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WO2016004168A1 - Nanoparticules sphériques en tant qu'agents antibactériens - Google Patents

Nanoparticules sphériques en tant qu'agents antibactériens Download PDF

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Publication number
WO2016004168A1
WO2016004168A1 PCT/US2015/038771 US2015038771W WO2016004168A1 WO 2016004168 A1 WO2016004168 A1 WO 2016004168A1 US 2015038771 W US2015038771 W US 2015038771W WO 2016004168 A1 WO2016004168 A1 WO 2016004168A1
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Prior art keywords
nanostructure
oligonucleotides
core
oligonucleotide
carrier
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Ceased
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PCT/US2015/038771
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English (en)
Inventor
Aleksandar Filip Radovic-Moreno
Askriti GOEL
Clayton RISCHE
Christopher C. MADER
Richard Kang
Subbarao NALLAGATLA
Sergei Gryaznov
Pinal PATEL
Weston Daniel
David A. Giljohann
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Exicure Inc
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Aurasense Therapeutics Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Antibiotics are agents that have the ability to either eliminate or inhibit the growth of microorganisms. Antibiotic development began as early as the 1920s and is still evolving due to rapid appearance of resistant strains. Resistance to antibiotics can be due to either spontaneous or induced gene mutations or acquisition of resistant genes from another bacterial species by horizontal gene transfer. Antibiotic resistant genes are, however, not limited to bacterial genomes. They can be transferred horizontally from one organism to another and even across species. Emergence of antibiotic resistance has been prevalent since only a few years after antibiotic development began. Once any antibiotic was widely used, resistant strains that were capable of inactivating the drug became prevalent. As a result, with evolution of antibiotics and their widespread use, resistance to antibiotics also became more common.
  • Antibiotic resistance can be explained by the following mechanisms- (1) inactivation of the antibiotic, (2) development of a resistant metabolic pathway, (3) alteration of the receptor for the antibiotic drug or/and (4) decrease in the amount of antibiotic that is able to reach the receptor by altering drug uptake. For example, tetracycline resistance, fosfomycin resistance, aminoglycoside resistance are all developed due to poor drug uptake. In addition, drug efflux, a mechanism that cycles toxins out of bacterial cells, often contributes to antibiotic resistance.
  • the invention in an aspect is a nano structure which includes a core, one or more carrier oligonucleotides positioned on the exterior of the core, and a therapeutic
  • the carrier oligonucleotides form an oligonucleotide shell on the exterior of the core and wherein the therapeutic pharmacophore is hybridized to the carrier oligonucleotides.
  • oligonucleotides and at least one other molecule.
  • the oligonucleotide shell is comprised entirely of oligonucleotides.
  • the oligonucleotides are comprised of single- stranded or double- stranded DNA oligonucleotides. In other embodiments the oligonucleotides are comprised of single- stranded or double- stranded RNA oligonucleotides. In other
  • the oligonucleotides are comprised of chimeric RNA-DNA oligonucleotides.
  • the carrier oligonucleotides are comprised of RNA-DNA or DNA- RNA oligonucleotide heteroduplexes.
  • the oligonucleotides are comprised of combinations of single- stranded or double- stranded DNA, RNA, or chimeric RNA-DNA oligonucleotides.
  • the oligonucleotides have structurally and nucleotide sequence identical oligonucleotides. In some embodiments the oligonucleotides have at least two structurally and nucleotide sequence different oligonucleotides.
  • the oligonucleotides have 2-10 different nucleotide sequences.
  • At least one of the carrier oligonucleotides is a modified oligonucleotide. In some embodiments at least 50% of the carrier oligonucleotides are modified oligonucleotides. In yet other embodiments all of the carrier oligonucleotides are modified oligonucleotides. In some embodiments the oligonucleotides have at least one phosphorothioate linkage. In other embodiments the oligonucleotides do not have a phosphorothioate linkage.
  • the nanostructure comprises a liposomal core having a lipid bilayer.
  • at least one oligonucleotide has its 5'- terminus exposed to the outside surface of the nano structure.
  • all of the oligonucleotides have their 5'- terminus exposed to the outside surface of the nano structure.
  • at least one carrier oligonucleotide has its 3'- terminus exposed to the outside surface of the nano structure. All of the carrier oligonucleotides have their 3'- terminus exposed to the outside surface of the nanostructure in other embodiments.
  • the oligonucleotides are directly linked to the core. In some embodiments the oligonucleotides are indirectly linked to the core through a linker. In other embodiments the oligonucleotides are indirectly linked to the core through more than one linker.
  • the carrier oligonucleotides may also be directly linked to the core. In some embodiments at least one carrier oligonucleotide is positioned laterally on the surface of the nanostructure. In other embodiments all of the carrier oligonucleotides are positioned laterally on the surface of the nanostructure.
  • the oligonucleotides are reversibly or irreversibly coupled to the core.
  • the carrier oligonucleotides are irreversibly coupled to the core.
  • the therapeutic pharmacophore is reversibly coupled to the core or carrier oligonucleotide.
  • the linker in some embodiments is a chemical structure containing one or more thiol groups, including various chain length alkane thiols, cyclic dithiol, lipoic acid, PEG-thiol, and other thiol group containing linkers.
  • the carrier oligonucleotides are linked to a liposomal core and the linker is one or more of the following linkers: tocopherols, sphingolipids such as sphingosine, sphingosine phosphate, methylated sphingosines and sphinganines, ceramides, ceramide phosphates, 1-0 acyl ceramides, dihydroceramides, 2- hydroxy ceramides, sphingomyelin, glycosylated sphingolipids, sulfatides, gangliosides, phospho sphingolipids, and phytosphingosines of various lengths and saturation states and their derivatives, phospholipids such as phosphatidylcholines, lysophosphatidylcholines, phosphatidic acids, lysophosphatidic acids, cyclic LPA, phosphatidylethanolamines, lysophosphatidylethanolamines, phospho
  • the oligonucleotides comprise 2-1,000 oligonucleotides.
  • the core in some embodiments is a solid or hollow core and may be inert, paramagnetic or supramagnetic.
  • the solid core is comprised of noble metals, including gold and silver, transition metals including iron and cobalt, metal oxides including silica, polymers or combinations thereof.
  • the core is a polymeric core and wherein the polymeric core is comprised of amphiphilic block copolymers, hydrophobic polymers including polystyrene, poly(lactic acid), poly(lactic co- glycolic acid), poly(glycolic acid), poly(caprolactone) and other biocompatible polymers.
  • the liposomal core is comprised of one type of lipid. In some embodiments the liposomal core is comprised of 2-10 different lipids.
  • the liposomal core in some embodiments is comprised of one or more lipids selected from: sphingolipids such as sphingosine, sphingosine phosphate, methylated sphingosines and sphinganines, ceramides, ceramide phosphates, 1-0 acyl ceramides, dihydroceramides, 2-hydroxy ceramides, sphingomyelin, glycosylated sphingolipids, sulfatides, gangliosides, phospho sphingolipids, and phytosphingosines of various lengths and saturation states and their derivatives, phospholipids such as phosphatidylcholines, lysophosphatidylcholines, phosphatidic acids, lysophosphatidic acids, cyclic LPA, phosphatidylethanolamines, lysophosphatidylethanolamines, phosphatidylglycerols, lysophosphatidy
  • the therapeutic pharmacophore may be a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the nanostructure further comprises an active agent.
  • the active agent is mixed together with the nanostructure.
  • the active agent is linked directly to the oligonucleotide shell and/or the therapeutic pharmacophore. In some embodiments the active agent is linked indirectly to the oligonucleotide shell and/or the therapeutic pharmacophore through a linker. In other embodiments the active agent is linked directly to the core. In yet another embodiment the active agent is linked indirectly to the core through a linker. In another embodiment an active agent -oligonucleotide conjugate is linked to the core through oligonucleotide hybridization. In some embodiments the active agent is associated with the core by being embedded within the core, optionally the liposomal core. In some embodiments the active agent is encapsulated within the liposomal core in an inner aqueous layer. In other embodiments the active agent is attached non-covalently to the oligonucleotide of the oligonucleotide shell.
  • the nanostructure is a self-assembling nanostructure.
  • the therapeutic pharmacophore is an RNA oligonucleotide, which is optionally an siRNA.
  • the nanostructure may include an antibacterial agent conjugated to the antisense oligonucleotide.
  • the therapeutic pharmacophore is an oligonucleotide having at least a region that is antisense to acyl carrier protein P (acpP).
  • the therapeutic pharmacophore is an oligonucleotide is coupled to a peptide.
  • the peptide may be a cell penetrating peptide and is conjugated to the oligonucleotide through a glycol linkage.
  • the cell penetrating peptide has the following sequence: KFFKFFKFFK (SEQ ID NO: 1).
  • a PEG is incorporated into the nanostructure.
  • the PEG may be 1,000-40,000 Daltons.
  • a method for treating a subject having a bacterial infection involves administering to a subject a nanostructure as described and claimed herein, in an effective amount to treat the bacterial infection.
  • the bacterial infection is selected from the group consisting of Escherichia coli, Acinetobacter baumannii,
  • Helicobacter pyloris e.g. M. tuberculosis, M. avium, M. intracellular, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,
  • Streptococcus pyogenes Group A Streptococcus
  • Streptococcus agalactiae Group B
  • Streptococcus Streptococcus
  • Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species.)
  • Streptococcus pneumoniae pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp.
  • the nanostructure is delivered by a route selected from the group consisting of oral, nasal, sublingual, intravenous, subcutaneous, mucosal, respiratory, direct injection, enema, and dermally.
  • the invention is a method for treating a surface by applying to a surface a nanostructure as described or claimed herein, in an effective amount to disrupt a bacterial infection on the surface.
  • the surface is a surgical device or implant.
  • the article is a device having a coating of a nanoparticle as described or claimed herein.
  • the device is a surgical device or implant.
  • the nanoparticle partially coats the device or fully coats the device.
  • the nanoparticle coats the device by being embedded in the surface of the device.
  • composition for use in the treatment of disease comprises the nanostructure and embodiments thereof.
  • Figures 1A-1B General structure of the spherical nucleic acids (SNAs) described herein.
  • SNAs spherical nucleic acids
  • A General structure of a co-loading model, wherein peptide-peptide nucleic acids (PNAs) (therapeutic pharmacophores) and carrier oligonucleotides (oligos) are both conjugated directly to the gold core.
  • PNAs peptide-peptide nucleic acids
  • oligos carrier oligonucleotides
  • FIGS 2A-2B Antibacterial efficacy of Peptide-peptide nucleic acid (PNA) spherical nucleic acid (SNA) construct (hybridization model) with KFF as the cell penetrating peptide (CPP) and PNA oligomer targeted towards the acpP gene.
  • PNA Peptide-peptide nucleic acid
  • SNA spherical nucleic acid
  • the negative control is a SNA construct with similar structure, with KFF as the CPP but scrambled PNA oligomer, not targeted towards any Escherichia coli gene.
  • the targeted SNA constructs Peptide-PNA:01igo 1: 1 (closed circles) and Peptide-PNA:01igo 2: 1 (closed squares), control SNA construct (CTL Peptide-PNA:01igo; 1: 1; closed up triangles) and free-peptide-PNA (Peptide-PNA Only; closed down triangles) were added to Escherichia coli K-12 (A) and Escherichia coli DH5a (B).
