower DNA densities (~150 strands/LSNA). The amount of observed FRET continued to decrease for the lipid-tail LSNAs at the 2 hour time point. After 24 hours of incubation, no significant differences in FRET were detected and only minimal FRET was observed for any of the structures, suggesting that the LSNAs gradually disassemble inside cells as a function of time.

[0075] Activation of toll-like receptors for immunotherapeutics The ability of both types of LSNAs to activate therapeutic targets inside cells was assessed. LSNAs were synthesized with immunostimulatory unmethylated CpG-rich DNA (1826 ODN) [Ramirez- Ortiz, et al. Infect. Immun. 2008, 76 (5), 2123-9]. These DNA sequences are known to activate toll-like receptor 9 (TLR9), which modulates innate immunity by stimulating cytokine production [Cooper, et al. J. Clin. Immunol. 2004, 24 (6), 693-701 ]. Exposing HEK-BLUE- mTLR9 cells, which are modified to secrete alkaline phosphatase upon activation of TLR9 for colorimetric readout of immune-stimulation, allows one to compare the immune stimulatory activity of both types of LSNAs. As shown in Figure 6A, the lipid-tail LSNAs shows increased activity at lower concentrations compared to cholesterol-tail analogs.

Pulse-chase experiments conducted at 250 nM concentration exhibited similar trends (Figure 6B) where earlier time points show much higher activity for the lipid-tail formulations.

[0076] To assess the ability of both types of LSNAs to activate therapeutic targets inside cells, LSNAs were synthesized with immunostimulatory unmethylated CpG-rich DNA (1826 ODN) [Ramirez-Ortiz et al., Infect. Immun. 2008, 76, 2123]. These DNA sequences are known to activate toll-like receptor 9 (TLR9), which modulates innate immunity by stimulating cytokine production [Cooper et al., J. Clin. Immunol. 2004, 24, 693]. Incubating these cells with RAW-Blue macrophages, which are modified to secrete alkaline phosphatase upon stimulation of TLRs for a colorimetric readout of immune-stimulation, allowed for comparison of the immune stimulatory activity of both types of LSNAs. As shown in Figure 14A, the lipid- tail LSNAs show modestly increased activity at lower concentrations compared to

cholesterol-tail analogs, but pulse-chase experiments conducted at 250 nM concentration reveal significantly faster activation of the macrophages (Figure 14B) by the lipid-tail LSNA formulations, presumably a consequence of more rapid uptake by cells (Figure 15B).

[0077] It was also demonstrated that the 1826 PS SNAs outperform 1826 PS Linear oligonucleotides (Figure 19), and that the SNA architecture improves immune activation of 1826 PS oligonucleotide compared to the lipid-oligonucleotide conjugate (Figure 20).

[0078] Accordingly, the disclosure provides methods of utilizing liposomal particles for modulating toll-like receptors. The method either up-regulates or down-regulates the Tolllike-receptor through the use of a TLR agonist or a TLR antagonist, respectively. The method comprises contacting a cell having a toll-like receptor with a liposomal particle. The toll-like receptors modulated 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 1 1 , toll-like receptor 12, and toll-like receptor 13. In any of the methods of the disclosure, a liposomal particle of the disclosure is administered to a mammal. In some embodiments, the mammal is a human.

[0079] This work presents a new strategy for synthesizing LSNAs through direct lipid functionalization. In general, LSNAs exhibit enhanced biocompatibility compared to inorganic core SNAs [Radovic-Moreno, et al. Proc. Natl. Acad. Sci. U. S. A. 2015, 1 12 (13), 3892- 3897; Banga, et al. J. Am. Chem. Soc. 2014, 136 (28), 9866-9]. However, the use of cholesterol and/or tocopherol anchoring groups in the conventional synthesis of LSNAs limits their stability and oligonucleotide loading density, which are critical for maximizing therapeutic potential. This new form of LSNA retains the biocompatibility of previous synthesis methods, while significantly enhancing the loading of DNA and stability of the particle.

[0080] The strain-promoted azide-alkyne coupling synthesis strategy of LSNAs utilized (e.g., use of a DBCO alkyne) is compatible with many types of nucleic acids and common phosphothioate backbone modifications that are incompatible with maleimide groups, a conventional method for lipid-DNA attachment. PS modifications are commonly used to reduce susceptibility to endonucleases, making this a generalizable approach for developing multiple therapeutic applications, particularly for immunomodulatory and gene regulatory therapies.

[0081] The stability and biological behavior of LSNAs synthesized with either lipid- or cholesterol-modified oligonucleotides is disclosed herein. This disclosure demonstrates that a synthetic route of directly modifying lipid-head groups on liposomes with DNA leads to higher nucleic acid shell densities and increased stability in physiological environments. These combined properties allow for enhanced interactions with cells and significant advantages in the context of sequence-specific immune modulation. Taken together, this disclosure provides structure-function principles for LSNAs that directly impact the design of nanomaterials for effective therapeutic platforms.

[0082] Uses of Particles in Gene Regulation/Therapy. Methods for inhibiting gene product expression provided herein include those wherein expression of the target gene product 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 LSNA. In other words, methods provided embrace those which results in essentially any degree of inhibition of expression of a target gene product.

[0083] The degree of inhibition is determined in vivo from a body fluid sample or from a biopsy sample or by imaging techniques well known in the art. Alternatively, the degree of inhibition is determined in a cell culture assay, generally as a predictable measure of a degree of inhibition that can be expected in vivo resulting from use of a specific type of liposomal SNA and a specific oligonucleotide.

