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Definitions

• Nanoparticles are in the submicron size domain and possess unique size-dependent properties that make the materials superior compared to their bulk forms.
• the advanced chemical and physical properties associated with nanoparticles have led to their extensive use in the fields of biology and medicine.
• High-density lipoproteins (HDL) are dynamic nanoparticles that circulate in the blood.
• the present disclosure presents the synthesis of soft-core, HDL-like nanoparticles with the size, shape, surface chemistry, composition, protein structure, and cholesterol transport properties that are consistent with mature human HDLs.
• Lipid-conjugated (LC) HDL NPs target relevant cell types to remove and deliver cellular cholesterol and reduce inflammation, recapitulating salient functions of human HDLs. Because synthesis of LC HDL NPs is straightforward and does not require time-intensive and costly enzymatic maturation steps, it is anticipated that these nanoparticles will prove to be strong candidates for next generation HDL-based therapeutics.
• the present disclosure provides methods for the synthesis and characterization of a spherical HDL mimic using lipid-conjugated organic core scaffolds.
• the core design motif constrains and orients phospholipid geometry to facilitate the assembly of soft-core nanoparticles that in some embodiments are approximately 10 nm in diameter and resemble human HDLs in their size, shape, surface chemistry, composition and protein secondary structure.
• the present disclosure provides a spherical high-density lipoprotein nanoparticle (HDL-NP) comprising: a core and a shell surrounding and attached to the core, wherein the core comprises a lipid conjugated organic core scaffold.
• the shell is a lipid shell.
• the shell is a lipid bilayer or monolayer.
• the spherical HDL-NP has a zeta potential of about -20 mV.
• the organic core scaffold comprises PL 4 .
• the organic core scaffold comprises 9-DNA-PL4.
• the organic core scaffold comprises 18-DNA-PL 4 .
• the shell has a zeta potential closer to human HDL than a synthetic HDL nanoparticle with an inorganic core.
• the spherical HDL-NP has a zeta potential closer to human HDL than a synthetic HDL nanoparticle with a gold core.
• the spherical HDL-NP has an apolipoprotein.
• the apolipoprotein is apolipoprotein A-I.
• the present disclosure provides a composition comprising any one of the spherical HDL-NP described above.
• the present disclosure provides a method of treating an inflammatory disorder comprising, administering to a subject an effective amount of a composition comprising any one of the spherical HDL-NP described above.
• the present disclosure provides a method of reducing NF-kB activity, comprising administering to a subject an effective amount of a composition comprising any one of the spherical HDL-NP described above.
• the disclosure relates to a spherical high-density lipoprotein nanoparticle (HDL-NP) comprising, a core and a shell surrounding and attached to the core, wherein the core comprises a lipid conjugated organic core scaffold.
• the shell is a lipid shell.
• the shell is a lipid bilayer or monolayer.
• the shell is a lipid bilayer.
• the shell is a lipid monolayer.
• the spherical HDL-NP has a surface zeta potential of about -20 millivolts (mV).
• the organic core scaffold comprises PL 4 . In some embodiments, the organic core scaffold comprises PL 4 .
• the organic core scaffold comprises 9-DNA-PL 4 . In some embodiments, the organic core scaffold comprises 18-DNA-PL4.
• the shell has a zeta potential closer to human HDL than a synthetic HDL nanoparticle with an inorganic core.
• the spherical HDL-NP has a zeta potential closer to human HDL than a synthetic HDL nanoparticle with a gold core.
• the spherical HDL-NP has a zeta potential of -16-26 mV.
• the spherical HDL-NP of the instant disclosure further comprise apolipoprotein.
• the apolipoprotein is apolipoprotein A-I.
• the spherical HDL-NP of the instant disclosure comprise a hydrodynamic diameter of greater than 8.7 nm. In some embodiments, the hydrodynamic diameter is 8.7 nm-17-7nm. In some embodiments, the hydrodynamic diameter is 10 nm- 15nm. In some embodiments, the hydrodynamic diameter is 12 nm-14nm.
• the organic core scaffold comprises a hydrophobic small molecule-phospholipid conjugate (PL 4 ).
• the PL 4 comprises a headgroup-modified phospholipid.
• the headgroup-modified phospholipid comprises a ring-strained alkyne, 1,2-dipalmitoyl-sn-glycero-3-phosphoethan- olamine-N-dibenzocyclooctyl.
• the phospholipid is coupled to the small molecule with a plurality of terminal functional groups.
• the small molecule is tetrakis(4-az-idophenyl)methane.
• the plurality of functional groups is 2-6 functional groups.
• the plurality of functional groups is 4 functional groups.
• the functional groups are terminal azides (SM-Az).
• the organic core scaffold comprises an amphiphilic DNA- linked small molecule-phospholipid conjugate (DNA- PL 4 ).
• the DNA is a double stranded oligonucleotide of 5-17 nucleotides in length. In some embodiments, the DNA is a double stranded oligonucleotide of 8-15 nucleotides in length. In some embodiments, the DNA is a double stranded oligonucleotide of 9 nucleotides in length. In some embodiments, a first single strand of the double stranded DNA is linked to a
• ssDNA-PL phospholipid and forms a ssDNA-phospholipid conjugate
• a second strand of the double stranded DNA, complementary to the first strand of the double stranded DNA is linked to a small molecule.
• the small molecule is a tetrahedral small molecule and the small molecule linked to the DNA forms a tetrahedral small molecule-DNA hybrid (SMDH 4 ).
• the SMDH 4 is linked to the ssDNA-PL through hydrogen bonding between the complementary single strands of DNA.
• the spherical HDL-NP has a more efficient cholesterol transport capacity than that of apolipoprotein or a synthetic HDL nanoparticle with a gold core.
• the spherical HDL-NP has a diameter of about 5-30 nm, 5-25 nm, 5-22 nm, 5-20 nm, 5-15 nm, 5-14 nm, 5-13 nm, 5-12 nm, 5-11 nm, 5-10 nm, 8-15 nm, 8- 14 nm, 8-13 nm, 8-12 nm, 8-11 nm, 8-10 nm, 10-12 nm, or 10 nm.
• the spherical HDL-NP further comprises a therapeutic agent linked to the HDL-NP.
• the therapeutic agent is a therapeutic nucleic acid.
• the therapeutic agent is an anti-cancer agent.
• the anti-cancer agent is chemotherapeutic agent.
• the therapeutic agent is an anti-inflammatory agent.
• the invention is a method for treating a cancer, comprising
• a spherical high-density lipoprotein nanoparticle comprising: a core and a shell surrounding and attached to the core, wherein the core comprises a lipid conjugated organic core scaffold in an effective amount to treat the cancer.
• a method of treating an inflammatory disorder by administering to a subject having an inflammatory disorder a spherical high-density lipoprotein nanoparticle (HDL-NP) comprising: a core and a shell surrounding and attached to the core, wherein the core comprises a lipid conjugated organic core scaffold in an effective amount to treat the inflammatory disorder is provided.
• the disclosure relates to a pharmaceutical composition comprising any of the spherical HDL-NP of the instant disclosure.
• the disclosure relates to method of treating cancer or an inflammatory disorder comprising, administering to a subject an effective amount of a composition comprising the spherical HDL-NP of the instant disclosure.
• the disclosure relates to a method of reducing NF-kB activity, comprising administering to a subject an effective amount of a composition comprising any of the spherical HDL-NP of the instant disclosure.
• the disclosure relates to a method for making a spherical high-density lipoprotein nanoparticle (HDL-NP) comprising, preparing a ssDNA-phospholipid conjugate (ssDNA-PL), preparing a tetrahedral small molecule-DNA hybrid (SMDH 4 ), wherein the ssDNA-PL and SMDH4 have complementary DNA sequences, incubating the ssDNA-PL and SMDH 4 such that the complementary DNA sequences bases pair with one another to form a DNA-PL core, and adding to the DNA-PL core phospholipid liposomes and apolipoprotein to produce the spherical HDL-NP.