  • the data are expressed in terms of estimated peptide-PNA concentration, assuming there is 100% hybridization of peptide PNA added to the complementary oligo.
  • Figures 4A-4B Antibacterial efficacy of Peptide- PNA SNAs (hybridization model) with KFF as the cell penetrating peptide and PNA oligomers targeted towards the acpP gene.
  • the control is peptide-PNAs hybridized to the oligos (Peptide-PNA:01igo Free; arrow point to closed circles), not functionalized on the gold core, to establish the role of gold core as a carrier of the peptide-PNA into the bacterial cell.
  • a bacterial turbidity assay was conducted and the data are expressed in terms of oligo concentration as Peptide-PNA:01igo 1: 1 (arrow points to closed circles), Peptide-PNA:01igo 2: 1 (closed squares), Peptide- PNA:01igo 3: 1 (closed up triangles), and Peptide-PNA:01igo 4: 1 (closed down triangles), based on the oligo loading that was identified using a fluorescence assay.
  • B The results of turbidity assay were confirmed by the colony forming unit (CFU)/mL viability assay and the data is presented.
  • Figures 5A-5B Time-dependent antimicrobial efficacy of nanostructures having a liposomal core functionalized with oligonucleotides.
  • the oligonucleotides are hybridized to peptide-peptide nucleic acids (pPNAs) via Watson-Crick hybridization in either duplex or triplex conformations.
  • pPNAs peptide-peptide nucleic acids
  • Each pPNA sequence is antisense to the acyl carrier protein in A. baumannii.
  • FIG. 6 Comparison of nanostructures having a liposomal core -SNAs to free peptide-peptide nucleic acids (pPNAs) and gold-core spherical nucleic acids functionalized with oligonucleotides.
  • the oligonucleotides are hybridized to pPNAs via Watson-Crick hybridization in either duplex or triplex conformations.
  • Each pPNA sequence is antisense to the acyl carrier protein in A. baumannii or E. coli.
  • the nanostructures having a liposomal core were administered to the bacteria on a 96-well plate, using a serial dilution, single-time dosage approach. Results indicate the percentage population as related to untreated cells after incubation overnight, with the liposomal pPNAs (LSNA-pPNAs; closed diamonds) compared to free pPNAs (closed squares).
  • Figures 7A-7B Antibacterial efficacy and general structure of the SNAs evaluated herein.
  • A Activity and structure of the co-loading model, wherein peptide-PNAs and stabilizing oligonucleotides (oligos) are both conjugated directly to the gold core via thiol linkage.
  • Antibacterial efficacy of Peptide-PNA SNAs (co-loading model) with KFF as the cell penetrating peptide and PNA oligomers targeted towards the acpP gene.
  • the Target Oligo & Peptide-PNA (closed diamonds), control construct CTL oligo & PeptidePNA
  • oligo concentration is expressed in terms of oligo concentration as Peptide-PNA:01igo 1: 1 (arrow points to closed circles), Peptide-PNA:01igo 2: 1 (closed squares), Peptide-PNA:01igo 3: 1 (closed up triangles), and Peptide-PNA:01igo 4: 1 (closed down triangles).
  • FIGS 8A-8B SNAs without carrier oligonucleotide and peptide nucleic acid do not show antibacterial efficacy.
  • T3 TAGTGCTCATACTCTT (SEQ ID NO: 4).
  • PO all phosphodiester backbone
  • FIG. 9 General structure of silver spherical nucleic acid (SNA) design and interaction described herein.
  • SNA silver spherical nucleic acid
  • a basic SNA is formulated with the adsorption of 3 '-thiol-oligonucleotide and methoxyl poly(ethylene glycol) thiol.
  • SNAs are known to adhere to or transfect across various types of cell membranes, which serve as the driving force behind a silver-derived SNA.
  • the unique properties of Ag-SNAs enable the use of these particles as potent antimicrobials.
  • Figure 10 TEM, DLS, and zeta-potential characterization of silver particles.
  • A The transmission electron microscopy (TEM) size analysis of silver nanoparticles (Ag-NPs) yielded a mean diameter of 25.8 + 4.8 nm.
  • B possess a mean diameter of 19.7 +4.05 nm.
  • C The dynamic light scattering (DLS) number size distributions display the peak sizes of the Ag-NPs and BT-Ag-SNAs. There is an upward shift of about 5-10 nm between Ag-NPs and BT-Ag-SNAs.
  • D The difference in zeta-potential indicates the presence of negatively charged oligonucleotides on the surface of BT-Ag-SNAs.
  • Figure 11 Growth curves for treated bacteria.
  • A. baumannii AYE treated with PS-BT-Ag-SNAs demonstrates an minimum inhibitory concentration (MIC) value of 0.3125 nM.
  • the corresponding PO-BT-Ag-SNAs and Ag-NPs displayed MICs of 0.625 and 5 nM, respectively.
  • A. baumannii UNT086-1 B) demonstrated MICs of 0.625, 1.25, and 5 nM when comparing PS-BT-Ag-SNAs, PO-BT-Ag-SNAs, and Ag-NPs, respectively.
  • MRSA MRSA showed a similar reaction to the particles, with MICs of 2.5, 5, and 10 nM for PS- BT-Ag-SNAs, PO-BT-Ag-SNAs, and Ag-NPs, respectively. Unconjugated oligonucleotide did not display activity against any bacteria tested.
  • D The time-dependent kill curve of BT- Ag-SNAs and Ag-NPs shows that the SNAs were able to completely eliminate bacteria within 7 hours, whereas Ag-NPs were only able to reduce the bacterial population at the same Ag concentration.
  • Figure 12 Interactions of BT-Ag-SNAs with bacteria. Fluorescence microscopy images of untreated (A) and fluorescent-BT-Ag-SNA treated (B) A. baumannii AYE demonstrate the association of particles with cells in a bacterial population. TEM images of untreated (C) and BT-Ag-SNA-treated (D) A. baumannii AYE cells display the
  • FIG. 13 Ampicillin acts synergistically with BT-Ag-SNAs and Ag-NPs. Graphs were obtained by comparing the fractional inhibitory concentrations of ampicillin with Ag- SNAs, where each point represents data that deviated from an additive relationship between the drugs (the dotted line). Both methicillin-resistant Staphylococcus aureus (MRSA) and A. baumannii UNT086-1 were challenged with a combination of ampicillin and BT-Ag-SNAs. Both strains of bacteria are resistant to ampicillin or more generally, ⁇ -lactam antibiotics.
  • MRSA methicillin-resistant Staphylococcus aureus
  • A. baumannii UNT086-1 were challenged with a combination of ampicillin and BT-Ag-SNAs. Both strains of bacteria are resistant to ampicillin or more generally, ⁇ -lactam antibiotics.
  • Figure 14 Toxicity of BT- Ag-SNAs with HFK cells.
  • human foreskin keratinocytes were treated with various concentrations of BT- Ag-SNAs and Ag- NPs.
  • FIG. 15 Topical Infection of A. baumannii UNT086-1 treated with BT- Ag-SNAs.
  • BT- Ag-SNAs displayed a decrease in CFUs after treatment, with a significant log 10 reduction of 1.51 (p ⁇ 0.05) compared to the untreated control.
  • Ag-NPs and unconjugated oligonucleotide did not demonstrate significant activity, with minor log 10 reductions of 0.63 and 0.58.
  • Figure 16 Growth curve analysis of P. aeruginosa and E. coli in vitro.
  • oligonucleotide reinforces the increased efficacy of the Ag-SNA, particularly against Gram- negative species.
  • the MIC of Ag-SNAs is 0.3125 nM, which is 8-fold lower than the MIC of Ag-NPs.
  • the MIC of Ag-SNAs was 0.16525 nM, a 16-fold reduction from Ag-NPs.
  • an ATP detection cell viability assay was used to verify the results of (B).
  • FIG. 17 Vancomycin and ciprofloxacin synergy with Ag-SNAs and Ag-NPs.
  • Ciprofloxacin's MIC value was 30 ⁇ g/mL, and displayed a respective 2- and 120-fold reduction in Ag-SNA and ciprofloxacin concentration at the point of greatest synergy.
  • FIG. 18 BT- Ag-SNA and silver ion growth curve analysis of A. baumannii UNT086-1.
  • A. baumannii UNT086-1 were treated with silver ions (Ag + ; closed triangles) or silver spherical nucleic acids (Ag-SNAs; closed squares) and the data is represented as percent of untreated (% UNT) cells as a function of increasing concentration of Ag atoms (nM). This comparison was made on an atomic basis, assuming that the entire outer layer of silver atoms on each SNA was free to interact with bacteria.
  • Methods and products for delivery of therapeutic molecules in a multivalent fashion are provided herein.
  • the methods and products may be used for, for example, antibacterial applications, prevention or treatment of infectious disease, modification or elimination of natural and artificial bacterial flora, disruption or elimination of biofilms, and modification of industrial bacterial cultures.
  • Nanostructures having a core and one or more carrier oligonucleotides and therapeutic pharmacophores positioned on the exterior of the core are provided herein.
  • This novel class of nanostructures has unexpectedly high anti-microbial activity.
  • These nanostructures are supra-molecular assemblies, which are spherical nucleic acids
  • SNAs can deliver combinations of anti-microbial agents in a highly spatiotemporally controlled manner to cells (Examples of the SNA structures are shown in Figure 1).
  • a distinctive feature of these nanostructures is the incorporation of at least two distinct sets of nucleic acids on the exterior of the core.
  • incorporación of the dual oligonucleotides in the SNA construct confers unique properties on the structure including but not limited to preferable chemical properties, enhanced bioavailablity, enhanced bacterial targeting, enhanced drug product efficacy, enhanced in vivo pharmacodynamics and pharmacokinetic properties. Importantly, these advantages as they relate to antibiotic development cannot be achieved by delivering either of the separate components individually or in simultaneous combination but not physically associated. Assembly of all of the components into a single structure is critical to obtaining the desired enhanced properties and effects.
  • MDR multi-drug resistant
  • Achieving specific delivery of a therapeutic pharmacophore to achieve bactericidal effects is a significant challenge.
  • the prior art typically relies heavily on significant chemical modification of nucleic acids, which results in relatively poor efficacy requiring very large dosages and high incidences of side effects and toxicity.
  • the methods of the invention achieve antibiotic effects that are enhanced relative to the use of single agents alone.
  • the nanostructure of the invention includes a core.
  • the core may be a solid or a hollow core, such as a liposomal core.
  • a solid core is a spherical shaped material that does not have a hollow center.
  • the term spherical as used herein refers to a general shape and does not imply or is not limited to a perfect sphere or round shape. It may include imperfections.
  • Solid cores can be constructed from a wide variety of materials known to those skilled in the art including but not limited to: noble metals (gold, silver), transition metals (iron, cobalt) and metal oxides (silica). In addition, these cores may be inert, paramagnetic, or supramagentic. These solid cores can be constructed from either pure compositions of described materials, or in combinations of mixtures of any number of materials, or in layered compositions of materials.
  • solid cores can be composed of a polymeric core such as amphiphilic block copolymers, hydrophobic polymers such as polystyrene, poly(lactic acid), poly(lactic co-glycolic acid), poly(glycolic acid), poly(caprolactone) and other biocompatible polymers known to those skilled in the art.
  • a polymeric core such as amphiphilic block copolymers, hydrophobic polymers such as polystyrene, poly(lactic acid), poly(lactic co-glycolic acid), poly(glycolic acid), poly(caprolactone) and other biocompatible polymers known to those skilled in the art.