[0084] In some aspects of the disclosure, it is contemplated that a liposomal particle performs both a gene inhibitory function as well as a therapeutic agent delivery function. In such aspects, a therapeutic agent is encapsulated in a liposomal particle of the disclosure and the particle is additionally functionalized with one or more oligonucleotides designed to effect inhibition of target gene expression. In further embodiments, a therapeutic agent is attached to a liposomal particle of the disclosure and the particle is additionally

functionalized with one or more oligonucleotides designed to effect inhibition of target gene expression..

[0085] In various aspects, the methods include use of an oligonucleotide which is 100% complementary to the target polynucleotide, i.e., a perfect match, while in other aspects, the oligonucleotide is at least (meaning greater than or equal to) about 95% complementary to the polynucleotide over the length of the oligonucleotide, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20% complementary to the polynucleotide over the length of the oligonucleotide to the extent that the oligonucleotide is able to achieve the desired degree of inhibition of a target gene product.

[0086] In some embodiments, the sequence of an antisense compound is 100% complementary to that of its target nucleic acid. It is understood in the art, however, that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event {e.g., a loop structure or hairpin structure). The percent complementarity is determined over the length of the oligonucleotide. For example, given an antisense compound in which 18 of 20 nucleotides of the antisense compound are complementary to a 20 nucleotide region in a target polynucleotide of 100 nucleotides total length, the oligonucleotide would be 90 percent complementary. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleotides. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0087] Accordingly, methods of utilizing a particle of the disclosure in gene regulation therapy are provided. This method comprises the step of hybridizing a polynucleotide encoding said gene product with one or more oligonucleotides complementary to all or a portion of said polynucleotide, said oligonucleotide being attached to a liposomal particle, wherein hybridizing between said polynucleotide and said oligonucleotide occurs over a length of said polynucleotide with a degree of complementarity sufficient to inhibit expression of said gene product. The inhibition of gene expression may occur in vivo or in vitro. [0088] The oligonucleotide utilized in this method is either RNA or DNA. The RNA can be an inhibitory RNA (RNAi) that performs a regulatory function, and in various embodiments is selected from the group consisting of a small inhibitory RNA (siRNA), an RNA that forms a triplex with double stranded DNA, and a ribozyme. Alternatively, the RNA is microRNA that performs a regulatory function. The DNA is, in some embodiments, an antisense-DNA.

EXAMPLES

[0089] Materials. Unless otherwise noted, all reagents were purchased from commercial sources and used as received. For oligonucleotide synthesis, all phosphoramidites and reagents were purchased from Glen Research, Co. (Sterling, VA, USA). All lipids were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA) either in dry powder form or chloroform and used without further purification. All other reagents were purchased from Sigma-Aldrich, Co. (St. Louis, MO, USA). Ultrapure deionized (Dl) H₂0 (18.2 MQ-cm resistivity) was obtained from a Milli-Q Biocel system (Millipore Co., Billerica, MA, USA).

[0090] Instrumentation. UV-vis absorbance spectra and thermal denaturation curves were collected on a Varian Cary 5000 UV-vis spectrometer (Varian Inc., Palo Alto, CA, USA) using quartz cuvettes with a 1 cm path length.

[0091] Matrix-assisted laser desorption/ionization time-of-f light (MALDI-ToF) mass spectrometry data were obtained on a Bruker AutoFlex III MALDI-ToF mass spectrometer (Bruker Daltonics Inc., Billerica, MA, USA). For MALDI-ToF analysis, the matrix was prepared by mixing an aqueous solution of ammonium hydrogen citrate (0.6 μί of a 35 wt % solution (15 mg in 30 μί of H₂0)) and 3-hydroxypicolinic acid (Sigma-Aldrich, 2 mg in H₂0:MeCN (30 μί of a 1 :1 v/v mixture). An aliquot of the DNA (-0.5 μί of a 150 μΜ solution) was then mixed with the matrix (1 :1 ) and the resulting solution was added to a steel MALDI-ToF plate and dried at 25 ^eC for 1 hour before analysis. Samples were detected as negative ions using the linear mode. The laser was typically operated at 10-20% power with a sampling speed of 10 Hz. Each measurement averaged for five hundred scans with the following parameters: ion source voltage 1 = 20 kV, ion source voltage 2 = 18.5 kV, lens voltage = 8.5 kV, linear detector voltage = 0.6 kV, deflection mass = 3000 Da.

[0092] Centrifugation was carried out in a temperature-controlled Eppendorf centrifuge 5430R (Eppendorf AG, Hauppauge, NY, USA). Dynamic light scattering (DLS) and zeta potential measurements were collected on a Zetasizer Nano ZS (Malvern Instruments, UK) equipped with a He-Ne laser (633 nm)

[0093] Oligonucleotide synthesis. Oligonucleotides were synthesized on CPG supports using an automated Expedite Nucleotide system (model: MM48 or MM12, BioAutomation Inc., Piano, TX, USA). Whenever a modified (i.e., non-nucleoside-bearing)

phosphoramidites is used, the coupling time is extended to 10 min compared to the usual 90 seconds. After synthesis, the completed DNA was cleaved off the CPG support through an overnight exposure to aqueous ammonium hydroxide (28-30 wt %). Excess ammonium hydroxide was removed from the cleaved DNA solution by passing a stream of dry nitrogen gas over the contents of the vial until the characteristic ammonia smell disappeared. The remaining solution was then passed through a 0.2 μηι cellulose acetate membrane filter to remove the solid support and then purified on a Varian ProStar 210 HPLC system (Agilent Technologies Inc., Palo Alto, CA, USA) equipped with reverse-phase semi-preparative Varian column ((Agilent Technologies, 250 mm ^χ 10 mm, Microsorb 300 A/10 μηιΛ34), gradient = 95:5 v/v 0.1 M TEAA (aq):MeCN (TEAA (aq) = triethylammonium acetate, aqueous solution), and increasing to pure acetonitrile in 45 min, flow rate = 3 mL/min for each 1 μηιοΙ DNA). The product fractions collected were concentrated using lyophilization. The lyophilized oligonucleotides were then re-suspended in ultrapure deionized water and their concentrations were measured using UV-vis spectroscopy. The purity of the synthesized oligonucleotides was assessed using MALDI-ToF mass spectrometry.