• HDL-NP spherical high-density lipoprotein nanoparticle
• the ssDNA-PL comprises an oligonucleotide of at least 9 nucleotides. In some embodiments, the oligonucleotide is 6-16 nucleotides in length. In some embodiments, the oligonucleotide comprises SEQ ID NO: 1. In some embodiments, the oligonucleotide is 18 nucleotides in length. In some embodiments, the oligonucleotide comprises SEQ ID NO: 2.
• the SMDH 4 comprises an oligonucleotide of at least 9 nucleotides. In some embodiments, the oligonucleotide is 9-15 nucleotides in length. In some embodiments, the oligonucleotide comprises SEQ ID NO: 3. In some embodiments, the oligonucleotide is 18 nucleotides in length. In some embodiments, the oligonucleotide comprises SEQ ID NO: 4.
• the apolipoprotein of any of the methods disclosed herein is apolipoprotein A-I.
• the disclosure relates to an organic core scaffold comprising, 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl (DBCO PE) linked to a tetrahedral small molecule core (tetrakis(4-azidophenyl)methane) with four terminal azides.
• DBCO PE 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl
• FIG.1 shows the canonical reverse cholesterol transport pathway.
• FC free cholesterol.
• CE cholesteryl esters.
• PL phospholipids.
• LCAT lecithin:cholesterol acyltransferase.
• FIGs.2A-2G show synthesis schemes for PL 4 and DNA-PL 4 core scaffolds, and LC HDL NP assembly.
• FIG.2D shows the composition of native and synthetic HDLs by mass percent (%). *Au HDL NP composition is reported from a previously published data set. 18
• FIG.2E shows circular dichroism spectra for LC HDL NPs and human HDLs.
• FIG.2F shows a schematic of native and synthetic HDL species which vary in their core materials.
• FIGs.3A-3D FIG.3A shows LC HDL NPs efflux H 3 -chol from cAMP-treated J774 macrophages.
• FIG.3B shows the quantification of H 3 -chol in all fractions from a tandem efflux-influx assay.
• FIG.3C shows the percent (%) Influx of cholesterol to hepatocytes in tandem assay.
• FIG.3D shows SR-B1 dependent cholesterol efflux.
• Statistical significance was determined using a two-tailed student’s t test. **p ⁇ 0.01. *** p ⁇ 0.001.
• FIG.4 LC HDL NPs facilitate rapid delivery of fluorescent cholesterol (NBD-chol) to hepatocytes (HepG2) in 30 min via flow cytometry (FIGs.4A-4B) and confocal microscopy (FIG.4C).
• NBD-chol fluorescent cholesterol
• FIG.4A-4B flow cytometry
• FIG.4C confocal microscopy
• FIGs.5A-5B FIG.5A shows the quantification of free cholesterol (FC) and cholesterol esters (CE) in an LCAT cholesterol esterification assay.
• FIG.5B shows LC HDL NP-mediated suppression of pro-inflammatory NF-kB signaling. Statistical significance was determined using a two-tailed student’s t test. *p ⁇ 0.05. ** p ⁇ 0.01.
• FIG.6 shows an analytical RP-HPLC trace of 9-SMDH4 (SEQ ID NO: 1) from the coupling reaction of the tetrakis(4-azidophenyl) methane with alkyne-functionalized 9-mer DNAs on the CPGs.
• the trace is the signal from the diode detector set at 260 nm.
• FIG.7 shows an analytical RP-HPLC trace of 18-SMDH4 (SEQ ID NO: 2) from the coupling reaction of the tetrakis(4-azidophenyl) methane with alkyne-functionalized 18-mer DNAs on the CPGs.
• the trace is the signal from the diode detector set at 260 nm.
• FIG.8 shows a semi-preparative RP-HPLC trace of crude 9-DNA-lipid (SEQ ID NO: 3).
• the trace is the signal from the diode detector set at 260 nm.
• FIG.9 shows a semi-preparative RP-HPLC trace of crude 18-DNA-lipid (SEQ ID NO: 4).
• the trace is the signal from the diode detector set at 260 nm.
• FIG.10 shows a denaturing PAGE-gel image (15%, 7 M Urea) of 9-SMDH 4 (SEQ ID NO: 1) and 18-SMDH4’s (SEQ ID NO: 2) and 9-DNA-lipid (SEQ ID NO: 3) conjugate and 18-DNA-lipid (SEQ ID NO: 4) conjugate.
• the gel experiment was carried out in 1 ⁇ TBE buffer at 180 V for 1 h and then the gels were stained with SYBR Gold (Thermo Fisher Scientific, Inc., Grand Island, NY) and their pictures were taken using a Typhoon 9400 (GE Healthcare, Pittsburgh, PA).
• FIG.11 PL 4 synthesis scheme.
• PL 4 core materials were synthesized by copper-free click chemistry conjugation of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- dibenzocyclooctyl (DBCO PE) with a tetrahedral small molecule core (tetrakis(4- azidophenyl)methane) with four terminal azides (FIG.11).
• the DBCO PE and tetrakis(4-azidophenyl)methane were each dissolved at 0.1 wt % in N,N- dimethylformamide (DMF, Sigma Aldrich) and mixed at a 10:1 molar ratio of DBCO PE to tetrakis(4-azidophenyl)methane in DMF.
• DMF N,N- dimethylformamide
• the reaction mixture was subjected to three rounds of alternating vortexing and bath sonication, and was then allowed to react at room temperature under vortex for 24 hour. HPLC and electrospray ionization mass spectrometry (FIG.12) was then used to characterize the resulting reaction mixture.
• FIG.12 only show a single species at the right mass for PL4, we conclude that there is no partially coupled product (PL3, PL2, etc.) and use the reaction mixture for the assembly step.
• the assembly step involves adding a large excess of DPPC lipids, there is no need to separate the excess DBCO PE molecule from the PL 4 core prior to its use in the assembly.
• FIG.12 shows an electrospray ionization mass spectrometry of PL4 core.
• Product m/z 4401.6 (Theoretical: 4401.7).
• FIG.13 shows an ultrastructural characterization of PL 4 HDL NPs via negative stain TEM. Imaging was performed with FEI Tecnai Spirit TEM. Inset (far right panel) is under 120,000 ⁇ magnification. Scale bars labeled on each image, from 50-200 nm. TEM diameter was determined to be 10 ⁇ 2 nm by measurement of > 50 particles using ImageJ software.
• FIG.14 shows an ultrastructural characterization of assembly attempt using 18-mer DNA-PL4 scaffolds.
• FIG.15 shows TEM imaging of Core + DPPC, w/o apoA-1 (top row: DNA-PL 4 core + DPPC; bottom row: PL 4 core + DPPC). Note: adequate visualization of Core + DPPC control samples without protein required uranyl acetate staining for longer time period (15 min stain) compared to protein-containing samples (20 s stains).
• FIG.16 Characterization of LC HDL NP assembly and apoA-1 oligomerization profiles.
• apoA-1 adopts higher order oligomerization states upon assembly with lipid conjugate core scaffolds for both PL 4 and DNA-PL 4 particles.
• High-density lipoproteins are native circulating nanoparticles that carry cholesterol, target specific cell types, and play important roles in a host of disease processes.
• synthetic HDL mimics have become promising therapeutic agents.
• approaches to date have been unable to reproduce key features of spherical HDLs, which are the most abundant HDL species, and are of particular clinical importance.
• Native HDLs are circulating nanoparticles ( ⁇ 8-13 nm in diameter) that transport cholesterol and play important roles in cancer and cardiovascular disease.
• Spherical HDLs are the most abundant HDL sub-species, and are particularly important clinically.
• previous HDL mimics have failed to reproduce features of spherical HDL (e.g. spherical HDL conformation) without relying on inorganic template materials (e.g. gold nanoparticles).
• inorganic template materials e.g. gold nanoparticles.
• These HDL-like nanoparticles of the present disclosure are the first of their kind, namely spherical HDL mimics with a soft material core.
• the HDL-like nanoparticles of the present disclosure mimic spherical HDL species using novel, lipid-conjugated organic core scaffolds.
• the core design motif constrains and orients phospholipid geometry to facilitate the assembly of soft-core nanoparticles that are, in some embodiments, approximately 10 nm in diameter and resemble human HDLs in their size, shape, surface chemistry, composition and protein secondary structure.
