WO2018175445A1 - Acides nucléiques sphériques de poly(acide lactique-co-glycolique) (plga) - Google Patents

Acides nucléiques sphériques de poly(acide lactique-co-glycolique) (plga) Download PDF

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WO2018175445A1
WO2018175445A1 PCT/US2018/023367 US2018023367W WO2018175445A1 WO 2018175445 A1 WO2018175445 A1 WO 2018175445A1 US 2018023367 W US2018023367 W US 2018023367W WO 2018175445 A1 WO2018175445 A1 WO 2018175445A1
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nanoparticle
toll
receptor
plga
oligonucleotide
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Chad A. Mirkin
Shengshuang ZHU
Hang XING
Jungsoo Park
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Northwestern University
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    • 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
    • A61K47/6931—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 the material constituting the nanoparticle being a polymer
    • A61K47/6935—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 the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—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 the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present disclosure relates to poly (lactic-co-glycolic acid)(PLGA) spherical nucleic acids (SNAs) having enhanced stability, methods of making the same, and uses thereof.
  • PLGA-SNAs are useful in gene regulation and drug delivery.
  • SNA Spherical nucleic acid
  • nucleic acid therapies provide privileged access at both the cellular and tissue levels.
  • SNAs are actively transported across cell membranes by engaging Class A scavenger receptors [Choi et al., Proc, Natl. Acad. Sci. USA 2013, 1 10, 7625; Wu et al., J. Am. Chem. Soc. 2014, 136, 7726] while unmodified linear nucleic acids do not enter cells in significant amounts without the use of transfection agents [Luo et al., Nat. Biotechnol. 2000, 18, 33; Opalinska et al., Nat. Rev. Drug Discov. 2002, 1 , 503].
  • the polyvalent, densely functionalized nucleic acid shell that defines an SNA can act as a high affinity binder for different classes of ligands, including certain receptor proteins [Choi et al, Proc, Natl. Acad. Sci. USA 2013, 1 10, 7625] and complementary nucleic acid sequences [Lytton-Jean et al., J. Am. Chem. Soc. 2005, 127, 12754]. Consequently, SNAs have emerged as a powerful platform for developing molecular diagnostic probes [Halo et al., Proc. Natl. Acad. Sci. USA 2014, 1 1 1 , 17104; Prigodich et al., Anal. Chem.
  • the disclosure provides a nanoparticle comprising poly (lactic-co- glycolic acid) (PLGA), an agent that facilitates escape of the nanoparticle from an endosome, and an oligonucleotide conjugated to the surface of the nanoparticle.
  • PLGA poly (lactic-co- glycolic acid)
  • the molecular weight of the nanoparticle is less than or equal to about 20,000 Daltons.
  • the oligonucleotide is a modified polynucleotide.
  • the oligonucleotide is a lipid-modified polynucleotide.
  • the lipid is cholesterol, tocopherol, or stearyl.
  • the oligonucleotide and the PLGA comprise complementary reactive moieties that together form a covalent bond.
  • the reactive moiety on the PLGA comprises an azide, an alkyne, a maleimide, a thiol, an alcohol, an amine, a carboxylic acid, an olefin, an isothiocyanate, a N-hydroxysuccinimide, a phosphine, a nitrone, a norbornene, an oxanorbornene, a transcycloctene, an s-tetrazene, an isocyanide, a tetrazole, a nitrile oxide, a quadricyclane, or a carbodiimide.
  • the reactive moiety on the oligonucleotide is on a terminus of the oligonucleotide.
  • the reactive moiety on the oligonucleotide comprises an alkyne, an azide, a maleimide, a thiol, an alcohol, an amine, a carboxylic acid, an olefin, an isothiocyanate, a N-hydroxysuccinimide, a phosphine, a nitrone, a norbornene, an oxanorbornene, a transcycloctene, an s-tetrazene, an isocyanide, a tetrazole, a nitrile oxide, a quadricyclane, or a carbodiimide.
  • the alkyne comprises dibenzocyclooctyl (DBCO) alkyne or a terminal alkyne.
  • DBCO dibenzocyclooctyl
  • the PLGA comprises an azide reactive moiety and the oligonucleotide comprises an alkyne reactive moiety, or vice versa.
  • the alkyne reactive moiety comprises a DBCO alkyne.
  • nanoparticle is at least about 2 pmol/cm 2 .