Table 4

Particle type Application Sequence SEQ ID NO:

T20 " Quantification 5'-T₂₀ -Cholesterol-3' 2

Cholesterol

T20 - DBCO Quantification 5'-T₂₀ -DBCO^fc-3' 3

T₂₀-BHQ2- Stability study 5' -T₂₀-BHQ^c-Cholesterol-3' 4

Cholesterol

T₂₀ -BHQ2- Stability study 5' -T₂₀-BHQ-DBCO-3' 5

DBCO

T₂₀-Cy5- Stability study 5' -T₂₀-Cy5-Cholesterol-3' 6

Cholesterol

T₂₀ -Cy5- Stability study 5' -T₂₀-Cy5-DBCO-3' 7

DBCO

IS-1826- Immunostimulation 5'-TCCATGACGTTCCTGACGTT- 8

cholesterol T₅-(Spacer18^a)₂-Cholesterol-3'

IS-1826- Immunostimulation 5'-TCCATGACGTTCCTGACGTT- 9

DBCO T₅-(Spacer18^a)₂-DBCO-3'

Scrambled- Immunostimulation 5'-T₂₀ -Cholesterol-3' 2

Cholesterol

Scrambled- Immunostimulation 5'-T₂₀-DBCO-3' 3

DBCO

aSpacer18 = 18-0-Dimethoxytritylhexaethyleneglycol,1 -[(2-cyanoethyl)-(A/,A/-diisopropyl)]- phosphoramidite. ^bDBCO = 5'-Dimethoxytrityl-5-[(6-oxo-6-(dibenzo[b,f]azacyclooct-4-yn-1 - yl)-capramido-A/-hex-6-yl)-3-acrylimido]-2'-deoxyuridine,3'-[(2-cyanoethyl)-(A/,A/-diisopropyl)]- phosphoramidite. ^cBHQ =5'-Dimethoxytrityloxy-5-[(A/-4"-carboxyethyl-4"-(A/-ethyl)-4'-(4-Nitro- phenyldiazo)-2'-methoxy-4'-methoxy-azobenzene)-aminohexyl-3-acrylimido]-2'-deoxyuridine- 3'-[(2-cyanoethyl)-(A/,A/-diisopropyl)]-phosphoramidite. [0094] Synthesis of small unilamellar vesicles (SUVs). All of the SUVs (shown in Tables 5 and 6) were synthesized using a previously published protocol (see Banga et al., J. Am. Chem. Soc. 2014, 136 (28), 9866-9). An aliquot from the lipid stock solution (1 ml_, 25 mg/mL concentration) was added into a 25 mL glass vial, and the solvent was carefully evaporated under a stream of nitrogen. The lipid monomer was lyophilized overnight to remove any residual chloroform. The resulting dried lipid film was hydrated with 1 ^χ HEPES- buffered saline (1 ^χ HBS; 20 mM HEPES, 150 mM NaCI, pH 7.5; 3.0 mL) to form a lipid- containing suspension, which was subjected to 5 freeze-thaw cycles. The SUV-containing supernatant were then subjected to membrane-extrusion process using two 50 nm pore-size membrane (Whatman, GE Healthcare Bio-Sciences, Pittsburgh, PA) for 10 cycles. The lipid concentration of the synthesized SUV suspension was determined by analyzing its phosphorus content by inductively coupled plasma mass spectrometry (ICP-MS, Thermo Fisher X Series II instrument, Thermo Fisher Scientific Inc., Waltham, MA, USA).

[0095] Synthesis and purification of different liposomal SNAs; cholesterol-tail liposomal SNAs. As described in a previously established protocol, a 3'-cholesterol-tail oligonucleotide (20 nmol) was added to the SUV colloids (1 .3 mM phospholipid

concentration, final volume 1 mL) and was allowed to shake overnight. The solution was purified via size-exclusion chromatography on a Sepharose CL-4B column (Sigma-Aldrich) and the particle size distribution was analyzed using DLS.

[0096] For cholesterol-tail LSNAs, a 3'-cholesterol-tail oligonucleotide was added to the SUV colloids (1 .3 mM phospholipid concentration, final volume 1 mL) and was shaken overnight. For lipid-tail LSNAs, an aliquot of the desired DBCO-tail oligonucleotides was added to a N₃-DPPE containing SUV (0.5 mM total phospholipid concentration, final volume 1 mL) with a DNA-DBCO:surface N₃-DPPE lipid molar ratio of 2:1 . The mixture was shaken overnight. Both structures were purified via size-exclusion chromatography on a Sepharose CL-4B column (Sigma-Aldrich) and the particle size distribution was analyzed using DLS. For details on the DLS measurements, see the discussion of the characterization of liposomal SNAs, below.

[0097] The density of the nucleic acid shell was determined by first dissociating the particles in sodium dodecyl sulfate (SDS) and then measuring the absorbance at 260 nm via UV-Vis spectroscopy to calculate the DNA concentration. The amount of lipid was determined by measuring the total phosphorus content via inductively coupled plasma mass spectroscopy (ICP-MS, Thermo Fisher X Series II instrument, Thermo Fisher Scientific Inc., Waltham, MA, USA) and subtracting the phosphorus content contributed by the DNA backbone. An alternative method to determine the density was also used. The absorbance spectrum of LSNAs synthesized with Cy5 labelled DNA and 1 mol% rhodamine labeled lipid were measured. The peak absorbance of the Cy5 and rhodamine (corrected to remove any Cy5 absorbance contribution) were used to measure the relative amount of DNA to lipid.