• the HDL-like nanoparticles mimic the structure of native HDL with respect to size ( ⁇ 10 nm), surface chemistry (-20 mV zeta potential), and HDL protein secondary structure as determined by circular dichroism. Synthetic HDLs have demonstrated promise as therapy for cardiovascular disease and cancer, among other indications.
• HDL-like nanoparticles of the present disclosure are designed to enable superior therapeutic efficacy when compared to previous synthetic platforms, none of which reproduce both of the features noted above.
• High-density lipoproteins are dynamic nanoparticles that circulate in the blood and transport cholesterol. Clinically, elevated HDL cholesterol levels are associated with reduced risk of atherosclerotic cardiovascular disease (ASCVD). 1-2 At the cellular level, HDLs target macrophages and hepatocytes to remove and deliver cholesterol, respectively, which has been shown to reduce inflammation and atherosclerotic burden. 3-4 Due to the beneficial association between HDL and ASCVD, cell-specific targeting properties, and the intrigue of using HDLs for targeted drug delivery, tremendous effort has focused on synthesizing HDLs that resemble their natural counterparts. 5 There are multiple HDL species in the blood that serve as synthetic targets.
• Immature HDLs are cholesterol poor and discoidal in shape, consisting mainly of the HDL-defining protein, apolipoprotein A-I (which may be referred to herein as apoA-I, apoA-1, and/or apolipoprotein A-1), and phospholipids.
• Spherical HDLs comprise the majority of HDLs in circulation. 6-7 Yet, synthetic forms of HDL used as therapy have exclusively resembled immature discoidal HDL.
• Spherical HDL species are most correlated with reduced ASCVD. 13 However, these HDLs are more difficult to synthesize due to the enzymatic steps required to mature discoidal HDLs into cholesterol- and cholesteryl ester-containing spherical HDLs. Previous studies have attempted to circumvent these biological maturation steps by using inorganic nanoparticles as templates. 14-16 The templates can be functionalized with phospholipids and apoA-I, and successfully restrict the size of HDL mimics to ⁇ 15 nm diameter. 17 However, these materials fail to recapitulate a critical feature of native spherical HDL, namely a robust, soft material core capable of dynamically loading and off-loading cholesterol and cholesteryl esters. Apart from inorganic template materials, soft-core discoidal HDL-like particles have also been synthesized using amphiphilic peptides. 22
• HDL mimetic nanoparticles using lipid-conjugated core scaffolds is accomplished in a two-step process: first, the core scaffolds are synthesized and purified; second, the particle is fabricated via supramolecular assembly of the core scaffold, free phospholipids, and the HDL-defining protein, apolipoprotein A1 (apo- A1).
• apolipoprotein A1 apo- A1
• a variety of lipid-conjugated organic cores can theoretically be used for particle assembly.
• successful particle fabrication using three different organic core scaffolds is demonstrated. Specifically, a tetrahedral small molecule-phospholipid hybrid, called PL4, and a tetrahedral ssDNA-phospholipid-small molecule hybrid, called DNA-PL4, of two different lengths are used.
• the present disclosure provides methods for the synthesis of spherical HDL-like nanoparticles with structural and functional properties of mature human HDLs using lipid- conjugated core scaffolds (LC HDL NP).
• LC HDL NP lipid- conjugated core scaffolds
• the first scaffold is a highly hydrophobic small molecule-phospholipid conjugate (PL 4 ) that was synthesized using copper-free click chemistry.
• a headgroup-modified phospholipid harboring a ring-strained alkyne, 1,2-dipalmitoyl-sn-glycero-3-phosphoethan-olamine-N- dibenzocyclooctyl was click coupled to tetrakis(4-az-idophenyl)methane, a small molecule with four terminal azides (SM-Az4) (FIGs.2A and FIGs.11-12).
• the second (9-DNA-PL4) and third (18-DNA-PL4) core scaffolds are amphiphilic DNA- linked PL4 cores (FIG.2B), with differing DNA linker length (9- vs.18-mer dsDNA).
• DNA-PL 4 cores were synthesized in a two-step fashion (see Synthesis section herein). First, DNA-phospholipid conjugates (ssDNA-PL), and tetrahedral small molecule-DNA hybrids (SMDH4), were synthesized (Table 2). Second, ssDNA-PL and SMDH 4 with complementary base-paired sequences were hybridized to yield the final DNA- PL 4 cores (FIG.2B and FIGs.6-9).
• LCAT lecithin:cholesterol acyl transferase
• PL4 HDL NPs exhibited a substantially greater capacity for esterification over Au HDL NPs and DNA- PL 4 HDL NPs, converting 87% of bound cholesterol to cholesteryl esters. This result is likely due to the increased flexibility and hydrophobicity of the PL 4 core over gold (Au) and DNA-PL4 cores, which enables efficient packing of cholesteryl esters.
• HDL-like As used herein, the terms“HDL-like”,“HDL-mimetic”, and“HDL mimic” are used interchangeably to refer to a synthetic HDL-NP.
• the present disclosure provides a spherical high-density lipoprotein nanoparticle (HDL-NP) comprising: a core and a shell surrounding and attached to the core, wherein the core comprises a lipid conjugated organic core scaffold.
• the shell may be a lipid shell that is linked to the components of the organic core scaffold at one or more points, as shown schematically in Fig.1.
• the shell is a lipid bilayer or monolayer.
• a structure described herein or a portion thereof, such as a shell of a structure includes one or more natural or synthetic lipids or lipid analogs (i.e., lipophilic molecules).
• One or more lipids and/or lipid analogues may form a single layer or a multi-layer (e.g., a bilayer) of a structure. In some instances where multi-layers are formed, the natural or synthetic lipids or lipid analogs interdigitate (e.g., between different layers).
• Non-limiting examples of natural or synthetic lipids or lipid analogs include fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids and polyketides (derived from condensation of ketoacyl subunits), and sterol lipids and prenol lipids (derived from condensation of isoprene subunits).
• a structure described herein includes one or more phospholipids.
• the one or more phospholipids may include, for example,
• phosphatidylcholine phosphatidylcholine, phosphatidylglycerol, lecithin, b, g-dipalmitoyl-a-lecithin,
• sphingomyelin phosphatidylserine, phosphatidic acid, N-(2,3-di(9-(Z)-octadecenyloxy))- prop-1-yl-N,N,N-trimethylammonium chloride, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylinositol, cephalin, cardiolipin, cerebrosides, dicetylphosphate, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine,
• a shell (e.g., a bilayer) of a structure includes 50-200 natural or synthetic lipids or lipid analogs (e.g., phospholipids).
• the shell may include less than about 500, less than about 400, less than about 300, less than about 200, or less than about 100 natural or synthetic lipids or lipid analogs (e.g., phospholipids), e.g., depending on the size of the structure.
• Non-phosphorus containing lipids may also be used such as stearylamine,
• docecylamine, acetyl palmitate, and fatty acid amides can be used to form portions of a structure described herein.
• other lipids such as fats, oils, waxes, cholesterol, sterols, fat-soluble vitamins (e.g., vitamins A, D, E and K), glycerides (e.g., monoglycerides, diglycerides, triglycerides) can be used to form portions of a structure described herein.
• a portion of a structure described herein such as a shell or a surface of a nanostructure may optionally include one or more alkyl groups, e.g., an alkane-, alkene-, or alkyne- containing species, that optionally imparts hydrophobicity to the structure.
• An“alkyl” group refers to a saturated aliphatic group, including a straight-chain alkyl group, branched-chain alkyl group, cycloalkyl (alicyclic) group, alkyl substituted cycloalkyl group, and cycloalkyl substituted alkyl group.
• the alkyl group may have various carbon numbers, e.g., between C 2 and C 40 , and in some embodiments may be greater than C 5 , C 10 , C 15 , C 20 , C 25 , C 30 , or C 35 .
• a straight chain or branched chain alkyl may have 30 or fewer carbon atoms in its backbone, and, in some cases, 20 or fewer.