  • the density of oligonucleotide on the surface of the nanoparticle is at least about 5 pmol/cm 2 .
  • the density of oligonucleotide on the surface of the nanoparticle is at least about 15 pmol/cm 2 .
  • the density of oligonucleotide on the surface of the nanoparticle is at least about 16 pmol/cm 2 , at least about 17 pmol/cm 2 , at least about 18 pmol/cm 2 , at least about 19 pmol/cm 2 , at least about 20 pmol/cm 2 , or higher.
  • the oligonucleotide comprises RNA or DNA.
  • the RNA is a non-coding RNA.
  • the non-coding RNA is an inhibitory RNA (RNAi).
  • the RNAi is selected from the group consisting of a small inhibitory RNA (siRNA), a single-stranded RNA (ssRNA) that forms a triplex with double stranded DNA, and a ribozyme.
  • the RNA is a microRNA.
  • the DNA is antisense-DNA.
  • diameter of said nanoparticle is less than or equal to about 50 nanometers.
  • the agent is encapsulated in the nanoparticle. In further embodiments, the agent is conjugated to the surface of the nanoparticle. In still further embodiments, the agent is encapsulated in the nanoparticle and conjugated to the surface of the nanoparticle.
  • the agent is an imidazole, poly or oligoimidazole, PEI, a peptide, a fusogenic peptide, a polycaboxylate, a polyacation, a masked oligo, a poly cation or anion, an acetal, a polyacetal, a ketal/polyketyal, an orthoester, a polymer with masked or unmasked cationic or anionic charges, or a dendrimer with masked or unmasked cationic or anionic charges.
  • a nanoparticle of the disclosure further comprises a therapeutic.
  • the therapeutic is a chemotherapeutic.
  • the therapeutic is encapsulated in the nanoparticle. In some embodiments, the therapeutic is conjugated to the surface of the nanoparticle. In still further embodiments, the agent is encapsulated in the nanoparticle and conjugated to the surface of the nanoparticle.
  • the disclosure provides a method of inhibiting expression of a gene comprising the step of hybridizing a polynucleotide encoding said gene product with one or more oligonucleotides complementary to all or a portion of said polynucleotide, said
  • oligonucleotide being attached to the nanoparticle of the disclosure, wherein hybridizing between said polynucleotide and said oligonucleotide occurs over a length of said
  • polynucleotide with a degree of complementarity sufficient to inhibit expression of said gene product.
  • expression of said gene product is inhibited in vivo. In further embodiments, expression of said gene product is inhibited in vitro.
  • the nanoparticle has a diameter about less than or equal to 50 nanometers.
  • the oligonucleotide comprises RNA or DNA.
  • the RNA is a non-coding RNA.
  • the non-coding RNA is an inhibitory RNA (RNAi).
  • the RNAi is selected from the group consisting of a small inhibitory RNA (siRNA), a single-stranded RNA (ssRNA) that forms a triplex with double stranded DNA, and a ribozyme.
  • the RNA is a microRNA.
  • the DNA is antisense-DNA.
  • a method for up-regulating activity of a toll-like receptor comprising contacting a cell having the toll-like receptor with a nanoparticle of the disclosure.
  • the method is performed in vitro. In some embodiments, the method is performed in vivo.
  • the oligonucleotide is a TLR agonist.
  • the toll-like receptor is chosen from the group consisting of toll-like receptor 1 , toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 6, toll-like receptor 7, toll-like receptor 8, toll-like receptor 9, toll-like receptor 10, toll-like receptor 1 1 , toll-like receptor 12, and toll-like receptor 13.
  • the disclosure provides a method for down-regulating activity of a toll-like receptor (TLR), comprising contacting a cell having the toll-like receptor with a nanoparticle of the disclosure.
  • TLR toll-like receptor
  • the method is performed in vitro. In some embodiments, the method is performed in vivo.
  • the oligonucleotide is a TLR antagonist.
  • the toll-like receptor is chosen from the group consisting of toll-like receptor 1 , toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 6, toll-like receptor 7, toll-like receptor 8, toll-like receptor 9, toll-like receptor 10, toll-like receptor 1 1 , toll-like receptor 12, and toll-like receptor 13.
  • Figure 1 depicts an example synthesis of PLGA-SNAs utilizing nanoprecipitation and Cu-free click chemistry.