[0098] Lipid-tail liposomal SNAs. An aliquot of desired DBCO-tail oligonucleotides (100 nmol) was added to a N₃-DPPE containing SUVs (0.5 mM phospholipid concentration, 0.05 mM of N₃ lipid concentration, final volume 1 mL) to achieve a molar ratio of 2:1 for DNA- DBCO:N₃-DPPE lipid in the solution. The mixture was allowed to shake overnight and purification was carried by size-exclusion chromatography on a Sepharose CL-4B column.

Table 5

Particle type Liposome type Liposome composition DNA type (5'-3')

FRET Cholesterol-particle Fluorescein Fluorescein PE ^a 1 % + T₂₀-Cholesterol pair 1 A SUV DOPC 99% (SEQ ID NO: 2)

FRET Cholesterol-particle Rhodamine Rhodamine PE ^S 1 % + T₂₀-Cy5- pair 2 B SUV DOPC 99% Cholesterol

(SEQ ID NO: 6)

FRET DBCO-particle A Fluorescein Fluorescein PE 1 % + N₃- T₂₀-DBCO (SEQ pair 1 SUV DPPE ^c 10% + DOPC ID NO: 3)

89%

FRET DBCO-particle B Rhodamine Rhodamine PE 1 % + N₃- T₂₀- Cy5-DBCO pair 2 SUV DPPE 10% + DOPC 89% (SEQ ID NO: 7)

Cholesterol-control Rhodamine Rhodamine PE ^a 1 % + T₂₀ -Cholesterol particle SUV DOPC 99% (SEQ ID NO: 2)

DBCO-control Rhodamine Rhodamine PE 1 % + N₃- T₂₀-DBCO particle SUV DPPE ^c 10% + DOPC (SEQ ID NO: 3)

89%

Cholesterol-FRET- Fluorescein Fluorescein PE 1 % + T₂₀ -Cholesterol positive-control and Rhodamine PE 1 %+ (SEQ ID NO: 2)

Rhodamine DOPC 89%

SUV

DBCO-FRET- Fluorescein Fluorescein PE 1 % + T₂₀-DBCO (SEQ positive-control and Rhodamine PE 1 % + N₃- ID NO: 3)

Rhodamine DPPE 10% + DOPC 88%

SUV

Cholesterol- SUV DOPC 100% IS-1826- immunostimulatory Cholesterol

(SEQ ID NO: 8)

DBCO- SUV DOPC 90% + N₃-DPPE IS-1826-DBCO immunostimulatory 10% (SEQ ID NO: 9)

Cholesterol- SUV DOPC 100% T₂₀-Cholesterol scrambled (SEQ ID NO: 2)

DBCO-scrambled SUV DOPC 90% + N₃-DPPE T₂₀-DBCO (SEQ

10% ID NO: 3) fluorescein PE: 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-A/-(carboxyfluorescein)

(ammonium salt). ^Rhodamine PE: 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-/V- (lissamine rhodamine B sulfonyl) (ammonium salt). ^CN₃-DPPE: 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-A/-(6-azidohexanoyl) (ammonium salt). Table 6

Particle type Liposome type Liposome composition DNA type (5'-3')

FRET Cholesterol-tail FRET Rhodamine SUV Rhodamine PE^b i % + T₂₀-Cy5-

Reporte reporter DOPC 99% Cholesterol r (SEQ ID NO: 6)

FRET Lipid-tail FRET Rhodamine SUV Rhodamine PE^fi 1 % + N₃- T₂₀- Cy5-DBCO

Reporte reporter DPPE^C 0.5-10% + DOPC (SEQ ID NO: 7) r 89-98.5%

Cholesterol-Control Rhodamine/Fluo Rhodamine PE? ϊ % + T₂₀-Cholesterol

Rhodamine rescein SUV DOPC 99% (SEQ ID NO: 2)

Cholesterol-Control Rhodamine Fluorescein PE^a 1 % + N₃- T₂₀-Cholesterol

Fluorescein SUVFIuorescein DPPE ^c 10% + DOPC 89% (SEQ ID NO: 2)

SUV

Cholesterol-tail Fluorescein and Fluorescein PE^a 1 % + T₂₀-Cholesterol

FRET-positive-control Rhodamine SUV Rhodamine PE^b ^ %+ (SEQ ID NO: 2)

DOPC 89%

Lipid-tail FRET- Fluorescein and Fluorescein PE^a 1 % + T₂₀-DBCO positive-control Rhodamine SUV Rhodamine PE^b 1 % + N₃- (SEQ ID NO: 3)

DPPE^C 10%^c + DOPC 89%

Cholesterol-tail SUV DOPC 1 00% IS-1826-Ts- immunostimulatory Cholesterol (SEQ

ID NO: 13)

Lipid-tail SUV DOPC 95% + N₃-DPPE^C 5% IS-1826-T₅ -DBCO limmunostimulatory (SEQ ID NO: 14)

Cholesterol-tail SUV DOPC 1 00% T₂₀-Cholesterol scrambled (SEQ ID NO: 2)

Lipid-tail scrambled SUV DOPC 95% + N₃-DPPE^C 5% T₂₀-DBCO (SEQ

SNA ID NO: 3) fluorescein PE: 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-A/-(carboxyfluorescein)

(ammonium salt). ^Rhodamine PE: 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-A/- (lissamine rhodamine B sulfonyl) (ammonium salt). ^CN₃-DPPE: 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-A/-(6-azidohexanoyl) (ammonium salt).