• a straight chain or branched chain alkyl may have 12 or fewer carbon atoms in its backbone (e.g., C 1 - C 12 for straight chain, C 3 -C 12 for branched chain), 6 or fewer, or 4 or fewer.
• cycloalkyls may have from 3-10 carbon atoms in their ring structure, or 5, 6 or 7 carbons in the ring structure.
• alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, hexyl, cyclochexyl, and the like.
• the spherical HDL-NP of the instant disclosure further comprise apolipoprotein.
• the apolipoprotein can be apolipoprotein A (e.g., apo A-I, apo A-II, apo A-IV, and apo A-V), apolipoprotein B (e.g., apo B48 and apo B100), apolipoprotein C (e.g., apo C-I, apo C-II, apo C-III, and apo C-IV), and apolipoproteins D, E, and H.
• apolipoprotein A e.g., apo A-I, apo A-II, apo A-IV, and apo A-V
• apolipoprotein B e.g., apo B48 and apo B100
• apolipoprotein C e.g., apo C-I, apo C-II, apo C-III, and apo C-IV
• apo A1 , apo A2 , and apo E promote transfer of cholesterol and cholesteryl esters to the liver for metabolism and may be useful to include in structures described herein.
• a structure described herein may include one or more peptide analogues of an apolipoprotein, such as one described above.
• proteins e.g., non-apolipoproteins
• the apolipoprotein is apolipoprotein A-I.
• the HDL-NP has an organic core scaffold.
• An organic core scaffold as used herein refers to non-metallic material, soft-core, having a 3 dimensional structure and charge sufficient to organize and hold a lipid layer in a spherical shape.
• A“spherical” shape or structure herein refers to a structure having a round or sphere-like structure. The structure does not need to be perfectly round or an exact sphere, but rather is an approximate sphere shape.
• the organic core scaffold comprises a hydrophobic small molecule-phospholipid conjugate (PL4).
• the hydrophobic small molecule-phospholipid conjugate comprises any small molecule capable of being linked to a phospholipid.
• the small molecule is tetrakis(4-az-idophenyl)methane.
• the phospholipid may be a headgroup-modified phospholipid.
• the headgroup-modified phospholipid comprises a ring-strained alkyne, 1,2-dipalmitoyl-sn-glycero-3-phosphoethan-olamine-N-dibenzocyclooctyl.
• the small molecule may be linked directly to the phospholipid or may be linked through the use of a functional group.
• the functional group may include any suitable end group that can be used to functionalize the phospholipid to the small molecule, e.g., an amino group (e.g., an unsubstituted or substituted amine), an amide group, an azide, an imine group, a carboxyl group, or a sulfate group.
• the functional group includes at least a second end group.
• the second end group may be a reactive group that can covalently attach to another functional group.
• the amino group e.g., an unsubstituted or substituted amine
• the phospholipid is coupled to the small molecule with a plurality of terminal functional groups.
• the plurality of functional groups is 2-6 functional groups. In some embodiments, the plurality of functional groups is 4 functional groups. In some
• the functional groups are terminal azides (SM-Az).
• the organic core scaffold comprises an amphiphilic DNA- linked small molecule-phospholipid conjugate (DNA- PL 4 ).
• the DNA (or any other nucleic acid, including modified and naturally occurring nucleic acids) provides a unique link between the phospholipid and small molecule. It is advantageous to use DNA because the size of the DNA and thus the core may be easily controlled by altering the length of the DNA strand.
• the DNA is 5-50 nucleotides in length
• the DNA is 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 6-45, 6-40, 6-35, 6-30, 6-25, 6-20, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6- 10, 6-9, 6-8, 6-7, 7-45, 7-40, 7-35, 7-30, 7-25, 7-20, 7-17, 7-16, 715, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-45, 8-40, 8-35, 8-30, 8-25, 8-20, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8- 10, 8-9, 9-45, 9-40, 9-35, 9-30, 9-25, 9-20, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9
• the DNA is a double stranded oligonucleotide. In some embodiments, the DNA is a double stranded oligonucleotide of 8-15 nucleotides in length. In some embodiments, the DNA is a double stranded oligonucleotide of 9 nucleotides in length. In some embodiments, a first single strand of the double stranded DNA is linked to a phospholipid and forms a ssDNA-phospholipid conjugate (ssDNA-PL). In some
• a second strand of the double stranded DNA, complementary to the first strand of the double stranded DNA is linked to a small molecule.
• the small molecule is a tetrahedral small molecule and the small molecule linked to the DNA forms a tetrahedral small molecule-DNA hybrid (SMDH4).
• the SMDH4 is linked to the ssDNA-PL through hydrogen bonding between the complementary single strands of DNA.
• the spherical HDL-NP further comprises a therapeutic agent linked to the HDL-NP.
• the therapeutic agent is a therapeutic nucleic acid.
• the therapeutic agent is an anti-cancer agent.
• the anti-cancer agent is chemotherapeutic agent.
• the therapeutic agent is an anti-inflammatory agent.
• a therapeutic nucleic acid may include any nucleic acid such as but not limited to a polynucleotide, a DNA sequence, a DNA sequence encoding a therapeutic protein, an RNA sequence, a small interfering RNA (siRNA), mRNA, a short-hairpin RNA (shRNA), a micro RNA (miRNA), an antisense oligonucleotide, a triplex DNA, a plasmid DNA (pDNA) or any combinations thereof.
• a therapeutic nucleic acid may be treated or chemically modified.
• a therapeutic nucleic acid may contain inter-nucleotide linkages other than phosphodiester bonds, such as phosphorothioate, methylphosphonate, methylphosphodiester, phosphorodithioate, phosphoramidate, phosphotriester, or phosphate ester linkages, which in some embodiments may confer increased stability.
• Nucleic acid stability may also be increased by incorporating 3'-deoxythymidine or 2'-substituted nucleotides (substituted with, e.g., an alkyl group) into the nucleic acid during synthesis or by providing the nucleic acid as phenylisourea derivatives, or by having other molecules, such as aminoacridine or poly-lysine, linked to the 3' end of the nucleic acid. Modifications of a RNA and/or a DNA may be present throughout the oligonucleotide or in selected regions of the nucleic acid, e.g., the 5' and/or 3' ends, for example by methylation.
• the anti-cancer agent is a chemotherapeutic drug such as Paclitaxel, Cisplatin, Carboplatin, Topotican and Doxoribicin.
• the disclosure relates to an organic core scaffold comprising, 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl (DBCO PE) linked to a tetrahedral small molecule core (tetrakis(4-azidophenyl)methane) with 2-6 terminal azides.
• DBCO PE 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl
• tetrahedral small molecule core tetrakis(4-azidophenyl)methane
• the structure has 4 terminal azides.
• the size, physical properties and functional properties of the HDL-NP of the instant disclosure are similar to that of naturally occurring HDL-NP and distinct from other synthetic HDL-NP. These properties include, for example, spherical shape, surface chemistry, size, hydrodynamic diameter, zeta potential, cholesterol efflux, cholesterol delivery, and therapeutic functions such as suppression of inflammation.
• the spherical HDL-NP of the instant disclosure can be assessed based on the hydrodynamic diameter.
• the hydrodynamic diameter of the spherical HDL-NP is similar to that of naturally occurring HDL-NP and distinct from other synthetic HDL-NP.
• Hydrodynamic diameter assesses the size of a hypothetical hard sphere that diffuses in the same manner as that of the particle being measured and provides an indication of the diffusional properties of the particle that will be indicative of the apparent size of the dynamic hydrated/solvated particle. It may be measured by Dynamic Light Scattering (DLS).
• DLS Dynamic Light Scattering
• the hydrodynamic diameter is greater than 8.7 nm.
• the hydrodynamic diameter is 8.7 nm-17.7nm.
• the hydrodynamic diameter is 10 nm-15nm.
• the hydrodynamic diameter is 12 nm-14nm.