  • the core may alternatively be a hollow core, which has at least some space in the center region of a shell material.
  • Hollow cores include liposomal cores.
  • a liposomal core as used herein refers to a centrally located core compartment formed by a component of the lipids or phospholipids that form a lipid bilayer.
  • "Liposomes" are artificial, self closed vesicular structure of various sizes and structures, where one or several membranes encapsulate an aqueous core. Most typically liposome membranes are formed from lipid bilayers membranes, where the hydrophilic head groups are oriented towards the aqueous environment and the lipid chains are embedded in the lipophilic core.
  • Liposomes can be formed as well from other amphiphilic monomeric and polymeric molecules, such as polymers, like block copolymers, or polypeptides. Unilamellar vesicles are liposomes defined by a single membrane enclosing an aqueous space. In contrast, oligo- or
  • multilamellar vesicles are built up of several membranes.
  • the membranes are roughly 4 nm thick and are composed of amphiphilic lipids, such as phospholipids, of natural or synthetic origin.
  • the membrane properties can be modified by the incorporation of other lipids such as sterols or cholic acid derivatives.
  • the lipid bilayer is composed of two layers of lipid molecules. Each lipid molecule in a layer is oriented substantially parallel to adjacent lipid bilayers, and two layers that form a bilayer have the polar ends of their molecules exposed to the aqueous phase and the non- polar ends adjacent to each other.
  • the central aqueous region of the liposomal core may be empty or filled fully or partially with water, an aqueous emulsion, oligonucleotides, or other therapeutic or diagnostic agent such as an antimicrobial agent.
  • Lipid refers to its conventional sense as a generic term encompassing fats, lipids, alcohol-ether- soluble constituents of protoplasm, which are insoluble in water. Lipids usually consist of a hydrophilic and a hydrophobic moiety. In water lipids can self organize to form bilayers membranes, where the hydrophilic moieties (head groups) are oriented towards the aqueous phase, and the lipophilic moieties (acyl chains) are embedded in the bilayers core. Lipids can comprise as well two hydrophilic moieties (bola amphiphiles). In that case, membranes may be formed from a single lipid layer, and not a bilayer.
  • lipids in the current context are fats, fatty oils, essential oils, waxes, steroid, sterols, phospholipids, glycolipids, sulpholipids, aminolipids, chromolipids, and fatty acids.
  • the term encompasses both naturally occurring and synthetic lipids.
  • Preferred lipids in connection with the present invention are: steroids and sterol, particularly cholesterol, phospholipids, including phosphatidyl, phosphatidylcholines and phosphatidylethanolamines and sphingomyelins.
  • fatty acids they could be about 12-24 carbon chains in length, containing up to 6 double bonds.
  • the fatty acids are linked to the backbone, which may be derived from glycerol.
  • the fatty acids within one lipid can be different
  • non-cationic lipids are derived from natural sources, such as lecithins (phosphatidylcholines) purified from egg yolk, bovine heart, brain, liver or soybean.
  • the liposomal core can be constructed from one or more lipids known to those in the art including but not limited to: sphingolipids such as sphingosine, sphingosine phosphate, methylated sphingosines and sphinganines, ceramides, ceramide phosphates, 1-0 acyl ceramides, dihydroceramides, 2-hydroxy ceramides, sphingomyelin, glycosylated sphingolipids, sulfatides, gangliosides, phospho sphingolipids, and phytosphingosines of various lengths and saturation states and their derivatives, phospholipids such as
  • phosphatidylcholines lysophosphatidylcholines, phosphatidic acids, lysophosphatidic acids, cyclic LPA, phosphatidylethanolamines, lysophosphatidylethanolamines,
  • phosphatidylglycerols phosphatidylglycerols, lysophosphatidylglycerols, phosphatidylserines,
  • lysophosphatidylserines phosphatidylinositols, inositol phosphates, LPI, cardiolipins, lysocardiolipins, bis(monoacylglycero) phosphates, (diacylglycero) phosphates, ether lipids, diphytanyl ether lipids, and plasmalogens of various lengths, saturation states, and their derivatives, sterols such as cholesterol, desmosterol, stigmasterol, lanosterol, lathosterol, diosgenin, sitosterol, zymosterol, zymostenol, 14-demethyl-lanosterol, cholesterol sulfate, DHEA, DHEA sulfate, 14-demethyl-14-dehydrlanosterol, sitostanol, campesterol, ether anionic lipids, ether cationic lipids, lanthanide chelating lipids, A
  • oligonucleotides including carrier oligonucleotides and therapeutic
  • pharmacophores are positioned on the exterior of the core.
  • An oligonucleotide that is positioned on the core is typically referred to as coupled to the core. Coupled may be direct or indirect.
  • One or more therapeutic pharmacophores may also be coupled to the carrier oligonucleotide. While the carrier may be reversibly or irreversibly coupled to the core, preferably the therapeutic pharmacophore is reversibly coupled to the core or carrier oligonucleotide.
  • Reversibly coupled compounds are associated with one another using a susceptible linkage.
  • a susceptible linkage is one which is susceptible to separation under physiological conditions. For instance Watson crick base pairing is a susceptible linkage.
  • Cleavable linkers are also susceptible linkages. It is describable for the therapeutic pharmacophore to become uncoupled from the carrier oligonucleotide in a cell or body. In some instances the therapeutic pharmacophore is hybridized to the carrier oligonucleot
  • At least two oligonucleotides are on the exterior, a carrier and a therapeutic pharmacophore oligonucleotide (referred to herein interchangeably with "therapeutic oligonucleotide”) .
  • a carrier and a therapeutic pharmacophore oligonucleotide referred to herein interchangeably with "therapeutic oligonucleotide”
  • at least 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1,000 oligonucleotides (carrier oligonucleotide and/or therapeutic pharmacophores) or any range combination thereof are on the exterior of the core.
  • 1-1000, 10-500, 50-250, or 50-300 oligonucleotides are present on the surface.
  • oligonucleotide refers to any nucleic acid containing molecule.
  • the nucleic acid may be DNA, RNA, PNA, LNA, ENA or combinations or modifications thereof. It may also be single, double or triple stranded.
  • the carrier oligonucleotide and/or therapeutic pharmacophore may be a wide variety of molecules including but not limited to: single- stranded deoxyribonucleotides, ribonucleotides, and other single- stranded
  • a carrier oligonucleotide is any oligonucleotide. It may or may not be a therapeutic pharmacophore. However, the carrier oligonucleotide is different from the therapeutic pharmacophore on any given nano structure. It may be different in nucleotide sequence, structure, modification, etc.
  • the carrier may have a therapeutic or diagnostic functionality or it may be non-functional. In some instance it is complementary fully or partially or with mismatches with the therapeutic pharmacophore.
  • a “therapeutic pharmacophore” or “therapeutic oligonucleotide” as used herein refers to a nucleic acid or partial nucleic acid having some therapeutic or diagnostic activity.
  • the pharmacophore may be, for instance, an inhibitory nucleic acid or molecule involved in gene knockdown or an activation oligonucleotide involved in upregulating gene expression.
  • the therapeutic pharmacophore may be a nucleic acid or other molecule that knocks down the expression of a bacterial mRNA and/or protein.
  • the therapeutic pharmacophores of the invention may target essential genes of bacteria in order to reduce or ameliorate a bacterial infection.
  • Essential genes that regulate or control bacterial growth, proliferation, virulence and synthesis of important living-dependent substances include but are not limited XofbpAJ fbpB/fbpC and glnAl in Mycobacterium tuberculosis, gyrA/ompA in Klebsiella pneumonia, inhA in Mycobacterium smegmatis, oxyR/ahpC in Mycobacterium avium, NPT/EhErd2 in Entamoeba histolytica, gtfB in Streptococcus mutans,fmhB/ gyrA/hmrB and fabl in
  • Staphylococcus aureus 23S rRNA, 16S rRNA plus lacZ/bla, and RNAse P in Escherichia coli, and acpP in Burkholderia cepacia, Escherichia coli as well as Salmonella enterica serovar Typhimurium, the marRAB operon in Escherichia coli, aac(6')-Ib, act in
  • Escherichia coli vanA in Enterococcus faecalis, cmeA in Campylobacter jejun, mecA in Staphylococcus aureus, metS/murB in Bacillus anthracis and oprM in Pseudomonas aeruginossa.
  • Therapeutic pharmacophores include but are not limited to antisense nucleic acids (single or double stranded), RNAi oligonucleotides, ribozymes, peptides, DNAzymes, peptide nucleic acids (PNAs), triple helix forming oligonucleotides, and aptamers and modified form(s) thereof directed to sequences in gene(s), RNA transcripts, or proteins.
  • Antisense nucleic acids include modified or unmodified RNA, DNA, or mixed
  • Antisense nucleic acid binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation
  • Antisense molecules may also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm.
  • antisense nucleic acid or "antisense oligonucleotide” describes a nucleic acid that is an oligoribonucleotide, oligodeoxyribonucleotide, modified
  • the antisense molecules are designed so as to interfere with transcription or
  • antisense oligonucleotides useful according to the invention are set forth in the Table below.
  • RNAi-based modalities could be employed to inhibit expression of a gene in a cell, such as siRNA-based oligonucleotides and/or altered siRNA-based
  • RNAi therapies may be desirable in knocking down gene expression in a eukaryotic cell that is exposed to a bacteria in order to remove an inhibitor of bacterial resistance.
  • Ribozymes have also been proposed as a means of both inhibiting gene expression of a mutant gene and of correcting the mutant by targeted trans-splicing. Ribozyme activity may be augmented by the use of, for example, non-specific nucleic acid binding proteins or facilitator oligonucleotides. Multitarget ribozymes (connected or shotgun) have been suggested as a means of improving efficiency of ribozymes for gene suppression. Triple helix approaches have also been investigated for sequence- specific gene suppression. Triple helix forming oligonucleotides have been found in some cases to bind in a sequence- specific manner. Similarly, peptide nucleic acids have been shown to inhibit gene expression
  • Minor-groove binding polyamides can bind in a sequence- specific manner to DNA targets and hence may represent useful small molecules for suppression at the DNA level.
  • the diverse array of suppression strategies that can be employed includes the use of DNA and/or RNA aptamers that can be selected to target a protein of interest.
  • the therapeutic pharmacophore is 100% identical to the nucleic acid target and/or to portions of the carrier oligonucleotide.
  • the term "percent identical" refers to sequence identity between two nucleotide sequences. Percent identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. Expression as a percentage of identity refers to a function of the number of identical amino acids or nucleic acids at positions shared by the compared sequences.
  • Various alignment algorithms and/or programs may be used, including FASTA, BLAST, or ENTREZ-FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings.
  • ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.
  • MPSRCH uses a Smith- Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.
  • An therapeutic pharmacophore may be designed to have partial or complete complementarity with one or more target genes. Depending on the particular target gene, the nature of the therapeutic pharmacophore and the level of expression of therapeutic pharmacophore (e.g. depending on copy number, promoter strength) the procedure may provide partial or complete loss of function for the target gene. Quantitation of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target mRNA or translation of target protein.
  • “Inhibition of gene expression” refers to the absence or observable decrease in the level of protein and/or mRNA product from a target gene. “Specificity” refers to the ability to inhibit the target gene without manifest effects on other genes of the cell.