[0099] Characterization of liposomal SNAs by dynamic light scattering (DLS) The particle size distribution and surface charge (ζ potential) of micellar SNAs were carried out via dynamic light scattering. To measure the size of nanoparticles, non-invasive backscatter method (detection at 173^Q scattering angle) was used. The collected data were fitted, using the method of cumulants, to the logarithm of the correlation function, yielding the diffusion coefficient D. The calculated diffusion coefficient was applied to the Stokes-Einstein equation (D_H = k_BT/3TTr|D, where k_B is the Boltzmann constant, T is the absolute

temperature, and η is the solvent viscosity (η = 0.8872 cP for water at 25 ^eC)), to obtain the hydrodynamic diameters (D_H) of the nanoparticles (NPs). The reported DLS size for each sample was based on at least six measurements, each of which was subjected to non- negative least squares analysis.

[0100] FRET-based DNA transfer studies in 1 x HBS buffer. In a typical experiment, a solution of liposomal SNA particle A (see Table 2, 0.1 μΜ of final [oligonucleotide], final volume = 1 mL) was aliquoted into a quartz cuvette at room temperature. An equivolume aliquot of liposomal SNA particle B (equal [oligonucleotide] concentration as particle A) was added and quickly pipetted up and down (within 3seconds) for uniform mixing. The fluorescence spectra for Particle A and Particle B were then monitored at room temperature at 1 min intervals for over 120 min. Oligonucleotide transfer was analyzed by monitoring two different FRET pairs. The increase in FRET pair 1 fluorescence (Fluorescein/Cy5, excitation at 480 nm, emission at 530 and 672 nm, slit width 3 nm) and the decrease in FRET pair 2 fluorescence (Rhodamine/Cy5 FRET pair, excitation at 560 nm, emission at 583 and 672nm, 3 nm slit width). The fluorescence studies were performed on a Fluorlog-3 system (HORIBA Jobin Yvon Inc., Edison, NJ, USA). In some experiments, an approximate 100-fold excess of DOPC liposomes was added and quickly pipetted up and down (within 3seconds) to mix uniformly, and the fluorescence of the FRET reporter particles was monitored over 3 hours.

[0101 ] For experiments measuring lipid dissociation, cholesterol-control rhodamine particles (Table 6), 0.1 μΜ by final [oligonucleotide], volume 1 ml_) were aliquoted into an Eppendorf tube at room temperature. An equimolar aliquot of cholesterol-control fluorescein particles (Table 6) was added and quickly pipetted up and down (within 3s) to mix uniformly. The fluorescence spectrum was then taken after incubation at 25 ^eC at 0, 0.25, and 24 h, with a plate reader (Synergy H4, BioTek Instruments, Inc., Winooski, VT, USA, 9.0 nm slit width, Excitation 480 nm). The FRET ratio was calculated using:

FRET Ratio =——

[0102] Where I_A is the acceptor intensity and I_D is the donor intensity measured at their respective peak wavelengths [Jiwpanich et al., J. Am. Chem. Soc. 2010, 132, 10683; Xie et al., Soft Matter 2016, 12, 6196].

[0103] FRET-based serum stability studies. In a typical experiment, particle B (100 nM final [DNA]) was added to a serum-containing solution (10 vol % fetal bovine serum (FBS) in 1 x HBS) and mixed well for 3 seconds. The fluorescence spectra and the intensity of FRET between rhodamine and Cy5 was then monitored as a function of time

(rhodamine/Cy5 FRET pair, excitation at 550 nm, emission at 583 and 672 nm, 3 nm slit width). Control experiments consisting of LSNAs assembled with 1 mol% fluorescein and rhodamine labeled lipids with unlabeled cholesterol-tail DNA (100 nM final [DNA]) were utilized to measure the exchange rate of the lipids in 10 vol% FBS. The fluorescence was monitored on a plate reader over 10 hours at 30 minute intervals (excitation at 480 nM, emission at 583 nm and 550 nm, 9 nm slit width). A final reading was performed after 36 h to observe the equilibrium fluorescence.

[0104] Experiments were also carried out in serum-containing media using a 96 well plate over a range of particle B concentration (final concentration of oligonucleotide in 100 μΙ_ = 500 nM, 50 nM, and 5 nM [DNA])). The solutions were mixed slowly for 10 s and monitored with a plate reader (Synergy H4, BioTek Instruments, Inc., Winooski, VT, USA, 9.0 nm slit width) at 37 °C using the same wavelengths as described above.

[0105] To assess the effects of FBS and LSNA concentrations on the dissociation rates, an aliquot of particle B (final [DNA]100 nM) was added separately to each well of a 96-well plate filled with serum-containing solution with different serum concentrations (10%, 20%, or 30% vol%, final volume = 100 μΙ_). After the addition of the particle B, the wells were mixed (using the slow mixing setting) for 2 seconds before each reading with a plate reader (Synergy H4, 9.0 nm slit width). The measurements were performed at 37° C using the same wavelengths as described above. The fluorescence intensities were collected for 8 hours at 10 minute sampling intervals. The results are shown in Figure 13.

[0106] A separate experiment was carried out to evaluate the effect of DNA density of a particle on dissociation rates in serum, FRET particle B liposomal SNAs were synthesized using separate rhodamine SUVs each doped with different concentration of N₃-DPPE lipid (0.5%, 1 %, 2.5%, 5%, and 10 mol%, of N₃-DPPE). These SUVs were functionalized with 5'- T₂₀- Cy5-DBCO-3' (SEQ ID NO: 7) oligonucleotide strand to form liposomal SNAs with varying DNA densities (See examples section for synthesis of liposomal SNAs). Liposomal SNA particle B synthesized with different concentrations of N₃-DPPE were added into individual wells of a 96 well plate filled with a serum-containing solution in triplicate (10 vol% fetal bovine serum (FBS) in 1 ^χ HBS) at 37° C. (final [DNA] = 100 nM in 100 μΙ_). The solutions were mixed slowly for 2 seconds before each read with a plate reader (Synergy H4, BioTek Instruments, Inc., Winooski, VT, USA, 9.0 nm slit width) at 37°C using the same wavelengths as previously described. The results are shown in Figures 7 and 9.