• the spherical HDL-like nanoparticles of the present disclosure can have a diameter with a largest cross-sectional dimension (or, sometimes, a smallest cross-section dimension) of, for example, less than or equal to about 500 nm, less than or equal to about 250 nm, less than or equal to about 100 nm, less than or equal to about 75 nm, less than or equal to about 50 nm, less than or equal to about 40 nm, less than or equal to about 35 nm, less than or equal to about 30 nm, less than or equal to about 25 nm, less than or equal to about 20 nm, less than or equal to about 15 nm, or less than or equal to about 5 nm.
• a largest cross-sectional dimension or, sometimes, a smallest cross-section dimension
• the spherical HDL-NP has a diameter of about 5-30 nm, 5-25 nm, 5-22 nm, 5-20 nm, 5-15 nm, 5- 14 nm, 5-13 nm, 5-12 nm, 5-11 nm, 5-10 nm, 8-15 nm, 8-14 nm, 8-13 nm, 8-12 nm, 8-11 nm, 8-10 nm, 10-12 nm, or 10 nm.
• the HDL-like nanoparticles of the present disclosure can have a core with a largest cross-sectional dimension (or, sometimes, a smallest cross-section dimension) of, for example, less than or equal to about 300 nm, less than or equal to about 250 nm, less than or equal to about 100 nm, less than or equal to about 75 nm, less than or equal to about 50 nm, less than or equal to about 40 nm, less than or equal to about 35 nm, less than or equal to about 30 nm, less than or equal to about 25 nm, less than or equal to about 20 nm, less than or equal to about 15 nm, or less than or equal to about 5 nm.
• a largest cross-sectional dimension or, sometimes, a smallest cross-section dimension
• the core has an aspect ratio of greater than about 1:1, greater than 3:1, or greater than 5:1.
• aspect ratio refers to the ratio of a length or a width, where length and width are measured perpendicular to one another, and the length refers to the longest linearly measured dimension.
• the shell has a zeta potential closer to human HDL than a synthetic HDL nanoparticle with an inorganic core.
• the spherical HDL-NP has a zeta potential closer to human HDL than a synthetic HDL nanoparticle with a gold core.
• the spherical HDL-NP has a zeta potential of -16-26 mV.
• the zeta potential of the HDL-like nanoparticles is about -20 millivolts (mV).
• the zeta potential of the HDL-like nanoparticles is selected from a group consisting of -10 mV, -12 mV, -14 mV, -16 mV, -18 mV, -20 mV, -22 mV, -24 mV, -26 mV, and -30 mV.
• the zeta potential of the HDL-like nanoparticles is greater than -20 mV.
• the zeta potential is less than -20 mV.
• the zeta potential is that of human HDL. Zeta potential may be assessed using methods known in the art, including the methods disclosed herein.
• the HDL-like nanoparticles of the present disclosure do not include a peptide-based scaffold material.
• compositions and methods of the present disclosure represent a significant step forward, toward a genuine synthetic functional mimic of spherical HDL.
• the HDL-like nanoparticles (also referred to as synthetic particles or synthetic HDL particle or HDL-NP) of the present disclosure are capable of executing salient HDL functions including cholesterol efflux from macrophages (or from lipid-laden macrophages), cholesterol delivery to hepatocytes, supporting lecithin:cholesterol acyltransferase activity, and suppression of inflammation.
• the spherical HDL-NP has a more efficient cholesterol transport capacity than that of apolipoprotein or a synthetic HDL nanoparticle with a gold core.
• Cholesterol transport capacity may be assessed using in vitro assays such as the assays described herein.
• the cholesterol transport capacity may be assessed relative to a standard known value of cholesterol transport or relative to a negative control such a synthetic HDL-NP particle having a gold core or APO-A1 or relative to a positive control such as naturally occurring HDL-NP.
• compositions and methods of the present disclosure can be used in applications including, but not limited to, cancer therapy, inflammatory disease,
• cardiovascular disease therapy bone disease therapy, and immune disease therapy.
• HDL-like nanoparticles of the present disclosure due to their novel elements, which include a soft core mimic of spherical HDL, represent strong candidates for substantially enhanced therapeutic effects of synthetic HDLs in the clinic.
• Inflammation promoted by macrophage NF-kB activity is a hall-mark of ASCVD that drives disease progression and increases morbidity and mortality.
• a human monocyte reporter cell line THP1-Dual
• lipopolysaccharide to stimulate NF-kB activity prior to treatment with LC HDL NPs or apoA-1 controls. While apoA-1 alone exhibited no capacity to reduce NF-kB activity, both PL 4 HDL NPs and DNA- PL 4 HDL NPs reduced NF-kB activity in a dose dependent manner, by 31% and 16% respectively at protein concentrations of 150 nM (FIG. 5B).
• the invention is a method for treating cardiovascular disease.
• Cardiovascular disease is a vascular endothelial cell dysfunction and certain symptoms begin, including as conventional or above the heart and vascular system-on, atherosclerosis, hypertension, gojihyeol, coronary heart disease (heart attack), cerebrovascular diseases (stroke, dementia), peripheral vascular disease, arrhythmia, heart failure, congestive heart disease, cardiac disease and for at least the name of the heart and blood vessels, including, but not limited thereto.
• Some of the main factors of cardiovascular disease include, but they are not limited to expression of genetic factors, lifestyle habits, and complications of diabetes
• Atherosclerosis is the mechanism by which arteries that supply the heart muscle with oxygen-rich blood become progressively narrowed with cholesterol-rich plaque.
• neointimal hyperplasia whereby smooth muscle cells grow through the stent, re-narrowing the vessel (i.e. restenosis).
• Drug-eluting stents (DES) coated with drugs to prevent cell growth, do reduce vessel restenosis by limiting the proliferation of smooth muscle cells; however, the drugs also inhibit the growth of endothelial cells that line naturally line blood vessels and prevent clot formation. Patients must remain on potent medications to prevent blood clots, predisposing them to life-threatening bleeding
• cardiovascular diseases included, but are not limited to, arteriosclerosis, coronary heart disease, ischemia, endothelium dysfunction, in particular those dysfunctions affecting blood vessel elasticity, restenosis, thrombosis, angina, high blood pressure, cardiomyopathy, hypertensive heart disease, heart failure, divermonale, cardiac
• dysrhythmias endocarditis, inflammatory cardiomegaly, myocarditis, myocardial infarction, valvular heart disease, stroke and cerebrovascular disease, aortic valve stenosis, congestive heart failure, and peripheral arterial disease
• the compositions of the invention will restore and/or improve cardiovascular parameters to normal ranges in a subject diagnosed with or at risk of a cardiovascular disease.
• Normal ranges of cardiovascular parameters include but are not limited to, an end-diastolic volume (EDV) from about 65-240 mL, an end-systolic volume (ESV) from about 16-143m L, a stroke volume from about 55-100 mL, an ejection fraction from about 55-70%, a heart rate from about 60-100 bpm, and/or cardiac output of about 4.0- 8.0 L/min.
• HDL-NP spherical high-density lipoprotein nanoparticle
• compositions of the invention may also be used to treat inflammatory diseases.
• inflammatory diseases include, but are not limited to acne vulgaris, asthma, autoimmune diseases (e.g., acute disseminated encephalomyelitis (ADEM), Addison's disease, agammaglbulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmunehemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Balo concentric sclerosis, Be
• encephalopathy Hashimoto's thyroiditis, Henoch-Schonlein purpura, herpes gestationis, hidradenitis suppurativa, Hughes-Stovin syndrome, hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic
• thrombocytopenic purpura IgA nephropathy, inclusion body myositis, chronic inflammatory demyelinating polyneuropathy, interstitial cystitis, juvenile idiopathic arthritis, Kawasaki's disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease, lupus erythematosus, Majeed syndrome, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, morphea, Mucha- Habermann disease, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica, neuromyotonia, ocular cicatricial pemphigoid, opsoclonus myoclonus syndrome, Ord's thyroiditis, palindromic rheumatism, PANDAS, paraneoplastic cerebellar degeneration, paroxysmal
• encephalomyelitis POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatic, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen's encephalitis, raynaud phenomenon, relapsing polychondritis, Reiter's syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatic fever, Schnitzler syndrome, scleritis, scleroderma, serum sickness, Sjogren's syndrome, spondyloarthropathy, stiff person syndrome, subacute bacterial endocarditis, Susac's syndrome, Sweet's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis, thrombocytopenia
• the invention is a method for treating a cancer, comprising
• HDL-NP spherical high-density lipoprotein nanoparticle
• Cancers are generally characterized by unregulated cell growth, formation of malignant tumors, and invasion to nearby parts of the body. Cancers may also spread to more distant parts of the body through the lymphatic system or bloodstream. Cancers may be a result of gene damage due to tobacco use, certain infections, radiation, lack of physical activity, obesity, and/or environmental pollutants. Cancers may also be a result of existing genetic faults within cells to cause diseases due to genetic heredity. Screenings may be used to detect cancers before any noticeable symptoms appear and treatment may be given to those who are at higher risks of developing cancers (e.g., people with a family history of cancers).