  • RNA solution hybridization nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and
  • FACS fluorescence activated cell analysis
  • the therapeutic pharmacophore is a peptide nucleic acid (PNA).
  • PNA oligomers have greater binding strength and specificity in the formation of a
  • PNA/DNA duplex or PNA/DNA/PNA triplex as compared to a DNA/DNA duplex.
  • PNAs also have increased stability to nucleases and proteases over a wide pH range, which makes them resistant to enzymatic degradation.
  • PNA oligomers as antimicrobial agents, there have been indications that in vivo dosages of these molecules tend to be insufficient for a prolonged drug effect. This is likely due to the host's ability to clear small molecules and toxins from the blood stream, resulting in a decrease of the effective time for each dose in the body. This may also be due to the lower solubility of PNAs in aqueous solution, enzymatic degradation, microbial uptake, the ability of PNAs to penetrate and dissipate throughout the host tissues, or a combination of these factors.
  • the nanostructures provided herein overcome these issues, thereby extending the lifetime of these types of biomolecules, resulting in the stabilization and/or enhancement of the effects of PNAs.
  • CPP cell penetrating peptide
  • the CPP is covalently coupled to a PNA.
  • CPP is believed to improve the bacterial cytosolic delivery of the therapeutic molecule. Coupling CPP to a therapeutic pharmacophore enhances stability and enables the penetration of the bacterial cell wall and membrane.
  • exemplary structure including a PNA was synthesized (as described in more detail in the Examples).
  • carrier phosphodiester oligonucleotides having the following sequence: 5'- AAAAAAAAAAGAGTATGAGAA -3' (SEQ ID NO: 20); acpP) were 5' modified with thiol and incorporated onto 13 nm diameter gold nanoparticles (AuNP).
  • AuNP gold nanoparticles
  • a thiol-modified polyethylene glycol (PEG) of 20 kDa average molecular weight was also incorporated into the nano structure.
  • PEG polyethylene glycol
  • peptide-PNA or pPNA conjugate containing a PNA active drug product coupled to a cell penetrating peptide was Watson-Crick hybridized to the carrier oligonucleotide.
  • the PNA sequence is complementary to the carrier oligonucleotide and antisense to the acyl carrier protein P (acpP) gene in E. coli and is coupled via a glycol linkage to the KFFKFFKFFK (SEQ ID NO: 1) cell penetrating peptide (5 '-3' or N-C: KFFKFFKFFK (SEQ ID NO: l)-0- CTCATACTCT (SEQ ID NO: 6)) ( Figure 1).
  • the PNA was selected on the basis of its ability to downregulate gene expression of the acpP gene in E. coli which is classified as an essential gene for bacterial growth.
  • the cell penetrating peptide was selected based on its ability to facilitate PNA function from a screen of peptides.
  • the carrier oligonucleotides and optionally the therapeutic oligonucleotides form an oligonucleotide shell.
  • An oligonucleotide shell is formed when at least 50% of the available surface area of the exterior surface of the core includes an oligonucleotide. In some embodiments at least 60%, 70%, 80%, 90%, 95%, 96%, 97% 98% or 99% of the available surface area of the exterior surface of the core includes an oligonucleotide.
  • the oligonucleotides of the oligonucleotide shell or therapeutic pharmacophores may be oriented in a variety of directions. In some embodiments the oligonucleotides are oriented radially outwards. The orientation of these oligonucleotides can be either 5' distal/3' terminal in relation to the core, or 3' distal/5 'terminal in relation to the core, or laterally oriented around the core. In one embodiment one or a multiplicity of different oligonucleotides are present on the same surface of a single SNA. In all cases, at least 1 oligonucleotide is present on the surface but up to 10,000 can be present.
  • the oligonucleotides may be linked to the core or to one another and/or to other molecules such an active agents either directly or indirectly through a linker.
  • the oligonucleotides may be conjugated to a linker via the 5' end or the 3' end, e.g. [Sequence, 5'-3']-Linker or Linker-[Sequence, 5'-3'].
  • Some or all of the oligonucleotides of the nanostructure may be linked to one another either directly or indirectly through a covalent or non-covalent linkage.
  • the linkage of one oligonucleotide to another oligonucleotide may be in addition to or alternatively to the linkage of that oligonucleotide to liposomal core.
  • One or more of the oligonucleotides may also be linked to other molecules such as an anti-microbial.
  • the oligonucleotides may be linked to the anti-microbial of the core either directly or indirectly through a covalent or non-covalent linkage.
  • the oligonucleotide shell formed of at least carrier oligonucleotides may be anchored to the surface of the core through one or multiple of linker molecules, including but not limited to: any chemical structure containing one or multiple thiols, such as the various chain length alkane thiols, cyclic dithiol, lipoic acid, or other thiol linkers known to those skilled in the art.
  • the oligonucleotide shell may be anchored to the surface of the liposomal core through conjugation to one or a multiplicity of linker molecules including but not limited to: tocopherols, sphingolipids such as
  • sphingosine sphingosine phosphate, methylated sphingosines and sphinganines
  • ceramides ceramide phosphates
  • 1-0 acyl ceramides dihydroceramides, 2-hydroxy ceramides, sphingomyelin, glycosylated sphingolipids, sulfatides, gangliosides, phosphosphingolipids, and phytosphingosines of various lengths and saturation states and their derivatives
  • phospholipids such as phosphatidylcholines, lysophosphatidylcholines, phosphatidic acids, lysophosphatidic acids, cyclic LPA, phosphatidylethanolamines,
  • lysophosphatidylethanolamines phosphatidylglycerols, lysophosphatidylglycerols, phosphatidylserines, lysophosphatidylserines, phosphatidylinositols, inositol phosphates, LPI, cardiolipins, lysocardiolipins, bis(monoacylglycero) phosphates, (diacylglycero) phosphates, ether lipids, diphytanyl ether lipids, and plasmalogens of various lengths, saturation states, and their derivatives, sterols such as cholesterol, desmosterol, stigmasterol, lanosterol, lathosterol, diosgenin, sitosterol, zymosterol, zymostenol, 14-demethyl- lanosterol, cholesterol sulfate, DHEA, DHEA sulfate, 14
  • the carrier oligonucleotide is positioned on the exterior of the core. It may be associated with the core by being embedded within the core (liposomal core) or it may be attached or linked, either indirectly (i.e. non-covalently or covalently through other molecules such a linkers) or directly (i.e. covalently).
  • oligonucleotide and “nucleic acid” are used interchangeably to mean multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)).
  • a substituted pyrimidine e.g., cytosine (C), thymidine (T) or uracil (U)
  • a substituted purine e.g., adenine (A) or guanine (G)
  • oligonucleotides are also include oligonucleosides (i.e., a oligonucleotide minus the phosphate) and any other organic base containing polymer. Oligonucleotides can be obtained from existing nucleic acid sources (e.g., genomic or cDNA), but are preferably synthetic (e.g., produced by nucleic acid synthesis).
  • the oligonucleotides may be single stranded or double stranded.
  • a double stranded oligonucleotide is also referred to herein as a duplex.
  • Double-stranded oligonucleotides of the invention can comprise two separate complementary nucleic acid strands.
  • duplex includes a double- stranded nucleic acid molecule(s) in which complementary sequences or partially complementary sequences are hydrogen bonded to each other.
  • the complementary sequences can include a sense strand and an antisense strand.
  • a double- stranded oligonucleotide can be double- stranded over its entire length, meaning it has no overhanging single- stranded sequences and is thus blunt-ended.
  • the two strands of the double- stranded oligonucleotide can have different lengths producing one or more single- stranded overhangs.
  • a double- stranded oligonucleotide of the invention can contain mismatches and/or loops or bulges. In some embodiments, it is double- stranded over at least about 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the length of the oligonucleotide. In some embodiments, the double- stranded oligonucleotide of the invention contains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.
  • Oligonucleotides associated with the invention can be modified such as at the sugar moiety, the phosphodiester linkage, and/or the base.
  • sugar moieties includes natural, unmodified sugars, including pentose, ribose and deoxyribose, modified sugars and sugar analogs. Modifications of sugar moieties can include replacement of a hydroxyl group with a halogen, a heteroatom, or an aliphatic group, and can include functionalization of the hydroxyl group as, for example, an ether, amine or thiol.
  • Modification of sugar moieties can include 2'-0-methyl nucleotides, which are referred to as "methylated.”
  • oligonucleotides associated with the invention may only contain modified or unmodified sugar moieties, while in other instances, oligonucleotides contain some sugar moieties that are modified and some that are not.
  • modified nucleomonomers include sugar- or backbone-modified ribonucleotides.
  • Modified ribonucleotides can contain a non-naturally occurring base such as uridines or cytidines modified at the 5'-position, e.g., 5'-(2-amino)propyl uridine and 5'- bromo uridine; adenosines and guanosines modified at the 8-position, e.g., 8-bromo guanosine; deaza nucleotides, e.g. , 7-deaza-adenosine; and N-alkylated nucleotides, e.g.
  • sugar-modified ribonucleotides can have the 2'-OH group replaced by an H, alkoxy (or OR), R or alkyl, halogen, SH, SR, amino (such as NH 2 , NHR, NR 2> ), or CN group, wherein R is lower alkyl, alkenyl, or alkynyl.
  • modified ribonucleotides can have the phosphodiester group connecting to adjacent ribonucleotides replaced by a modified group, such as a phosphorothioate group.
  • 2'-0-methyl modifications can be beneficial for reducing undesirable cellular stress responses, such as the interferon response to double- stranded nucleic acids.
  • the sugar moiety can also be a hexose.
  • alkyl includes saturated aliphatic groups, including straight-chain alkyl groups (e.g. , methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • straight-chain alkyl groups e.g. , methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decy
  • a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g. , Q-C 6 for straight chain, C3-C 6 for branched chain), and more preferably 4 or fewer.
  • preferred cycloalkyls have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure.
  • C C 6 includes alkyl groups containing 1 to 6 carbon atoms.
  • alkyl includes both "unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • alkenyl includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond.
  • alkenyl includes both "unsubstituted alkenyls” and “substituted alkenyls,” the latter of which refers to alkenyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • base includes the known purine and pyrimidine heterocyclic bases, deazapurines, and analogs (including heterocyclic substituted analogs, e.g., aminoethyoxy phenoxazine), derivatives (e.g. , 1-alkyl-, 1-alkenyl-, heteroaromatic- and 1-alkynyl derivatives) and tautomers thereof.
  • purines include adenine, guanine, inosine, diaminopurine, and xanthine and analogs (e.g., 8-oxo-N 6 -methyladenine or 7-diazaxanthine) and derivatives thereof.
  • Pyrimidines include, for example, thymine, uracil, and cytosine, and their analogs (e.g., 5-methylcytosine, 5-methyluracil, 5-(l-propynyl)uracil, 5-(l- propynyl)cytosine and 4,4-ethanocytosine).
  • suitable bases include non- purinyl and non-pyrimidinyl bases such as 2-aminopyridine and triazines.
  • nucleoside includes bases which are covalently attached to a sugar moiety, preferably ribose or deoxyribose.
  • examples of preferred nucleosides include ribonucleosides and deoxyribonucleosides.
  • Nucleosides also include bases linked to amino acids or amino acid analogs which may comprise free carboxyl groups, free amino groups, or protecting groups. Suitable protecting groups are well known in the art (see P. G. M. Wuts and T. W. Greene, "Protective Groups in Organic Synthesis", 2 nd Ed., Wiley- Interscience, New York, 1999).