[0107] Cell culture studies. HEK-Blue™-mTLR9 cells (InvivoGen, CA, USA), derivatives of HEK-293 cells stably expressing a secreted alkaline phosphatase (SEAP) inducible by NF-κΒ, were cultured as recommended by the supplier supplemented with fetal bovine serum (10 vol%), penicillin (0.2 units/mL), and streptomycin (0.1 g/mL), Normocin™ (100 Mg/m), L-glutamine (2 mM concentration); 200 μί of media/well. Confocal imaging was performed on U-87 MG cells (epithelial, glioblastoma) for uptake studies (DMEM, FBS (10% vol.), penicillin (0.2 units/mL), and streptomycin (0.1 μg/mL)). Confocal imaging and flow cytometry were performed on U-87 MG cells (epithelial, glioblastoma) using the

recommended culture conditions in complete growth media (Minimum Essential Medium (MEM) supplemented with FBS (10 vol%), penicillin (0.2 units/mL), and streptomycin (0.1 Mg/mL)).

[0108] RAW-Blue cells (InvivoGen, CA, USA), which are derivatives of RAW 264.7 macrophage cells stably expressing a secreted alkaline phosphatase (SEAP) under a NF-KB promoter, were cultured as recommended by the supplier in complete growth media (Dulbecco's Modified Eagle Medium supplemented with 10 vol% heat inactivated FBS, penicillin (0.2 units/mL), streptomycin (0.1 g/mL), Normocin (100 Mg/m), and L-glutamine (2 mM)).

[0109] Confocal Microscopy. U-87 MG cells were seeded in an 8 well chamber slide (German 1 .5, LabTek II, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 10,000 cells/well and incubated overnight. Following overnight incubation, Cy5-labeled Liposomal SNAs (0.1 μΜ DNA) were incubated with the cells in complete growth media for 1 , 2, 4, and 24 hours. Cells were rinsed with PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. The paraformaldehyde was removed and the cells were then rehydrated in PBS for imaging. The cell nuclei were stained with Hoechst 3342 (Invitrogen, Thermo Fisher Scientific Inc., Carlsbad, CA, USA) following the manufacturer's protocol. Confocal microscopy imaging of these cells were carried out on a Zeiss LSM 800 inverted laser- scanning confocal microscope (Carl Zeiss, Inc., Thornwood, NY, USA) at 40χ and 63χ magnification. Acceptor photobleaching experiments were performed by zooming into and exciting the Cy5 dye in a small region of interest (ROI) at 640 nm with 100% power for 40 cycles. Rhodamine and Cy5 fluorescence intensities were measured before and after photobleaching of the Cy5 dye. The FRET efficiencies were determined by comparing the intensity of the rhodamine fluorescence before and after Cy5 photobleaching using ImageJ software (Available free of charge through https://imagej.nih.gov/ij/). Approximately 10 cells per ROI were imaged for each condition for calculating FRET efficiencies. FRET efficiency was determined using the following equation after subtraction of the background

fluorescence:

^Bleached ^— ^0

Efficiency =—

r Bleached

[0110] ln-vitro cell stimulation studies HEK-Blue™-mTLR9 cells were plated in 96-well plates at a density of 50,000-60,000 cells per well for HEK-Blue cells in DMEM media (see examples for details on media, 200 μΙ_ of media well). Immediately after plating, the cells were treated with cholesterol-tail or lipid-tail liposomal SNAs and incubated at 37 °C for 16 hours. The Quanti-Blue assay was developed using the manufacturers recommended protocol, which is described below.

[0111 ] For analysis, a 180 μΙ_ aliquot of QUANTI-Blue™ solution (InvivoGen, prepared according to the manufacturer's protocol) was added to each well in a separate 96 well plate the following day. A 20 μΙ_ aliquot of the supernatant of treated HEK-Blue cells (20 μΙ_ supernatant of untreated HEK-cells was used as a negative control) was then added to each respective well. After 4 hours incubation, the change in color due to SEAP activity was quantified by reading the OD at 655 nm using a BioTek Synergy H4 Hybrid Reader. [0112] For the pulse-chase experiments, HEK-Blue™-mTLR9 were plated as described above. Immediately after plating, the cells were treated with the cholesterol-tail or lipid-tail LSNAs (See Table 3) at 250 nM final DNA concentration per well. The cells were incubated with the different LSNAs for 15, 30, 45, 60, 90, and 180 min. After each time point, the media was removed and replaced with fresh media and further incubated at 37 °C for 16 hours. The Quanti-Blue analysis then proceeded as described below.

[0113] Flow cytometry experiments. A comparative cell-uptake study between the two types of liposomal SNAs (cholesterol-tail and lipid-tail constructs) was carried out using HEK-Blue™-mTLR9 cells or U87MG cells. Cells were plated on a 96-well plate in complete growth media and incubated with either liposomal SNAs(final DNA concentration 0.25 μΜ) for different times (15, 30, 60, 90, 120, or 240 minutes). At the end of incubation period, the cells were washed with 1 ^χ PBS. The resulting cell suspension was subjected to flow cytometry using the Cy5 intensity channel on a Guava easyCyte 8HT instrument (Millipore, Billerica, MA, USA). The fluorescence was normalized using untreated cells as a negative control for these time-points. The error-values were calculated using the standard error of the mean of median signal from different wells representing one type of sample.