• screening techniques for cancer include but are not limited to physical examination, blood or urine tests, medical imaging, and/or genetic testing.
• Non-limiting examples of cancers include: bladder cancer, breast cancer, colon and rectal cancer, endometrial cancer, kidney or renal cell cancer, leukemia, lung cancer, melanoma, Non- Hodgkin lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, stomach cancer, wasting disease, and thyroid cancer.
• nanostructures described herein are useful for treating a cancer that expresses or overexpresses scavenger receptor class B type I (SR-BI).
• SR-BI scavenger receptor class B type I
• Non-limiting examples of cancers that express or overexpress SR-BI include human prostate cancer, breast cancer, and renal cell carcinoma. Additional non-limiting examples of cancers and cancer cell lines that overexpress SR-BI are listed in Rajora et al. Front Pharmacol. (2016) 7:326.
• the term“overexpression” or“increased expression,” refers to an increased level of expression of a given gene product in a given cell, cell type or cell state, as compared to a reference cell, for example, a non-cancer cell or a cancer cell that does not overexpress SR-BI.
• the cancer cell expresses any level of SR- BI.
• the nanostructures are also useful for treating and preventing autoimmune disease or disorder.
• Autoimmune disease or disorder is a class of diseases in which an subject’s own antibodies react with host tissue or in which immune effector T cells are autoreactive to endogenous self peptides and cause destruction of tissue.
• an immune response is mounted against a subject’s own antigens, referred to as self antigens.
• Autoimmune diseases or disorders include, but are not limited to, rheumatoid arthritis, Crohn's disease, multiple sclerosis, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto’s thyroiditis, Goodpasture’s syndrome, pemphigus (e.g., pemphigus vulgaris), Grave’s disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis, pernicious anemia, idiopathic Addison’s disease, autoimmune-associated infertility, glomerulonephritis (e.g., crescentic glomerulonephritis, proliferative glomerulonephritis), bullous pemphigoid, Sjögren’s syndrome, insulin resistance, and autoimmune diabetes mellitus.
• compositions can be administered to a patient with a clinically determined predisposition or increased
• compositions of the invention can be administered to the patient (e.g., a human) in an amount sufficient to delay, reduce, or preferably prevent the onset of the clinical disease.
• compositions are administered to a patient (e.g., a human) already suffering from a cardiovascular disease, hyperproliferative diseases (e.g., cancer) , an inflammatory disease, and other pathological conditions associated with cholesterol, in an amount sufficient to cure or at least partially arrest the symptoms of the condition and its complications.
• a therapeutically effective dose an amount of a compound sufficient to substantially improve some symptom associated with a disease or a medical condition.
• a therapeutically effective amount of an agent or composition is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, or the disease or condition symptoms are ameliorated, or the term of the disease or condition is changed or, for example, is less severe or recovery is accelerated in an individual.
• the disclosure relates to a method of reducing NF-kB activity, comprising administering to a subject an effective amount of a composition comprising any of the spherical HDL-NP of the instant disclosure.
• a“subject” or a“patient” refers to any mammal (e.g., a human).
• subjects or patients include a human, a non-human primate, a cow, a horse, a pig, a sheep, a goat, a dog, a cat or a rodent such as a mouse, a rat, a hamster, or a guinea pig.
• the invention is directed toward use with humans.
• the HDL-like nanoparticles of the present disclosure are in a pharmaceutical compositions.
• These“pharmaceutical compositions” or“pharmaceutically acceptable” compositions may comprise a therapeutically effective amount of one or more of the structures described herein, formulated together with one or more pharmaceutically acceptable carriers, additives, and/or diluents.
• the pharmaceutical compositions described herein may be useful for treating sepsis or other related diseases. It should be understood that any suitable structures described herein can be used in such pharmaceutical compositions, including those described in connection with the figures.
• compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream or foam; sublingually;
• oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption,
• phrases“pharmaceutically acceptable” is employed herein to refer to those structures, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• phrases“pharmaceutically-acceptable carrier” as used herein means a
• composition or vehicle such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
• a liquid or solid filler such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
• Each carrier must be“acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
• materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ring
• wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
• antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid,
• EDTA ethylenediamine tetraacetic acid
• sorbitol sorbitol
• tartaric acid tartaric acid
• phosphoric acid and the like.
• the structures described herein may be orally administered, parenterally
• a structure or pharmaceutical preparation is administered orally. In other embodiments, the structure or pharmaceutical preparation is administered intravenously.
• Alternative routes of administration include sublingual, intramuscular, and transdermal administrations.
• the disclosure relates to a method for making a spherical high-density lipoprotein nanoparticle (HDL-NP) comprising, preparing a ssDNA-phospholipid conjugate (ssDNA-PL), preparing a tetrahedral small molecule-DNA hybrid (SMDH 4 ), wherein the ssDNA-PL and SMDH4 have complementary DNA sequences, incubating the ssDNA-PL and SMDH 4 such that the complementary DNA sequences bases pair with one another to form a DNA-PL core, and adding to the DNA-PL core phospholipid liposomes and apolipoprotein to produce the spherical HDL-NP.
• HDL-NP spherical high-density lipoprotein nanoparticle
• the ssDNA-PL comprises an oligonucleotide of at least 9 nucleotides. In some embodiments, the oligonucleotide is 6-16 nucleotides in length. In some embodiments, the oligonucleotide comprises SEQ ID NO: 1. In some embodiments, the oligonucleotide is 18 nucleotides in length. In some embodiments, the oligonucleotide comprises SEQ ID NO: 2.
• the SMDH 4 comprises an oligonucleotide of at least 9 nucleotides. In some embodiments, the oligonucleotide is 9-15 nucleotides in length. In some embodiments, the oligonucleotide comprises SEQ ID NO: 3. In some embodiments, the oligonucleotide is 18 nucleotides in length. In some embodiments, the oligonucleotide comprises SEQ ID NO: 4.
• the apolipoprotein of any of the methods disclosed herein is apolipoprotein A-I.
• DNA-PL 4 cores were synthesized in a two-step fashion (see Synthesis section herein).
• Liposomes were prepared from 1,2-dipalmitoyl phosphatidyl choline (DPPC) by re-suspending a thin film (1 mM DPPC) in phosphate-buffered saline (PBS: 10 mM
• PL4 core scaffolds were then prepared as thin films, while improved aqueous solubility enabled beginning with DNA- PL 4 core scaffolds already in aqueous buffer.
• the other NP components were then added to the core scaffolds: first, DPPC liposomes, and second, apoA-1, each pre-suspended in PBS. The mixture was subjected to three rounds of sonication, allowed to relax on ice, and then filtered and concentrated using a 50 kDa MWCO spin column, with alternating benchtop centrifugation to remove aggregates (FIG.2C).
• PL 4 HDL NPs and DNA-PL 4 HDL NPs were found to closely resemble human HDLs in secondary structure, while Au HDL NPs by contrast, exhibited excess alpha helicity and substantially less beta sheet and turn content (Table 1) (FIG.2E).
• FIG.2E To further characterize LC HDL NP structure, the oligomerization state of apoA-1 was investigated by cross-linking LC HDL NPs with bissulfosuccinimidyl suberate followed by immunoblot. 18 ApoA-1 interaction with LC cores was found to induce stable oligomer formation (FIG.16).