  • Figure 2 depicts an example synthesis of lipid-modified PLGA-SNAs comprising lipid- modified oligonucleotides.
  • Figure 3 shows results of experiments performed to characterize the PLGA-SNAs comprising lipid-modified oligonucleotides, (a) the size of PLGA-SNA can be tuned by changing initial polymer concentration, (b) PLGA nanoparticle core size is approximately 46 nanometers (nm) measured by TEM with 2.5 mg/ml initial polymer concentration, (c) PLGA-SNAs synthesized with three lipophilic linkers are resolved by 1 % agarose gel electrophoresis.
  • Lane descriptions - 1 PLGA-SNAs synthesized with tocopherol-modified DNA; 2: tocopherol- modified free DNA; 3: PLGA-SNAs synthesized with stearyl-modified DNA; 4: stearyl-modified free DNA; 5: PLGA-SNAs synthesized with cholesterol-modified DNA; 6: cholesterol-modified free DNA. (d) maximizing loading by changing polymer to DNA ratio.
  • Figure 4 shows results of further studies characterizing the PLGA-SNAs.
  • Lane description - 1 PLGA-SNAs synthesized with tocopherol-modified DNA; 2: tocopherol-modified free DNA; 3: PLGA-SNAs synthesized with stearyl-modified DNA; 4: stearyl-modified free DNA; (d) surface loading of PLGA-SNAs synthesized with stearyl and tocopherol-modified DNA.
  • Figure 5 shows results of experiments testing the cooperative binding and cellular uptake abilities of PLGA-SNAs.
  • (red) labeled PLGA-SNAs and Cy5 labeled free DNA incubated at 0.5, 2, 8, and 24 hours.
  • Figure 6 shows the size distribution of PLGA-SNAs in water, 1 X PBS, and 1 X PBS containing 0.1 % Tween 20.
  • Figure 7 shows (a) titration of PLGA-SNAs synthesized with Cy3 labeled DNA. (b) visualization of PLGA-SNAs hybridized with AuSNAs synthesized with a complementary sequence, (c) and (d) melting profile of PLGA-SNA.
  • Figure 8 describes experiments that utilize PLGA-SNAs comprising an agent that enhances or facilitates endosomal escape.
  • Figure 9 shows the size distribution of PLGA-PEG-N 3 as a function of PLGA concentration measured by DLS.
  • Figure 10 shows (A) Linear DNA and PLGA-SNA resolved by 1 % agarose gel electrophoresis (B) TEM image of PLGA-N 3 -PEG NPs.
  • Figure 11 shows: (A, C) (Insets) Images of PLGA-PEG-N 3 NPs and PLGA-SNAs acquired by AFM. Histograms were fit to Gaussian distributions with an average height of 49 ⁇ 13 nm for PLGA-PEG-N 3 NPs and 66 ⁇ 19 nm for PLGA-SNAs.
  • D Cooperative melting profile of PLGA-SNAs. Green particles: 13-nm Au SNAs synthesized with complementary sequences. Inset: Optical image of red precipitates after Au SNAs were incubated with PLGA-SNAs that bear a complementary sequence.
  • Figure 12 shows (A) PLGA-SNA nanoparticle concentration measured by NTA; (b) Cy5 fluorescence calibration curve with a concentration range from 0 to 100 nM.
  • Figure 13 shows the first derivative of SNA melting profile.
  • Figure 14 shows (A) PLGA-PEG-N 3 NP size as a function of polymer composition measured by DLS. (B) Drug encapsulation efficiency of RG 504 as a function of mass percent of coumarin 6.
  • Figure 15 depicts (A) Scheme of a FRET PLGA-SNA and FRET turn-on experiment. Rhodamine was excited at 530 nm and the emission spectrum was recorded from 550 to 700 nm. (B) Representative fluorescence kinetics landscape of FRET PLGA-SNAs. (C) Release profiles of nucleic acids on the surface of the SNAs in 10% FBS. [0040] Figure 16 shows: (A) Coumarin 6 was utilized as a fluorescent model drug encapsulated inside the PLGA matrix for the evaluation of drug release kinetics. (B) Release kinetics of coumarin 6 from PLGA-SNAs prepared from the three polymer compositions in 10% FBS.
  • Figure 18 depicts DNase I resistance of PLGA-SNAs (left) and free DNA (right).
  • the PLGA-SNAs and linear DNA were loaded onto the gel at the same concentration as their controls.