[0114] For U87MG cells, cells were plated at 20,000 cells per well in a 96 well plate in complete growth media. The cells were placed in the incubator to recover overnight. The following day, the cells were treated with both types of LSNA, cholesterol- and lipid-tail, cholesterol-DNA, and DBCO-DNA with a final DNA concentration 0.5 μΜ for 1 , 2, and 4 h. After each time point, the cells were triple washed with 1 ^χ PBS, trypsinized (5% trypsin, 30 μΙ_ for 5 min at 37 ^eC), and fixed in 200 μΙ_ of 4% paraformaldehyde. Flow cytometry was performed on the cells using the red laser and red fluorescence channel on a Guava easyCyte 8HT instrument (Millipore, Billerica, MA, USA). The distribution of cell fluorescence of the gated-cells was collected and the MFI was calculated. Error-values were determined using the standard deviation of the median signal from three different wells. For the scavenger receptor inhibition studies, the cells were incubated with Fucoidan (50 μg/ml) for 30 minutes prior to the addition of the respective oligonucleotide structures, and flow cytometry was performed after 1 hour of incubation with the LSNAs using the

aforementioned protocol.

[0115] ln-vitro cell stimulation studies RAW-Blue™ were plated in 96-well plates at a density of 10,000 cells per well in DMEM media (200 μί of media/well). Immediately after plating, the cells were treated with the cholesterol-tail or lipid-tail liposomal SNAs (See Table 4) at 250 nM of final DNA concentration per well. The cells were incubated with the different liposomal SNAs for 15, 30, 60, and 90 minutes. After each time point, the media was removed and replaced with fresh media and further incubated at 37 °C for 16 hours. The following day a 20 μΐ aliquot of the supernatr"t of treated RAW-Blue cells was removed and transferred to separate 96-well plate. Twenty microliters of supernatant of untreated RAW- Blue cells was used as a negative control. One hundred and eighty microliters of QUANTI- Blue™ solution (Invivogen, prepared as according to the manufacturer's protocol) was added to each well of the plate containing the transferred 20 μΙ_ aliquots. After 4 hours incubation, the change in color due to SEAP activity was quantified by reading the OD at 655 nm using a BioTek Synergy H4 Hybrid Reader.

[0116] Cytotoxicity. Cytotoxicity studies were performed on U87MG cells to determine whether the lipid-tail LSNAs would damage cells. U87MG cells were plated in a 96 well format at 20,000 cells per well. The cells were then allowed to recover overnight. The following day, the cells were treated with lipid-tail LSNAs at 5 and 1 μΜ for 24 hours.

Following overnight incubation, an AlamarBlue Assay (Thermo Fisher Scientific, Waltham, MA) was performed according to the manufacturer's protocol with a 2 hour incubation period. As a positive control, cells were treated with 10 vol% DMSO immediately before performing the assay. Results of this study are shown in Figure 16.

[0117] Quanti-Blue Assay. For analysis, a 180 μΙ_ aliquot of QUANTI-Blue™ solution (InvivoGen, prepared according to the manufacturer's protocol) was added to each well in a different 96 well plate. A 20 μΙ_ aliquot of the supernatant from the treated HEK-Blue cells was then added to each respective well. As a negative control, supernatant of untreated HEK-cells was used. After 4 hours of incubation, the change in color due to SEAP activity was quantified by reading the absorbance at 630 nm using a BioTek Synergy H4 Hybrid Reader.

ADDITIONAL REFERENCES

1 . Mirkin, et al. Nature 1996, 382 (6592), 607-9.

2. Radovic-Moreno, et al. Proc. Natl. Acad. Sci. U. S. A. 2015, 1 12 (13), 3892-3897.

3. Giljohann, et al. J. Am. Chem. Soc. 2009, 131 (6), 2072-3.

4. Zheng, et al. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (30), 1 1975-1 1980.

5. Banga, et al. J. Am. Chem. Soc. 2014, 136 (28), 9866-9.

6. Sprangers, et al. Small 2016.

7. Chinen, et al. Angew. Chem., Int. Ed. 2015, 54 (2), 527-31 .

8. Cutler, et al. J. Am. Chem. Soc. 2012, 134 (3), 1376-91 .

9. Calabrese, et al. Angew. Chem., Int. Ed. 2015, 54 (2), 476-80.

10. Li, et al. Nano Lett. 2004, 4 (6), 1055-1058.

1 1 . Zelphati, et al J. Controlled Release 1996, 41 (1 -2), 99-1 19. 12. Reddy, et al. Biochim. Biophys. Acta 2012, 1818 (9), 2271 -81 .

13. Dayani, et al. Biomacromolecules 2013, 14 (10), 3380-5.

14. Mclean, et al. Biochemistry 1981 , 20 (10), 2893-2900.

15. Frykholm, et al. Langmuir 2009, 25 (3), 1606-161 1 .

16. Frykholm, et al. Febs J. 2010, 277, 234-235.

17. Kim, et al. Arch. Pharmacal Res. 1991 , 14 (4), 336-341 .

18. Hurst, et al. Anal. Chem. 2006, 78 (24), 8313-8.

19. Baskin, et al. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (43), 16793-16797.

20. Filippov, et al. Biophys. J. 2003, 84 (5), 3079-3086.

21 . Machan, et al. Biochim. Biophys. Acta, Biomembr. 2010, 1798 (7), 1377-1391 .

22. Dave, et al. ACS Nano 201 1 , 5 (2), 1304-12.

23. Yu, et al. Nucleic Acids Res. 2002, 30 (7), 1613-9.

24. Baldwin, et al. Bioconjugate Chem. 201 1 , 22 (10), 1946-53.

25. Reulen, et al. Bioconjugate Chem. 2010, 21 (5), 860-6.

26. Wolfrum, et al. Nat. Biotechnol. 2007, 25 (10), 1 149-57.

27. Zelphati, et al. Biochim. Biophys. Acta, Lipids Lipid Metab. 1998, 1390 (2), 1 19-133.

28. Choi, et al. Proc. Natl. Acad. Sci. U. S. A. 2013, 1 10 (19), 7625-30.

29. Choi, et al. Proc. Natl. Acad. Sci. U. S. A. 2013, 1 10 (19), 7625-30.

30. Giljohann, et al. Nano Lett. 2007, 7 (12), 3818-21 .

31 . Luo, et al. Nat. Biotechnol. 2000, 18 (1 ), 33-7.