• LC HDL NPs were found to closely mirror HDL 2 and HDL 3 with respect to the relative mass contributions of each component, with the exception that LC HDL NPs do not contain cholesterol or cholesteryl esters (FIG.2D; Table 4).
• LC HDL NPs Complementary biological assays were performed to investigate whether LC HDL NPs could recapitulate salient HDL functions.
• a critical property for HDL mimicry is the ability to efflux cholesterol from lipid-laden macrophages.
• Efflux efficiency of LC HDL NPs was examined using an in vitro radiolabeled cholesterol efflux assay (see Oligomerization Assay section). 24 Briefly, J774 macrophages were loaded with tritium-labeled cholesterol (H 3 -chol), cultured with cAMP to upregulate cholesterol efflux receptors, and then treated with nanoparticles or controls for 4 hours. The media supernatant was subjected to liquid scintillation counting to quantify percent effluxed H 3 -chol.
• LC HDL NPs In addition to efflux, cholesterol delivery is a specialized function carried out in the native setting by mature HDLs.
• cultured hepatocytes HepG2
• NBD-chol fluorescent cholesterol
• LC HDL NPs facilitated efficient delivery of NBD-chol in only 30 min (FIG.4), with 23% of PL4 HDL NP-treated cells being NBD-positive compared to 5.7% and 6.8% for Au HDL NP and apoA-1 respectively.
• lipids 1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-dibenzocyclooctyl (DBCO PE) and 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC)– and fluorescent cholesterol (22-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol- 4-yl)Amino)-23,24-Bisnor-5-C noir-3b-Ol ((NBD)-cholesterol)),were obtained from Avanti Polar Lipids (Alabaster, AL).
• DBCO PE 1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-dibenzocyclooctyl
• DPPC 1,2-dipalmitoyl-sn-glycero-3- phosphocholine
• fluorescent cholesterol 22-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-
• ApoA-I protein was obtained from MyBioSource (San Diego, CA). All chemicals were used without further purification. Ultrapure deionized (DI) H 2 O (18.2 MW ⁇ cm resistivity) was obtained from a Millipore system (Milli-Q Biocel). Tetrakis(4- azidophenyl)methane 1 and lipid phosphoramidite 2 were synthesized according to previously published procedures.
• DNA products were purified and analyzed on an Agilent 1100 HPLC equipped with reverse-phase (RP) semi-preparative (Dynamax, 250 ⁇ 10 mm, Microsorb 300 ⁇ /10 mm/C18, Agilent # R083213C10) and analytical (Dynamax, 100 ⁇ 4.6 mm, Microsorb 100 ⁇ /3 mm/C18, Agilent # R0080200E3) columns, respectively.
• RP reverse-phase
• Thermomixer R 5355 Eppendorf AG North America, Hauppauge, NY
• Negative stain transmission electron microscopy (TEM) images were acquired using a FEI Tecnai Spirit TEM operating at 120 kV, using 300-mesh carbon-coated copper grids (Electron Microscopy Services). Confocal imaging was carried out with a Nikon A1R Spectral microscope. Flow cytometry was performed using a BD LSRFortessa cell analyzer. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometric data were collected as negative ions using the linear mode on a Bruker AutoFlex III MALDI-ToF mass spectrometer (Bruker Daltonics, Billerica, MA).
• MALDI-ToF Matrix-assisted laser desorption/ionization time-of-flight
• the instrument was equipped with SmartbeamTM laser technology operated at 30-40% power with a sampling speed of 10 Hz. One thousand scans were averaged for each mass spectrum. Data from Agilent HPLC and Bruker MALDI-ToF instruments were processed using MestreNova software version 8.1.1-11591. Synthesis
• SMDHs Small molecule-DNA hybrids with 9 and 18 mer DNA arms (9-SMDH 4 (SEQ ID NO: 1) and 18-SMDH 4 ’s (SEQ ID NO: 2), respectively) were synthesized and purified according to a previously published procedure 3 and DNA sequences used in this study are listed in the Table 2.
• TEAA(aq):MeCN over 35 min (at a ramp of +1 vol% MeCN/min), with a flow rate of 1 mL/min. Then, the whole sample was subjected to purification using a semi-preparative RP- HPLC column (see Materials and Instrumentation section herein) and a gradient method beginning with 95:5 v/v 0.1 M TEAA (aq):MeCN and increasing to 60:40 v/v 0.1 M
• the CPG beads were placed in a 1 mmol synthesis column and 3 ⁇ - phosphoramidites (Glen Research, dA-CE phosphoramidite # 10-1000-C5, Ac-dC-CE phosphoramidite # 10-1015-C5, dmf-dG-CE phosphoramidite # 10-1029-C5, dT-CE phosphoramidite # 10-1030-C5) were then added using the standard 1 mmol protocol on an Expedite 8909 synthesizer to make the CPG-3 ⁇ -ssDNA (see Table 2 for sequences).
• a lipid phosphoramidite was added to the 5 ⁇ end of ssDNA strand and then the beads were dried with a stream of dried nitrogen gas and placed in a vial containing aqueous fresh AMA solution (1 mL of a 1:1 v/v mixture of 30 wt % aqueous ammonium hydroxide solution and 40 wt % aqueous methylamine solution).
• the vial was then capped and heated at 65 °C for 15 min to cleave DNA-lipid conjugates from the solid supports.
• the ammonia and methyl amine byproducts were then removed by passing a stream of dry nitrogen gas over the content of the vial until the characteristic ammonia smell disappears.
• the extract was combined with the initial solution of crude DNA-lipid conjugates (affording a total volume of 0.4 mL at the end) and filtered through a 0.45 mm nylon syringe filter (Acrodisc® 13 mm syringe filter # PN 4426T).
• the collected sample of crude product was subjected to purification using analytical RP-HPLC (FIGs.8-9) and a gradient method beginning with 95:5 v/v 0.1 M TEAA
• Tetrakis(4-azidophenyl)methane was synthesized according to a previously published procedure 1 .
• PL4 core materials were synthesized by copper-free click chemistry conjugation of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl (DBCO PE) with a tetrahedral small molecule core (tetrakis(4-azidophenyl)methane) (FIG.11).
• DBCO PE 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl
• FOG.11 tetrahedral small molecule core
• Nanoparticle assembly was carried out by first preparing 1,2-dipalmitoyl-sn-glycero- 3-phosphocholine (DPPC) liposomes.
• DPPC 1,2-dipalmitoyl-sn-glycero- 3-phosphocholine
• DPPC 1,2-dipalmitoyl-sn-glycero- 3-phosphocholine
• a thin film was then generated by evaporating the solvent with N2 gas. The film was further dried under reduced pressure in a desiccator for > 2 h.
• PBS phosphate-buffered saline
• the PL4 core scaffold was prepared by generating a thin film from a 0.1wt% solution in DMF, in the same manner described above for DPPC.
• the non- scaffold nanoparticle components were then added sequentially to the core scaffolds.
• lipid conjugate core scaffold either PL4 or DNA-PL4
• apoA-1 lipid conjugate core scaffold
• the suspensions were then diluted in PBS to a final core concentration of 10 uM in PBS.
• the suspensions were subjected to three rounds of alternating bath sonication (90 s ON, 30 s OFF) and vortexing, and were allowed to relax on ice for > 30 min.
• the particles were then filtered and concentrated using 0.5 mL 50 kDa MWCO spin columns (MilliPore).
• the columns were first rinsed in PBS for 10 min at 10,000 x g at 4°C, prior to three rounds of spin filtration of the particles under the same conditions. 500 ul of PBS were added to the column after each spin, such that the solvent of each particle suspension was PBS after spin filtration, with TAMg buffer removed from solution for the DNA-PL4 HDL NPs. After each round of filtration, the particles were centrifuged by desktop centrifugation to remove any aggregates.
• Control samples were prepared to investigate the LC HDL NPs assembly process, and its dependence on the lipid conjugated core scaffold and apoA-1.