  • linkage used in the context of an internucleotide linkage includes a naturally occurring, unmodified phosphodiester moiety (-0-(PO )-0-) that covalently couples adjacent nucleomonomers.
  • substitute linkage or “modified linkage” or modified internucleotide linkage” includes any analog or derivative of the native phosphodiester group that covalently couples adjacent nucleomonomers.
  • Substitute linkages include phosphodiester analogs, e.g. , phosphorothioate,
  • non-hydrolizable linkages are preferred, such as phosphorothioate linkages.
  • oligonucleotides of the invention comprise 3' and 5' termini (except for circular oligonucleotides).
  • the 3' and 5' termini of a oligonucleotide can be substantially protected from nucleases, for example, by modifying the 3' or 5' linkages (e.g. , U.S. Pat. No. 5,849,902 and WO 98/13526).
  • Oligonucleotides can be made resistant by the inclusion of a "blocking group.”
  • the term "blocking group” as used herein refers to substituents (e.g.
  • oligonucleotides or nucleomonomers that can be attached to oligonucleotides or nucleomonomers, either as protecting groups or coupling groups for synthesis (e.g. , FITC, propyl (CH 2 -CH 2 -CH 3 ), glycol (-0-CH 2 -CH 2 -0-) phosphate (PO 3 " ), hydrogen phosphonate, or phosphoramidite).
  • “Blocking groups” also include "end blocking groups” or “exonuclease blocking groups” which protect the 5' and 3' termini of the oligonucleotide, including modified nucleotides and non-nucleotide exonuclease resistant structures.
  • Exemplary end-blocking groups include cap structures (e.g., a 7-methylguanosine cap), inverted nucleomonomers, e.g., with 3'-3' or 5 '-5' end inversions (see, e.g. , Ortiagao et al. 1992. Antisense Res. Dev. 2: 129), methylphosphonate, phosphoramidite, non-nucleotide groups (e.g. , non-nucleotide linkers, amino linkers, conjugates) and the like.
  • the 3' terminal nucleomonomer can comprise a modified sugar moiety.
  • the 3' terminal nucleomonomer comprises a 3'-0 that can optionally be substituted by a blocking group that prevents 3'- exonuclease degradation of the oligonucleotide.
  • the 3'-hydroxyl can be esterified to a nucleotide through a 3' ⁇ 3' internucleotide linkage.
  • the alkyloxy radical can be methoxy, ethoxy, or isopropoxy, and preferably, ethoxy.
  • the 3' ⁇ 3'linked nucleotide at the 3' terminus can be linked by a substitute linkage.
  • the 5' most 3' ⁇ 5' linkage can be a modified linkage, e.g., a phosphorothioate or a P-alkyloxyphosphotriester linkage.
  • the two 5' most 3' ⁇ 5' linkages are modified linkages.
  • the 5' terminal hydroxy moiety can be esterified with a phosphorus containing moiety, e.g., phosphate, phosphorothioate, or P- ethoxypho sphate .
  • oligonucleotides can be chimeric RNA-DNA oligonucleotides which include both DNA and RNA or DNA-RNA or RNA-DNA duplexes.
  • oligonucleotides are preferably in the range of 2 to 1000, 2-500, 2-100, 5-500, 5- 100, 10- 500, 10-100, 10-50, 15-500, 15-100, 15-50, 20-500, 20-100, 20-50, or 20-40 bases in length.
  • nucleic acids of other sizes are useful.
  • the oligonucleotides have a modified backbone such as a phosphorothioate (PS) backbone. In other embodiments the oligonucleotides have a phosphodiester (PO) backbone. In yet other embodiments oligonucleotides have a mixed or chimeric PO and PS backbone. In some embodiments, PEG of different sizes is incorporated into the structure to alter the in vivo properties including but not limited to sizes from 1,000 Da to 40,000 Da.
  • the nanostructure may also include an active agent.
  • An active agent as used herein is a molecule capable of providing some therapeutic or diagnostic advantage to a cell or subject. Active agents include, for instance, anti-microbial agents.
  • Active agents can be attached to the structures by the externally-facing
  • oligonucleotides through covalent or non-covalent, e.g. Watson/Crick hybridization.
  • the active agents may be incorporated into a liposomal bilayer via conjugation to a hydrophobic moiety.
  • active agent may be incorporated inside the inner aqueous layer of the liposome.
  • active agent is conjugated to the liposomal nanostructure via interactions with the oligonucleotide shell.
  • the active agent - oligonucleotide conjugate is linked to the core through oligonucleotide hybridization.
  • the oligonucleotide is hybridized to a complementary or partially
  • oligonucleotide to form a duplex or partial duplex.
  • One or both of the oligonucleotides of the duplex is linked directly to the core and the active agent which is external facing (on the outside of the lipid bilayer) or which is internal (in the inner aqueous layer) and not directly linked to the core is linked to one or both of the oligonucleotides in the duplex.
  • active agent is conjugated to the liposomal
  • the active agent can be anchored to the surface of the liposomal core through conjugation to one or a multiplicity of linker molecules including but not limited to: tocopherols, sphingolipids such as sphingosine, sphingosine phosphate, methylated sphingosines and sphinganines, ceramides, ceramide phosphates, 1-0 acyl ceramides, dihydroceramides, 2-hydroxy ceramides, sphingomyelin, glycosylated sphingolipids, sulfatides, gangliosides, phosphosphingolipids, and
  • phospholipids such as phosphatidylcholines, lysophosphatidylcholines, phosphatidic acids, lysophosphatidic acids, cyclic LPA, phosphatidylethanolamines,
  • lysophosphatidylethanolamines phosphatidylglycerols, lysophosphatidylglycerols, phosphatidylserines, lysophosphatidylserines, phosphatidylinositols, inositol phosphates, LPI, cardiolipins, lysocardiolipins, bis(monoacylglycero) phosphates, (diacylglycero) phosphates, ether lipids, diphytanyl ether lipids, and plasmalogens of various lengths, saturation states, and their derivatives, sterols such as cholesterol, desmosterol, stigmasterol, lanosterol, lathosterol, diosgenin, sitosterol, zymosterol, zymostenol, 14-demethyl- lanosterol, cholesterol sulfate, DHEA, DHEA sulfate, 14
  • the invention also encompasses the use of the nanostructures for the treatment of a subject having an infectious disease.
  • a "subject having an infectious disease” is a subject that has had contact with a microorganism. Thus the microorganism has invaded the subjects body.
  • the word "invade” as used herein refers to contact by the microorganism with the external surface of the subject, e.g., skin or mucosal membranes and/or refers to the penetration of the external surface of the subject by the microorganism.
  • infectious disease refers to a disorder arising from the invasion of a host, superficially, locally, or systemically, by an infectious microorganism.
  • Infectious microorganisms include bacteria, viruses, and fungi.
  • Bacteria are unicellular organisms which multiply asexually by binary fission. They are classified and named based on their morphology, staining reactions, nutrition and metabolic requirements, antigenic structure, chemical composition, and genetic homology. Bacteria can be classified into three groups based on their morphological forms, spherical (coccus), straight-rod (bacillus) and curved or spiral rod (vibrio, Campylobacter, spirillum, and spirochaete).
  • Bacteria are also more commonly characterized based on their staining reactions into two classes of organisms, gram-positive and gram-negative. Gram refers to the method of staining which is commonly performed in microbiology labs. Gram-positive organisms retain the stain following the staining procedure and appear a deep violet color. Gram-negative organisms do not retain the stain but take up the counter-stain and thus appear pink.
  • Bacteria have two main structural components, a rigid cell wall and protoplast (material enclosed by the cell wall).
  • the protoplast includes cytoplasm and genetic material. Surrounding the protoplast is the cytoplasmic membrane which includes some of the cell respiratory enzymes and is responsible for the permeability of bacteria and transport of many small molecular weight substances.
  • the cell wall surrounding the cytoplasmic membrane and protoplast is composed of mucopeptides which include complex polymers of sugars cross-linked by peptide chains of amino acids.
  • the wall is also composed of polysaccharides and teichoic acids.
  • Infectious bacteria include, but are not limited to, gram negative and gram positive bacteria.
  • Gram positive bacteria include, but are not limited to Pasteurella species,
  • Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species.
  • infectious bacteria include but are not limited to: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellular e, M. kansaii, M.
  • Streptococcus Streptococcus
  • Streptococcus agalactiae Group B Streptococcus
  • Streptococcus viridans group
  • Streptococcus faecalis Streptococcus bovis
  • Streptococcus anaerobic species.
  • Streptococcus pneumoniae pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp.,
  • Erysipelothrix rhusiopathiae Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema per pneumonia, Leptospira, Rickettsia, and Actinomyces israelii.
  • the methods of the invention involve nanostructures that optionally further include an anti-microbial agent for the treatment or prevention of infectious disease.
  • An antimicrobial agent refers to a naturally-occurring or synthetic compound which is capable of killing or inhibiting infectious microorganisms.
  • the type of anti-microbial agent useful according to the invention will depend upon the type of microorganism with which the subject is infected or at risk of becoming infected.
  • One type of anti-microbial agent is an antibacterial agent.
  • Antibacterial agents kill or inhibit the growth or function of bacteria.
  • a large class of antibacterial agents is antibiotics.
  • Antibiotics which are effective for killing or inhibiting a wide range of bacteria, are referred to as broad spectrum antibiotics.
  • Other types of antibiotics are predominantly effective against the bacteria of the class gram-positive or gram-negative. These types of antibiotics are referred to as narrow spectrum antibiotics.
  • Other antibiotics which are effective against a single organism or disease and not against other types of bacteria, are referred to as limited spectrum antibiotics.
  • Antibacterial agents are sometimes classified based on their primary mode of action.
  • antibacterial agents are cell wall synthesis inhibitors, cell membrane inhibitors, protein synthesis inhibitors, nucleic acid synthesis or functional inhibitors, and competitive inhibitors.
  • Cell wall synthesis inhibitors inhibit a step in the process of cell wall synthesis, and in general in the synthesis of bacterial peptidoglycan.
  • Cell wall synthesis inhibitors include ⁇ -lactam antibiotics, natural penicillins, semi- synthetic penicillins, ampicillin, clavulanic acid, cephalosporins, and bacitracin.
  • the ⁇ -lactams are antibiotics containing a four-membered ⁇ -lactam ring which inhibits the last step of peptidoglycan synthesis
  • ⁇ -lactam antibiotics can be synthesized or natural.
  • the natural antibiotics are generally produced by two groups of fungi, penicillium and cephalosporium molds.
  • the ⁇ -lactam antibiotics produced by penicillium are the natural penicillins, such as penicillin G or penicillin V. These are produced by fermentation of penicillium chrysogenum.
  • the natural penicillins have a narrow spectrum of activity and are generally effective against streptococcus, gonococcus, and staphylococcus.
  • Other types of natural penicillins, which are also effective against gram-positive bacteria include penicillins F, X, K, and O.
  • Semi-synthetic penicillins are generally modifications of the molecule 6- aminopenicillanic acid produced by a mold.
  • the 6-aminopenicillanic acid can be modified by addition of side chains which produce penicillins having broader spectrums of activity than natural penicillins or various other advantageous properties.
  • Some types of semisynthetic penicillins have broad spectrums against gram-positive and gram-negative bacteria, but are inactivated by penicillinase. These semi- synthetic penicillins include ampicillin, carbenicillin, oxacillin, azlocillin, mezlocillin, and piperacillin.