32. Ramirez-Ortiz, et al. Infect. Immun. 2008, 76 (5), 2123-9.

33. Cooper, et al. J. Clin. Immunol. 2004, 24 (6), 693-701 .

Claims

WHAT IS CLAIMED IS:

1 . A liposomal particle comprising:

a lipid bilayer comprising a plurality of lipids; and

a plurality of oligonucleotides,

wherein each oligonucleotide is covalently bound to a lipid, and the oligonucleotide and the lipid comprise complementary reactive moieties that together form a covalent bond.

2. The liposomal particle of claim 1 , wherein the plurality of lipids comprises

(a) a fatty acid chain portion comprising (1 ) 10 to 22 carbons and (2) 0 to 5 carbon- carbon double bonds; and

(b) a hydrophilic head portion, wherein the hydrophilic head portion can be neutral, cationic, or anionic.

3. The liposomal particle of claim 2, wherein the fatty acid chain portion comprises 15 to 22 carbons and 0 to 2 carbon-carbon double bonds.

4. The liposomal particle of any one of claims 1 -3, wherein the plurality of lipids comprises a lipid selected from the group consisting of the phosphatidylcholine,

phosphatidylglycerol, and phosphatidylethanolamine family of lipids.

5. The liposomal particle of claim 4, wherein the lipid is selected from the group consisting of dioleoylphosphatidylcholine; dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine; dilauroylphosphatidylcholine,

dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and diarachidonylphosphatidylcholine (DAPC), dioleoylphosphatidylethanolamine,

dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE) distearoylphosphatidylethanolamine (DSPE), and a modified analog thereof.

6. The liposomal particle of any one of claims 1 -5, wherein the reactive moiety on the lipid comprises an azide, an alkyne, a maleimide, a thiol, an alcohol, an amine, a carboxylic acid, an olefin, an isothiocyanate, a N-hydroxysuccinimide, a phosphine, a nitrone, a norbornene, an oxanorbornene, a transcycloctene, an s-tetrazene, an isocyanide, a tetrazole, a nitrile oxide, a quadricyclane, or a carbodiimide.

7. The liposomal particle of any one of claims 1 -6, wherein the reactive moiety on the oligonucleotide is on a terminus of the oligonucleotide.

8. The liposomal particle of any one of claims 1 -7, wherein the reactive moiety on the oligonucleotide comprises an alkyne, an azide, a maleimide, a thiol, an alcohol, an amine, a carboxylic acid, an olefin, an isothiocyanate, a N-hydroxysuccinimide, a phosphine, a nitrone, a norbornene, an oxanorbornene, a transcycloctene, an s-tetrazene, an isocyanide, a tetrazole, a nitrile oxide, a quadricyclane, or a carbodiimide.

9. The liposomal particle of any one of claims 6-8, wherein the alkyne comprises dibenzocyclooctyl (DBCO) alkyne or a terminal alkyne.

10. The liposomal particle of any one of claims 1 -9, wherein the lipid comprises an azide reactive moiety and the oligonucleotide comprises an alkyne reactive moiety, or vice versa.

1 1 . The liposomal particle of claim 10, wherein the alkyne reactive moiety comprises a DBCO alkyne.

12. The liposomal particle of any one of claims 1 -1 1 , wherein the oligonucleotide comprises RNA or DNA.

13. The liposomal particle of claim 12, wherein the RNA is a non-coding RNA.

14. The liposomal particle of claim 13, wherein the non-coding RNA is an inhibitory RNA.

15. The liposomal particle of claim 13 or claim 14, wherein the inhibitory RNA is selected from the group consisting of a small inhibitory RNA (siRNA), a single-stranded RNA (ssRNA) that forms a triplex with double stranded DNA, and a ribozyme.

16. The liposomal particle of claim 13 or claim 14, wherein the RNA is a microRNA.

17. The liposomal particle of claim 12, wherein the DNA is antisense-DNA.

18. The liposomal particle of any one of claims 1 -17, wherein diameter of the liposomal particle is less than or equal to about 50 nanometers.

19. The liposomal particle of any one of claims 1 -18, wherein the particle comprises 50 to 500 oligonucleotides.

20. The liposomal particle of claim 19, wherein the particle comprises 150 to 350 oligonucleotides.

21 . The liposomal particle of claim 19, wherein the particle comprises 200 to 300 oligonucleotides.

22. The liposomal particle of any one of claims 1 -21 , wherein the oligonucleotide is a modified oligonucleotide.

23. The liposomal particle of any one of claims 1 -22, wherein the oligonucleotide further comprises a fluorescent tag.

24. A method of inhibiting expression of a gene comprising hybridizing a polynucleotide encoding a gene product expressed from the gene with one or more oligonucleotides complementary to all or a portion of the polynucleotide, the complementary oligonucleotide comprising the oligonucleotide of the liposomal particle of any one of claims 1 -23, wherein hybridizing between the polynucleotide and the complementary

oligonucleotide occurs over a length of the polynucleotide with a degree of complementarity sufficient to inhibit expression of the gene product.

25. The method of claim 24, wherein expression of the gene product is inhibited in vivo.

26. The method of claim 24, wherein expression of the gene product is inhibited in vitro.

27. A method for up-regulating activity of a toll-like receptor (TLR), comprising contacting a cell having the TLR with the liposomal particle of any one of claims 1 -23.

28. The method of claim 27, wherein the oligonucleotide is a TLR agonist.

29. The method of claim 27 or claim 28, wherein the TLR is 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 1 1 , toll-like receptor 12, or toll-like receptor 13.