  • a new class of polymer spherical nucleic acid (SNA) conjugates comprised of poly(lactic-co-glycolic acid) (PLGA) nanoparticle (NP) cores is disclosed herein.
  • the nucleic acid shell that defines the PLGA-SNA exhibits a half-life of more than two hours in fetal bovine serum.
  • the ideal SNA should exhibit the following properties: First, it should consist of a biocompatible organic core capable of carrying and temporally releasing drugs. Second, it should contribute to the long-term stability of the nucleic acid shell so that the chemical and biophysical properties of such a construct can be retained under challenging physiological conditions. Finally, it should be straightforward to synthesize in a scalable manner.
  • poly(lactic-co-glycolic acid) is an attractive material as the core for SNAs. It is biocompatible and biodegradable [Danhier et al., J. Control. Release 2012, 161 , 505; Gilding et al., Polymer 1979, 20, 1459], relatively stable [Zolnik et al., J. Control. Release 2007, 122, 338], and exhibits composition-dependent and therefore tunable release kinetics for encapsulated cargos [Farokhzad et al., Proc. Natl. Acad. Sci. USA 2006, 103, 6315; Astete et al., J. Biomater. Sci. Polym. Ed.
  • PLGA-SNAs from PLGA particle cores [Fessi et al., Int. J. Pharm. 1989, 55, R1 ].
  • the PLGA core is terminated with azides and oligonucleotides are terminated with the dibenzocyclooctyne (DBCO) group via Cu-free click chemistry.
  • the PLGA-SNAs are produced from 50 nm diameter PLGA particle cores.
  • aptamer-PLGA conjugates for targeting purposes were produced [Farokhzad et al., Proc. Natl. Acad. Sci. USA 2006, 103, 6315; Cheng et al., Biomaterials 2007, 28, 869].
  • the aptamer-PLGA conjugates did not comprise an agent that facilitates escape of the nanoparticle from an endosome.
  • the PLGA- SNAs disclosed herein demonstrate polymer composition-dependent release from an oligonucleotide-polymer nanoparticle conjugate ⁇ i.e., SNAs).
  • PLGA-SNAs of the present disclosure are utilized to encapsulate a hydrophobic drug, which can then be released in a polymer composition- dependent tunable manner, while the dissociation rate of the nucleic acid shell remains relatively constant, regardless of core composition.
  • the nanoparticles disclosed herein provide a means for controlling the release kinetics of encapsulated cargos in the context of the SNA platform, which is useful for developing combination therapeutics.
  • PLGA-SNAs may be synthesized using several strategies, including but not limited to the following. First, PLGA-SNAs may be synthesized by conjugating lipid-modified
  • oligonucleotides to the surface of PLGA nanoparticles via hydrophobic-hydrophobic interactions.
  • PLGA-SNAs may be synthesized by conjugating oligonucleotide and the
  • PLGA which comprise complementary reactive moieties that together form a covalent bond.
  • DBCO-modified DNA strands are then covalently conjugated to, e.g., azide groups through Cu-free click chemistry [Baskin, et al. Proc. Natl. Acad. Sci. U. S. A. 2007,
  • alkyne moieties can be used instead, including a terminal alkyne (HC ⁇ C-) or an internal alkyne (RC ⁇ C-, where R comprises an alkyl).
  • the alkyne moiety can also be attached to the oligonucleotide via a linker.
  • the reactive moiety on the polymer comprises an azide, an alkyne, a maleimide, a thiol, an alcohol, an amine, a carboxylic acid, an olefin, an isothiocyanate, a N- hydroxysuccinimide, a phosphine, a nitrone, a norbornene, an oxanorbornene, a
  • the reactive moiety on the oligonucleotide is on a terminus of the oligonucleotide.
  • the reactive moiety on the oligonucleotide comprises an alkyne, an azide, a maleimide, a thiol, an alcohol, an amine, a carboxylic acid, an olefin, an isothiocyanate, a N-hydroxysuccinimide, a phosphine, a nitrone, a norbornene, an oxanorbornene, a transcycloctene, an s-tetrazene, an isocyanide, a tetrazole, a nitrile oxide, a quadricyclane, or a carbodiimide.
  • the alkyne comprises dibenzocyclooctyl (DBCO) alkyne or a terminal alkyne.
  • the polymer ⁇ e.g., PLGA or PLGA-PEG) comprises an azide reactive moiety and the oligonucleotide comprises an alkyne reactive moiety, or vice versa.