  • Other types of semi- synthetic penicillins have narrower activities against gram-positive bacteria, but have developed properties such that they are not inactivated by penicillinase.
  • ⁇ -lactamase inhibitors such as clavulamic acids and sulbactam.
  • the ⁇ -lactamase inhibitors do not have anti-microbial action but they function to inhibit penicillinase, thus protecting the semi- synthetic penicillin from degradation.
  • Cephalosporins are produced by cephalolsporium molds, and have a similar mode of action to penicillin. They are sensitive to degradation by bacterial ⁇ -lactamases, and thus, are not always effective alone. Cephalosporins, however, are resistant to penicillinase. They are effective against a variety of gram-positive and gram-negative bacteria.
  • Cephalosporins include, but are not limited to, cephalothin, cephapirin, cephalexin, cefamandole, cefaclor, cefazolin, cefuroxine, cefoxitin, cefotaxime, cefsulodin, cefetamet, cefixime, ceftriaxone, cefoperazone, ceftazidine, and moxalactam.
  • Bacitracin is another class of antibiotics which inhibit cell wall synthesis. These antibiotics, produced by bacillus species, prevent cell wall growth by inhibiting the release of muropeptide subunits or peptidoglycan from the molecule that delivers the subunit to the outside of the membrane. Although bacitracin is effective against gram-positive bacteria, its use is limited in general to topical administration because of its high toxicity. Since lower effective doses of bacitracen can be used when the compound is administered with the nanostructures of the invention, this compound can be used systemically and the toxicity reduced.
  • Carbapenems are another broad spectrum ⁇ -lactam antibiotic, which is capable of inhibiting cell wall synthesis.
  • Examples of carbapenems include, but are not limited to, imipenems.
  • Monobactems are also broad spectrum ⁇ -lactam antibiotics, and include, euztreonam.
  • An antibiotic produced by streptomyces, vancomycin, is also effective against gram-positive bacteria by inhibiting cell membrane synthesis.
  • Another class of anti-bacterial agents is the anti-bacterial agents that are cell membrane inhibitors. These compounds disorganize the structure or inhibit the function of bacterial membranes. Alteration of the cytoplasmic membrane of bacteria results in leakage of cellular materials from the cell.
  • Polymyxin produced by Bacillus polymyxis. Polymyxins interfere with membrane function by binding to membrane phospholipids. Polymyxin is effective mainly against Gram- negative bacteria and is generally used in severe Pseudomonas infections or Pseudomonas infections that are resistant to less toxic antibiotics. It is also used in some limited instances topically. It' s limited use is due to the severe side effects associated with systemic administration, such as damage to the kidney and other organs.
  • cell membrane inhibitors include Amphotericin B and Nystatin produced by the bacterium Streptomyces which are also anti-fungal agents, used predominantly in the treatment of systemic fungal infections and Candida yeast infections respectively.
  • Imidazoles produced by the bacterium Streptomyces, are another class of antibiotic that is a cell membrane inhibitor. Imidazoles are used as bacterial agents as well as anti-fungal agents, e.g., used for treatment of yeast infections, dermatophytic infections, and systemic fungal infections. Imidazoles include but are not limited to clotrimazole, miconazole, ketoconazole, itraconazole, and fluconazole.
  • Anti-bacterial agents are protein synthesis inhibitors. These compounds prevent bacteria from synthesizing structural proteins and enzymes and thus cause inhibition of bacterial cell growth or function or cell death. In general these compounds interfere with the processes of transcription or translation.
  • Anti-bacterial agents that block transcription include but are not limited to Rifampins, produced by the bacterium Streptomyces and Ethambutol, a synthetic chemical. Rifampins, which inhibit the enzyme RNA polymerase, have a broad spectrum activity and are effective against gram-positive and gram-negative bacteria as well as Mycobacterium tuberculosis. Ethambutol is effective against
  • Anti-bacterial agents which block translation interfere with bacterial ribosomes to prevent mRNA from being translated into proteins.
  • this class of compounds includes but is not limited to tetracyclines, chloramphenicol, the macrolides (e.g.
  • erythromycin and the aminoglycosides (e.g. streptomycin).
  • the aminoglycosides are a class of antibiotics which are produced by the bacterium Streptomyces, such as, for instance streptomycin, kanamycin, tobramycin, amikacin, and gentamicin. Aminoglycosides have been used against a wide variety of bacterial infections caused by Gram-positive and Gram- negative bacteria. Streptomycin has been used extensively as a primary drug in the treatment of tuberculosis.
  • Gentamicin is used against many strains of Gram-positive and Gram-negative bacteria, including Pseudomonas infections, especially in combination with Tobramycin.
  • Kanamycin is used against many Gram-positive bacteria, including penicillin- resistant staphylococci.
  • One side effect of aminoglycosides that has limited their use clinically is that at dosages which are essential for efficacy, prolonged use has been shown to impair kidney function and cause damage to the auditory nerves leading to deafness.
  • the tetracyclines bind reversibly to the 30s ribosomal subunit and interfere with the binding of charged tRNA to the bacterial ribosome.
  • the tetracyclines are a class of antibiotics, produced by the bacterium Streptomyces, that are broad-spectrum and are effective against a variety of gram-positive and gram-negative bacteria.
  • Examples of tetracyclines include tetracycline, minocycline, doxycycline, and chlortetracycline. They are important for the treatment of many types of bacteria but are particularly important in the treatment of Lyme disease.
  • Anti-bacterial agents such as the macrolides bind reversibly to the 50s ribosomal subunit and inhibits elongation of the protein by peptidyl transferase or prevents the release of uncharged tRNA from the bacterial ribosome or both.
  • the macrolides contain large lactone rings linked through glycoside bonds with amino sugars. These compounds include erythromycin, roxithromycin, clarithromycin, oleandomycin, and azithromycin.
  • Erythromycin is active against most Gram-positive bacteria, Neisseria, Legionella and Haemophilus, but not against the Enterobacteriaceae. Lincomycin and clindamycin, which block peptide bond formation during protein synthesis, are used against gram-positive bacteria.
  • chloramphenicol Another type of translation inhibitor is chloramphenicol.
  • Chloramphenicol binds the 70S ribosome inhibiting the bacterial enzyme peptidyl transferase thereby preventing the growth of the polypeptide chain during protein synthesis.
  • Chloramphenicol can be prepared from Streptomyces or produced entirely by chemical synthesis.
  • One serious side effect associated with chloramphenicol is aplastic anemia. Aplastic anemia develops at doses of chloramphenicol which are effective for treating bacteria in a small proportion (1/50,000) of patients.
  • Chloramphenicol which was once a highly prescribed antibiotic is now seldom uses as a result of the deaths from anemia. Because of its effectiveness it is still used in life- threatening situations (e.g. typhoid fever).
  • these compounds can again be used as anti-bacterial agents because nanostructures allow a lower dose of the chloramphenicol to be used, a dose which may avoid side effects.
  • Some anti-bacterial agents disrupt nucleic acid synthesis or function, e.g., bind to
  • nalidixic acid is in treatment of lower urinary tract infections (UTI) because it is effective against several types of Gram- negative bacteria such as E. coli, Enterobacter aerogenes, K. pneumoniae and Proteus species which are common causes of UTI.
  • UTI lower urinary tract infections
  • Co-trimoxazole is a combination of
  • Rifampicin is a derivative of rifamycin that is active against Gram-positive bacteria (including Mycobacterium tuberculosis and meningitis caused by Neisseria meningitidis) and some Gram-negative bacteria. Rifampicin binds to the beta subunit of the polymerase and blocks the addition of the first nucleotide which is necessary to activate the polymerase, thereby blocking mRNA synthesis.
  • Another class of anti-bacterial agents is compounds that function as competitive inhibitors of bacterial enzymes.
  • the competitive inhibitors are mostly all structurally similar to a bacterial growth factor and compete for binding but do not perform the metabolic function in the cell.
  • These compounds include sulfonamides and chemically modified forms of sulfanilamide which have even higher and broader antibacterial activity.
  • the sulfonamides e.g. gantrisin and trimethoprim
  • Streptococcus pneumoniae beta-hemolytic streptococci and E. coli, and have been used in the treatment of uncomplicated UTI caused by E. coli, and in the treatment of
  • a major problem associated with anti-microbial drug resistance is that the particular anti-microbial agent is then useless in the treatment of the infection by the microorganism. As this resistance develops, additional therapies need to be identified or the infection, which was once manageable will become serious and untreatable.
  • the nanostructures of the invention are useful for the prevention of anti-microbial resistance.
  • the nanostructures are administered optionally with an anti-microbial agent resistant strains may be prevented from developing.
  • the nanostructures may be administered before, at the same time as, or after the anti-microbial agent as long as it is within a time period that is sufficient to effectively treat the disease or prevent the drug resistance.
  • the anti-microbial is incorporated as part of the nano structure.
  • the nanostructures of the invention may also be coated with or administered in conjunction with an anti-microbial agent.
  • An anti-microbial agent refers to a naturally- occurring or synthetic compound which is capable of killing or inhibiting infectious microorganisms.
  • the type of anti-microbial agent useful according to the invention will depend upon the type of microorganism with which the subject is infected or at risk of becoming infected.
  • the nanostructure can be combined with other therapeutic agents.
  • the nanostructure and/or other therapeutic agent may be administered simultaneously or sequentially.
  • the other therapeutic agents are administered simultaneously they can be administered in the same or separate formulations, but are administered at the same time.
  • the other therapeutic agents are administered sequentially with one another and with the nanostructure antimicrobial agent, when the administration of the other therapeutic agents and the
  • nanostructure and anti-microbial agent is temporally separated.
  • the separation in time between the administration of these compounds may be a matter of minutes or it may be longer.
  • an effective amount of an nanostructure refers to the amount necessary or sufficient to realize a desired biologic effect.
  • an effective amount of an nanostructure for treating or preventing infectious disease is that amount necessary to prevent the infection with the microorganism if the subject is not yet infected or is that amount necessary to prevent an increase in infected cells or microorganisms present in the subject or that amount necessary to decrease the amount of the infection that would otherwise occur in the absence of the nanostructure.
  • an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject.
  • the effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular nanostructure being administered the size of the subject, or the severity of the disease or condition.
  • One of ordinary skill in the art can empirically determine the effective amount of a particular nanostructure without necessitating undue experimentation.
  • Subject doses of the compounds described herein typically range from about 0.1 ⁇ g to 10,000 mg, more typically from about 1 g/day to 8000 mg, and most typically from about 10 ⁇ g to 100 ⁇ g. Stated in terms of subject body weight, typical dosages range from about 0.1 ⁇ g to 20 mg/kg/day, more typically from about 1 to 10 mg/kg/day, and most typically from about 1 to 5 mg/kg/day.
  • the nanostructure is administered with a sub-therapeutic dosage of an anti-microbial agent.
  • the two classes of drugs may be administered in sub-therapeutic doses in order to produce a desirable therapeutic result.
  • a "sub-therapeutic dose” as used herein refers to a dosage which is less than that dosage which would produce a therapeutic result in the subject.
  • the sub-therapeutic dose of an antimicrobial agent is one which would not produce the desired therapeutic result in the subject in the absence of the nanostructure.