• Core + scaffold w/o apoA-1 controls were prepared by making DPPC liposomes as described above (see Synthesis section herein). DPPC liposomes were then added to core scaffolds at the same concentration and molar ratio as in LC HDL NPs, with PL 4 scaffolds thin filmed and DNA-PL 4 scaffolds in aqueous buffer (PBS). The mixtures were then subjected to three rounds of alternating sonication and vortexing, and were filtered and concentrated through 50 kDa MWCO spin columns as above. apoA-1 and DPPC alone controls were assembled in an identical manner without core scaffolds. Negative Stain Transmission Electron Microscopy
• Circular dichroism was performed using a JASCO J-815 CD spectrometer. All samples were diluted to 75 ug/mL protein in distilled water. Spectra were derived from three accumulations of a single sample. Secondary structure data was acquired using CDPro software package running the analysis algorithm, CONTIN. Three distinct analyses were performed using different soluble protein reference sets. The values reported in Table 1 reflect the mean ⁇ SEM results of these three analyses. Composition Characterization
• BCA bovine serum albumin
• Phospholipid quantification Phospholipid content of nanoparticles and native HDL was quantified using a colorimetric Phospholipid Assay Kit (Sigma-Aldrich) per the manufacturer’s instructions. Briefly, a phospholipid standard curve was prepared from 15 mM to 200 mM via serial dilution. Samples were serially diluted in PBS to obtain one or more dilutions within the range of the standard curve. Samples and standards were then incubated with the enzyme reaction mixture for 30 min at rt and then absorbance at 572 nm was determined using a Synergy plate reader. c. Cholesterol, Cholesteryl ester, and Core quantifications.
• Cholesterol and cholesteryl ester concentrations were determined using an Amplex Red Cholesterol Assay (ThermoFisher) per the manufacturer’s instructions. Briefly, cholesterol standard curves were prepared in kit-provided aqueous buffer from 8 ug/mL to 125 ng/mL. Two sets of samples were prepared in triplicate and diluted into 1x Reaction Buffer. Two reaction mixtures were then prepared containing hydrogen peroxide, resorufin, and the enzymes horseradish peroxidase, cholesterol oxidase, with or without cholesterol esterase to enable quantification of both free cholesterol and cholesteryl esters.
• DNA-PL4 HDL NPs was determined by measuring absorbance at 260 nm via UV-Vis spectroscopy. Because PL4 core concentration could not be determined directly, the core concentration was estimated using the experimentally determined core:protein molar ratio obtained for DNA-PL 4 particles.
• LC HDL NPs were prepared as described above and diluted to 50 mg/mL protein in PBS.
• BS3 cross-linking agent was then added to LC HDL NPs for a final BS3 concentration of 2.5 mM, and the reaction was allowed to proceed for 30 min at room temperature.
• lipid-free pure apoA-1 MyBioSource was dialyzed in PBS, and then subjected to cross-linking with BS3 (0.25 mM) at elevated protein
• PVDF polyvinylidine fluoride
• Efflux assays For efflux experiments, we used the gold standard assay in the field, efflux of tritium-labeled cholesterol (H 3 -chol) from J774 macrophages. J774 macrophages were cultured for at least two passages prior to seeding at 150,000 cells per well in 24 well plates in RPMI, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (PenStrep) on Day 1. Ethanol stocks of H 3 -chol were handled under sterile conditions. H 3 -chol stocks were evaporated, redissolved in 1 mL of ethanol and incubated at 37 °C for 60 min.
• FBS fetal bovine serum
• PenStrep penicillin-streptomycin
• Efflux samples containing nanoparticles and controls were prepared in serum-free media (MEM, 25 mM HEPES, 1% PenStrep) to reduce non-specific H 3 -chol efflux by serum cholesterol carriers. Cells were washed twice in MEM, 25 mM HEPES and efflux media was added to each well according to the treatment regimen. Efflux was allowed to proceed for 4 h. Efflux media was then removed, vacuum filtered, and added to 3 mL UltimaGold scintillation fluid for scintillation counting.
• MEM serum-free media
• Tandem efflux-influx assays For efflux-influx experiments, efflux was conducted identically as above. Hepatocytes (HepG2 cells) were plated at 100,000 cells/well in DMEM, 10% FBS, 1% PenStrep in 24 well plates on Day 3. On Day 4, immediately after 4 h of efflux, HepG2 cells were washed twice in serum-free MEM, 25 mM HEPES, and then efflux media from J774 macrophages was removed and added directly to HepG2 cells. Influx was allowed to proceed for 4 h prior to harvesting. Influx media supernatant was then collected and processed identically as efflux media described above.
• H 3 -chol was then extracted from HepG2 cells by incubating in 500 mL isopropanol for 4 hours at rt, sealed in Parafilm. H 3 - chol-containing isopropanol was then collected, evaporated, and redissolved in xylene prior to addition to UltimaGold for scintillation counting. NBD-Cholesterol Delivery Experiments
• LC HDL NPs and Au HDL NPs (250 nM) suspended in PBS were incubated with LCAT (10 nM) and free cholesterol (100 mg/mL) in PBS at 37 °C for 15 h in a ThermoMixer shaking at 300 rpm. Samples were then centrifuged three times at 10,000 g for 10 min through a 50 kDa spin column to remove excess unbound cholesterol. Samples were then diluted 100 times in 1 ⁇ Reaction Buffer prior to quantification with Amplex Red Cholesterol Assay (Thermo Fisher). Amplex Red Cholesterol Assay was performed per manufacturer’s instructions.
• a cholesterol standard curve was prepared by serially diluting cholesterol in kit- provided aqueous buffer from 8 ug/mL to 125 ng/mL and adding 50 ul to a 96-well black- bottom microplate.50 ml of sample were also added to each well of the microplate in triplicate. Two sets of Amplex Red reaction mix were prepared, with and without cholesterol esterase.50 ml of Amplex Red reaction mix were then added to each well. Microplates were incubated at 37 °C for 1 h or until fluorescent signal began to decline. NF-kB Activity Assays
• THP1-Dual cells were used in conjunction with a QUANTI- Blue secreted embryonic alkaline phosphatase (SEAP) detection kit (Invivogen).
• SEAP embryonic alkaline phosphatase
• THP1-Dual cells were cultured in suspension in RPMI with 10% FBS and passaged at least twice prior to use in experiments.
• THP1-Dual cells were plated at 100,000 cells per well in 96 well plates. Lipopolysaccharide (LPS) (5 ng/mL) was used to stimulate NF-kB activity.
• Experimental wells were treated with LPS 1 h prior to addition of nanoparticles or controls. Cells were then incubated with particles or controls for 24 h prior to detection.
• LPS Lipopolysaccharide
• QUANTI-Blue solution was prepared by dissolving the contents of the packet in endotoxin-free water and incubating at 37 °C for 30 min.
• QUANTI-Blue solution 180 mL was added to each well in 96-well plates.
• THP1-Dual cells supernatant (20 mL) was then added to QUANTI-Blue solution, and the plate was incubated at 37 °C for 2-4 h.
• SEAP levels were the quantified by detecting absorbance at 650 nm using a Synergy plate reader. Table 1: Structural Analysis of Native and Synthetic HDLs via Circular Dichroism
• Table 2 List of DNA Sequences of SMDH4 DNA Arms and DNA-Lipid Congugates
• DNA sequences of SMDH 4 DNA arms are complementary to those of DNA-lipid conjugates (i.e., 9-SMDH4 (SEQ ID NO: 1) to 9-DNA-lipid (SEQ ID NO: 3) and 18-SMDH4 (SEQ ID NO: 2) to 18-DNA-lipid (SEQ ID NO: 4)).
• Table 3 Dynamic Ligh Scattering Results for LC HDL NPs and Controls
• inventive embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.
• inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein.
• references to“A and/or B”, when used in conjunction with open-ended language such as“comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
• “or” should be understood to have the same meaning as“and/or” as defined above.
• “or” or“and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as“only one of” or“exactly one of,” or, when used in the claims,“consisting of,” will refer to the inclusion of exactly one element of a number or list of elements.
• the phrase“at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
• This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase“at least one” refers, whether related or unrelated to those elements specifically identified.
• “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

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