  • Therapeutic doses of anti-microbial agent are well known in the field of medicine for the treatment of infectious disease. These dosages have been extensively described in references such as Remington's Pharmaceutical Sciences, 18th ed., 1990; as well as many other medical references relied upon by the medical profession as guidance for the treatment of infectious disease.
  • the nanostructures of the invention may be delivered to a subject in vivo or ex vivo for therapeutic and/or diagnostic use or may be used in vitro, ex vivo or in vivo for research purposes.
  • the nanostructures may be administered alone or in any appropriate
  • nanostructures may be formulated or unformulated.
  • the formulations of the invention can be administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
  • nanostructures are mixed with a substance such as a lotion (for example, aquaphor) and are administered to the skin of a subject, whereby the
  • nanostructures are delivered through the skin of the subject.
  • the nanostructures may also be sterile.
  • the nanostructure is administered on a routine schedule.
  • the routine schedule may encompass periods of time which are identical or which differ in length, as long as the schedule is predetermined.
  • the routine schedule may involve administration of the nano structure on a daily basis, every two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks there-between, every two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, etc.
  • the predetermined routine schedule may involve administration of the nanostructure on a daily basis for the first week, followed by a monthly basis for several months, and then every three months after that. Any particular combination would be covered by the routine schedule as long as it is determined ahead of time that the appropriate schedule involves administration on a certain day.
  • an effective amount of the nanostructures can be administered to a subject by any mode that delivers the nanostructures to the desired cell.
  • Administering pharmaceutical compositions may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to oral, parenteral,
  • a subject shall mean a human or vertebrate animal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, turkey, chicken, primate, e.g., monkey, and fish (aquaculture species), e.g. salmon.
  • the invention can also be used to treat infections in non-human subjects.
  • the term treat, treated, or treating when used with respect to an disorder such as an infectious disease refers to a prophylactic treatment which increases the resistance of a subject to development of the disease (e.g., to infection with a bacteria) or, in other words, decreases the likelihood that the subject will develop the disease (e.g., become infected with the bacteria) as well as a treatment after the subject has developed the disease in order to fight the disease (e.g., reduce or eliminate the bacteria) or prevent the disease from becoming worse.
  • kits typically defines a package or an assembly including one or more of the compositions of the invention, and/or other compositions associated with the invention, for example, as previously described.
  • kits typically defines a package or an assembly including one or more of the compositions of the invention, and/or other compositions associated with the invention, for example, as previously described.
  • Each of the compositions of the kit if present, may be provided in liquid form (e.g., in solution), or in solid form (e.g., a dried powder).
  • some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species, which may or may not be provided with the kit.
  • a kit associated with the invention includes one or more components of the nano structure.
  • the kit may include liposomes for forming a liposome core or a metal for forming a solid core, and or carrier or therapeutic
  • kits can also include one or more anti-microbials and or other therapeutic agents.
  • a kit of the invention may, in some cases, include instructions in any form that are provided in connection with the compositions of the invention in such a manner that one of ordinary skill in the art would recognize that the instructions are to be associated with the compositions of the invention.
  • the instructions may include instructions for the use, modification, mixing, diluting, preserving, administering, assembly, storage, packaging, and/or preparation of the compositions and/or other compositions associated with the kit.
  • the instructions may also include instructions for the use of the compositions, for example, for a particular use, e.g., to a sample.
  • the instructions may be provided in any form recognizable by one of ordinary skill in the art as a suitable vehicle for containing such instructions, for example, written or published, verbal, audible (e.g., telephonic), digital, optical, visual (e.g., videotape, DVD, etc.) or electronic communications (including Internet or web-based communications), provided in any manner. Examples
  • Gold nanoparticles were synthesized by initially dissolving gold chloride trihydrate and trisodium citrate separately into nanopure water. The gold chloride trihydrate solution was then added to a larger volume of nanopure water while stirring the mixture using a stirrer plate. After waiting for this solution to reflux rapidly, trisodium citrate solution was added to the reaction mixture. Upon addition of this solution, the reaction mixture changed color from clear to black and then red. Color change is an indication of a successful reaction. The solution was allowed to stir and reflux for 30 minutes with heat before it was allowed to cool down. After the solution cools down, the gold nanoparticles were filtered using a 0.45 ⁇ filter paper and a UV-vis reading was taken to verify the size of the nanoparticles to be 13 nm.
  • a surfactant (10% Tween-20) was added at 0.01% volume/volume to the desired volume of gold nanoparticles.
  • the next step was adding phosphate buffer (pH 7.4), which was followed by addition of carrier phosphodiester oligonucleotides of sequence 5'- AAAAAAAAAAGAGTATGAGAA -3' ((SEQ ID NO: 20); acpP) that were 5' modified with thiol.
  • the oligonucleotides were added up to 500 fold excess of the gold nanoparticle amount.
  • oligonucleotide sequence was complementary to the carrier oligonucleotide and antisense to the acyl carrier protein P (acpP) gene in E.
  • KFFKFFKFFK SEQ ID NO: 1
  • cell penetrating peptide 5 '-3' or N-C: KFFKFFKFFK(SEQ ID NO: l)-0- CTCATACTCT(SEQ ID NO: 6)
  • the number of oligonucleotide strands per gold nanoparticle was measured using a characterization method developed previously, wherein a fluorescent dye was used to bind to the oligonucleotide bases, dye was excited and a measurement of emission predicted the oligonucleotide concentration.
  • the functionalized gold nanoparticles were allowed to incubate overnight at room temperature again. The following day, functionalized SNAs were washed with water after which they were ready to treat bacteria.
  • PEG of different molecular weights may be incorporated into the structure, PEG of average molecular weight ranging from 1000 Da to 40,000 Da were added to the gold nanoparticles at varying concentrations after addition of the surfactant.
  • Liposomes were synthesized using l,2-Dioleoyl-sn-glycero-3-Phosphatidylcholine (DOPC) via an extrusion process which utilized filtration to obtain a relatively
  • DOPC dichloromethane
  • DCM dichloromethane
  • HEPES 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid
  • sodium chloride buffer was used to resuspend the dried DOPC. The contents were then subjected to sonication and vortexing until the DOPC was completely resuspended in the buffer solution.
  • This solution was then freeze-fractured using liquid nitrogen via shell freezing method multiple times, allowing for room-temperature thaws between freezing. After freeze-fracture, the particles were then subjected to extrusion and filtration through a 100 nm filter followed by progressively smaller filters up to, but not limited to, 20 nm in size. Liposomes were stored at 4 °C following extrusion and filtration, until addition and functionalization with oligonucleotides and peptide-PNAs.
  • Carrier phosphodiester oligonucleotides of the sequence 5'- AAAAAAAAAAATGAGGAGAAT -3' (SEQ ID NO: 21) were 5' modified with thiol and coupled to a reactive l,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine-polyethylene glycol-maleimide (DMPE-PEG-Mal) molecule.
  • DMPE-PEG-Mal reactive l,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine-polyethylene glycol-maleimide
  • a peptide-peptide nucleic acid (peptide-PNA or pPNA) of the composition (5 '-3' or N-C: KFFKFFKFFK(SEQ ID NO: l)-0-ATTCTCCTCAT(SEQ ID NO: 15)) was Watson-Crick hybridized to this coupled oligonucleotide via duplex or triplex, and the combined hybrid was then added to liposomes in aqueous suspension.
  • KFFKFFKFFK (SEQ ID NO: 1) was selected based on its ability to facilitate PNA function from a screen of peptides.
  • the PNA sequence is antisense to the carrier oligonucleotide and antisense to the acyl carrier protein P (acpP) gene in A. baumannii, which was classified as an essential gene for bacterial growth.
  • the PNA sequence was selected based on its ability to downregulate expression of the acpP gene in the bacteria A. baumannii.
  • the PEG at 5,000 Da in molecular weight was added. It should be noted that hybridization of the pPNAs to the oligonucleotide may be conducted before or after functionalization of the liposome.
  • the co-loading model involves peptide-PNAs (therapeutic pharmacophores) and stabilizing oligonucleotides (carrier oligonucleotides) both conjugated directly to the gold core.
  • the 13nm gold core was functionalized with stabilizing oligos with phosphodiester linkages (PO) and densely functionalized with peptide-PNAs that had a thiol group at the end for direct conjugation with the gold core that targeted the acpP gene (see Table 1 for sequences).
  • peptide-PNA was loaded on the gold core via hybridization to a complementary carrier oligo. A two-step process was used to build these constructs.
  • the 13nm gold core was functionalized with an oligo that was complementary to the PNA portion of the peptide-PNA. Based on the loading of the oligo, peptide-PNA was added in different fold excess of the carrier oligo loading to achieve maximum loading density and 100% hybridization of the peptide-PNA. Addition of a PEG molecule was also proposed to increase the stability and circulation time of the SNA construct.
  • Table 1 Sequences of oligonucleotides and peptides used in these experiments.
  • AS antisense oligo
  • CPP cell penetrating peptide.
  • the Peptide-PNA sequences used for co-loading are modified with a -SH (thiol) group at the 3' end/C terminal.
  • the complementary oligo was modified with one spacer 18 molecule and a 5' thiol group to conjugate to the gold core.
  • the hybridized model SNAs appeared to be more stable compared to the other co- surface loaded orientation under the conditions tested. It is possible that the slightly positive charge of the peptide-PNA reduces the nanoparticle's colloidal stability by reducing the electrostatic charge density. As a result, the latter SNA construct was chosen for further experimentation to evaluate antibacterial activity.
  • the antibacterial efficacy of the peptide-PNA SNAs was assessed using the dose- response behavior in preventing the growth of Escherichia coli.
  • the results of testing antibacterial efficacy of Peptide-PNA SNA construct (hybridization model) with KFF as the CPP and PNA oligomer targeted towards the acpP gene are shown in Figure 2.
  • Different SNA formulations have different fold excess of peptide-PNA added during synthesis, for example, a 1: 1 and 2: 1 ratio of peptide-PNA to oligo, to ensure maximum loading of peptide-PNA by formation of PNA/DNA duplex.
  • the negative control was a SNA construct with similar structure, with KFF as the CPP but scrambled PNA oligomer, not targeted towards any Escherichia coli gene.
  • the targeted SNA constructs, control SNA construct and free-peptide-PNA were treated to Escherichia coli K-12 (A) and Escherichia coli DH5a (B).
  • the data are expressed in terms of estimated peptide-PNA concentration, assuming there was 100% hybridization of peptide PNA added to the complementary oligo. The results show in this case that Peptide-PNAs were able to achieve complete growth inhibition of bacteria at higher concentrations of Peptide-PNA.
  • the peptide-PNA SNA construct has increased antimicrobial activity at higher
  • FIG. 3 shows the data for antibacterial efficacy of Peptide-PNA SNA construct (hybridization model) with KFF as the CPP and PNA oligomer targeted towards the acpP gene.
  • the bacteria treated with highest concentrations of peptide-PNA SNA construct, negative control and free peptide-PNA ( Figure 2) as well as the untreated bacteria were diluted to various dilutions and plated on Tryptic Soy Broth (TSB) agar plates.
  • the CFU assay was performed for treated and untreated Escherichia coli K-12 (A) and Escherichia coli DH5a (B). The data are expressed in terms of colony forming units counted per volume of the bacterial cells. The results of this assay confirm increased antibacterial activity of the targeted peptide-PNA SNA construct as compared to the negative control and the free peptide-PNA.
  • FIG. 4 demonstrates the antibacterial efficacy of Peptide-PNA SNAs (hybridization model) with KFF as the cell penetrating peptide and PNA oligomers targeted towards the acpP gene.
  • Different SNA formulations have different fold excess of peptide-PNA added during synthesis (1: 1, 2: 1, 3: 1 and 4: 1 ratio of peptide-PNA to oligo) to ensure maximum loading of peptide-PNA by formation of PNA/DNA duplex.
  • the control is peptide-PNAs hybridized to the oligos, not functionalized on the gold core, to establish the role of gold core as a carrier of the peptide-PNA into the bacterial cell.
  • the targeted SNA constructs and control construct were tested against Escherichia coli K-12.
  • the bacterial turbidity assay data are expressed in terms of oligo concentration, based on the oligo loading that was identified using a fluorescence assay. The results show that Peptide-PNAs were able to achieve complete growth inhibition of bacteria at higher concentrations of Peptide-PNA for lower peptide-PNA densities. But in case of higher peptide-PNA density, complete growth inhibition was observed at even lower SNA concentrations.
  • the peptide-PNA SNA constructs lead to complete growth inhibition at
  • Each pPNA sequence is antisense to the acyl carrier protein in A. baumannii.
  • the oligonucleotides were anchored into the surface of the liposomes via a conjugation to a strand of polyethylene glycol that was conjugated to the phospholipid 2-Dimyristoyl-sn- glycero-3-phosphoethanolamine (DMPE).
  • DMPE 2-Dimyristoyl-sn- glycero-3-phosphoethanolamine
  • the liposomes were administered to the bacteria on a 96-well plate, using a serial dilution, single-time dosage approach.
  • results indicate the percentage population as related to untreated cells after incubation overnight, with the liposomal pPNAs compared to free pPNAs.
  • the stabilizing oligos have phosphodiester linkages (PO) and are targeted/non-targeted oligos towards the acpP gene (see Table 1 for sequences).
  • PO phosphodiester linkages
  • the control is free peptide-PNA not conjugated to the gold core, to establish the role of the SNA contrsuct as a carrier of the peptide-PNA into the bacterial cell.
  • the targeted and control SNA constructs were tested against Escherichia coli K-12.
  • the bacterial turbidity assay data are expressed in terms of Peptide PNA
  • FIG. 7B The structure of peptide-PNA loaded on the gold core via hybridization to a complementary oligo (hybridization model) is shown in Figure 7B.
  • the structure also contains PEG molecules to increase the stability and circulation time of the SNA construct.
  • Antibacterial efficacy of Peptide-PNA SNAs (hybridization model) with KFF as the cell penetrating peptide and PNA oligomers targeted towards the acpP gene is shown in the graph of Figure 7B.
  • Different SNA formulations have different fold excess of peptide-PNA added during synthesis (1: 1, 2: 1, 3: 1 and 4: 1 ratio of peptide-PNA to oligo) to ensure maximum loading of peptide-PNA by formation of PNA/DNA duplex.
  • the control is peptide-PNAs hybridized to the oligos, not functionalized on the gold core, to establish the role of gold core as a carrier of the peptide-PNA into the bacterial cell.
  • the targeted SNA constructs and control construct were tested against Escherichia coli K-12.
  • the bacterial turbidity assay data are expressed in terms of oligo concentration, based on the oligo loading that was identified using a fluorescence assay.
  • the results show that Peptide-PNAs are able to achieve complete growth inhibition of bacteria only at higher concentrations of Peptide- PNA for lower peptide-PNA densities. But in the case of higher peptide-PNA density, complete growth inhibition is observed at even lower SNA concentrations.
  • PO all phosphodiester backbone
  • 2'0Me 2'0Me modifications to all bases
  • G/PS Gap-mer with 18 total bases, from 5' to 3' containing six 2'OMe modified bases followed by six 2'deoxy nucleobases followed by six 2'OMe modified bases (all linked by phosphorothioate backbone).
  • SNAs were prepared as known in the art, see for example Giljohann D et al. Nano Letters 2007 7:3818-3821, and/or Patel P et al. Proc. Natl. Acad. Sci. USA. 2008 105: 17222-17226. with oligonucleotides arranged around a gold core without incorporating the specific chemical changes that are shown to be important in this application.
  • Example 4 Design and Synthesis of BT-Ag-SNAs
  • Pre-treating the oligonucleotide to be loaded onto SNA with dithiothreitol (DTT) allowed us to increase the binding availability of oligonucleotides to Ag-NPs. Notably, this method produced greater yields of Ag-SNAs while using less oligonucleotide as compared with prior work. 13 BT- Ag-SNAs used in the experiments contained approximately 45 oligonucleotide strands per particle. In addition, our synthesis allows for the formulation of Ag-SNAs with variable oligonucleotide backbone chemistries while potentially improving particle stability.
  • DTT dithiothreitol
  • the functionalized Ag-SNAs were measured to have a mean diameter of 19.7
  • oligonucleotide strands alone is largely responsible for the antimicrobial efficacy.
  • Figure 1 ID displays a time-dependent CFU kill curve of A. baumannii UNT086-1 challenged with BT-Ag-SNAs, Ag-NPs, and a gentamicin control.
  • BT-Ag-SNAs effectively eliminated the bacteria after 5 hours of incubation, with no detectable growth via CFU by the 7-hour mark.
  • Ag-NPs did not manage to eliminate the bacteria by the ending time point of the experiment and were slower to treat the infection than the Ag-SNAs.
  • FIG. 16A-B shows the growth curves of P. aeruginosa and E.coli K- 12 as detected via optical density using a 96- well plate reader at 900 nm. While P.
  • aeruginosa -targeted Ag-SNAs exhibited an 8-fold increase in efficacy over Ag-NPs
  • E.coli K-12-targeted Ag-SNAs exhibited a 16-fold increase in efficacy over Ag-NPs
  • PS unconjugated oligonucleotide failed to display any activity when used alone against the bacterial cells.
  • an Adenosine triphosphate (ATP) assay was also performed to verify the results of the optical density assay.
  • Figure 16C indicates the results of the ATP assay, where in E.coli K- 12 -targeted Ag-SNAs again exhibit a 16-fold increase in efficacy over Ag-NPs.
  • BT-Ag-SNAs have been shown to display synergy with ampicillin against A.
  • A. baumannii UNT086-1 was treated with BT-Ag- SNAs and vancomycin or ciprofloxacin.
  • Example 10 Reduced Toxicity of BT-Ag-SNAs as Compared to Ag-NPs in Human Foreskin Keratinocytes (HFKs)
  • BT-Ag-SNAs represent a new strategy in antibiotics with applicability against DR Gram- positive and DR Gram-negative organisms.
  • Our data suggest that the BT-Ag-SNA constructs with oligonucleotides on the surfaces of these particles offer means for Ag-NPs to better interact with the surfaces of bacterial cells, while improving efficacy and decreasing the toxic effects for the host.
  • the BT-Ag-SNA also represents the possibility of low-dose silver to be used synergistically with existing antibiotics, as ampicillin worked
  • BT-Ag-SNAs exhibit a functional, stable, and broad spectrum approach to antimicrobials.
  • Our data suggests that the polyvalent nature of the Ag-SNA combined with the potency of silver nanoparticles brings closer to the possibility of a safe and powerful silver-based antibiotic.
  • These specialized nanoparticles differentiate themselves through their antimicrobial efficacy, potent antibiotic synergy, and improved control over unwanted toxicity as compared to Ag nanoparticles.
  • Example 12 Enhanced bacterial toxicity of Ag-SNAs relative to silver ions.
  • a comparison of silver ions was made with Ag-SNAs in A. baumannii UNT086-1, and the results indicated that SNAs possessed greater efficacy (Figure 18).
  • This comparison was made on an atomic basis, assuming that the entire outer layer of silver atoms on each SNA was free to interact with bacteria. We made this assumption on the basis that no significant color change occurred in the SNAs during the experiment (color change would indicate a significant change in particle size and concentration). Together, these results demonstrate that the SNA construct can be used to couple therapeutics and that this action generates new and unexpected properties to the conjugate structure.

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Abstract

L'invention concerne des nanoparticules sphériques pour le traitement de bactéries. Les nanoparticules peuvent être des particules s'auto-assemblant ayant un noyau décoré par des oligonucléotides et des pharmacophores thérapeutiques. Les nanoparticules sont utiles pour des applications prophylactiques et thérapeutiques ainsi que pour des indications de recherche et de diagnostic.
PCT/US2015/038771 2014-07-01 2015-07-01 Nanoparticules sphériques en tant qu'agents antibactériens Ceased WO2016004168A1 (fr)

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US10568898B2 (en) 2013-08-13 2020-02-25 Northwestern University Lipophilic nanoparticles for drug delivery
EP3487536A4 (fr) * 2016-07-20 2020-04-01 Guild Biosciences Nanostructures d'acides nucléiques fonctionnalisées pour l'administration d'arn
WO2020181144A1 (fr) * 2019-03-06 2020-09-10 Northwestern University Acide nucléique sphérique conjugué à un oligonucléotide de type épingle à cheveux
US10837018B2 (en) 2013-07-25 2020-11-17 Exicure, Inc. Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use
EP3789046A1 (fr) * 2019-09-05 2021-03-10 Nanordica Medical Oü Nanoparticules biocompatibles avec diverses fonctionnalisations de surface pour des applications antimicrobiennes
US11696954B2 (en) 2017-04-28 2023-07-11 Exicure Operating Company Synthesis of spherical nucleic acids using lipophilic moieties
US11896612B2 (en) 2019-03-29 2024-02-13 Board Of Trustees Of Michigan State University Resurrection of antibiotics that MRSA resists by silver-doped bioactive glass-ceramic particles

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Cited By (11)

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US10837018B2 (en) 2013-07-25 2020-11-17 Exicure, Inc. Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use
US10894963B2 (en) 2013-07-25 2021-01-19 Exicure, Inc. Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use
US10568898B2 (en) 2013-08-13 2020-02-25 Northwestern University Lipophilic nanoparticles for drug delivery
EP3487536A4 (fr) * 2016-07-20 2020-04-01 Guild Biosciences Nanostructures d'acides nucléiques fonctionnalisées pour l'administration d'arn
US11696954B2 (en) 2017-04-28 2023-07-11 Exicure Operating Company Synthesis of spherical nucleic acids using lipophilic moieties
WO2020181144A1 (fr) * 2019-03-06 2020-09-10 Northwestern University Acide nucléique sphérique conjugué à un oligonucléotide de type épingle à cheveux
US20220175956A1 (en) * 2019-03-06 2022-06-09 Northwestern University Hairpin-like oligonucleotide-conjugated spherical nucleic acid
US11896612B2 (en) 2019-03-29 2024-02-13 Board Of Trustees Of Michigan State University Resurrection of antibiotics that MRSA resists by silver-doped bioactive glass-ceramic particles
CN110483706A (zh) * 2019-07-11 2019-11-22 江苏大学 一种基于寡核苷酸双亲性温敏性嵌段聚合物双功能荧光探针的制备方法及应用
CN110483706B (zh) * 2019-07-11 2021-10-12 江苏大学 一种基于寡核苷酸双亲性温敏性嵌段聚合物双功能荧光探针的制备方法及应用
EP3789046A1 (fr) * 2019-09-05 2021-03-10 Nanordica Medical Oü Nanoparticules biocompatibles avec diverses fonctionnalisations de surface pour des applications antimicrobiennes

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