  • the alkyne reactive moiety comprises a DBCO alkyne.
  • the PLGA-SNAs of the disclosure may contain a polymer selected from the group consisting of diblock poly(lactic) acid-poly(ethylene)glycol (PLA-PEG) copolymer, diblock poly(lactic acid-co-glycolic acid)-poly(ethylene)glycol (PLGA-PEG) copolymer, and combinations thereof.
  • a polymer selected from the group consisting of diblock poly(lactic) acid-poly(ethylene)glycol (PLA-PEG) copolymer, diblock poly(lactic acid-co-glycolic acid)-poly(ethylene)glycol (PLGA-PEG) copolymer, and combinations thereof.
  • Nanoparticles disclosed herein include one, two, three or more biocompatible and/or biodegradable polymers.
  • a contemplated nanoparticle may include about 35 to about 99.75 weight percent in some embodiments; about 50 to about 99.75 weight percent, in some other embodiments; about 50 to about 99.5 weight percent, in some embodiments; about
  • a contemplated nanoparticle may include 35 to 99.75 weight percent in some embodiments; 50 to 99.75 weight percent, in some other embodiments; 50 to 99.5 weight percent, in some embodiments; 50 to 99 weight percent in still other embodiments; 50 to 98 weight percent in further embodiments; 50 to 97 weight percent in still further embodiments; 50 to 96 weight percent in additional embodiments; 50 to 95 weight percent in other embodiments, 50 to 94 weight percent in still other embodiments; 50 to 93 weight percent in other embodiments; 50 to 92 weight percent in still other embodiments; 50 to 91 weight percent, in some embodiments 50 to 90 weight percent; in some embodiments, 50 to 85 weight percent; in some embodiments 60 to 85 weight percent; in some embodiments, 65 to 85 weight percent; and in some embodiments, 50 to 80 weight percent of one or more block copolymers that include a biodegradable polymer and poly(ethylene glycol) (PEG), and 0 to 50 weight percent of a biodegradable homopolymer
  • contemplated biocompatible polymers may be biodegradable, i.e., the polymer is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body.
  • biodegradable polymers are those that, when introduced into cells, are broken down by the cellular machinery (biologically degradable) and/or by a chemical process, such as hydrolysis, (chemically degradable) into components that the cells can either reuse or dispose of without significant toxic effect on the cells.
  • the biodegradable polymer and their degradation byproducts can be biocompatible.
  • Particles disclosed herein may or may not contain PEG.
  • a contemplated polymer may be one that hydrolyzes spontaneously upon exposure to water (e.g., within a subject), or the polymer may degrade upon exposure to heat ⁇ e.g., at temperatures of about 37° C). Degradation of a polymer may occur at varying rates, depending on the polymer or copolymer used. For example, the half-life of the polymer (the time at which 50% of the polymer can be degraded into monomers and/or other nonpolymeric moieties) may be on the order of minutes, hours, days, weeks, months, or years, depending on the polymer.
  • polymers may be polyesters, including copolymers comprising lactic acid and glycolic acid units, such as poly(lactic acid-co-glycolic acid) and poly(lactide-co- glycolide), collectively referred to herein as "PLGA”; and homopolymers comprising glycolic acid units, referred to herein as "PGA,” and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as "PLA.”
  • exemplary polyesters include, for example, polyhydroxyacids; PEGylated polymers and copolymers of lactide and glycolide (e.g.,
  • polyesters include, for example, polyanhydrides, poly(ortho ester) PEGylated poly(ortho ester), poly(caprolactone), PEGylated poly(caprolactone), polylysine, PEGylated polylysine, poly(ethylene imine), PEGylated poly(ethylene imine), poly(L-lactide-co-L-lysine), poly(serine ester), poly(4-hydroxy-L-proline ester), poly[.alpha.-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.
  • a polymer may be PLGA.
  • PLGA is a biocompatible and biodegradable co-polymer of lactic acid and glycolic acid, and various forms of PLGA can be characterized by the ratio of lactic acid:glycolic acid.
  • Lactic acid can be L-lactic acid, D-lactic acid, or D, L-lactic acid.
  • the degradation rate of PLGA can be adjusted by altering the lactic acid-glycolic acid ratio.
  • PLGA can be characterized by a lactic acid:glycolic acid molar ratio of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15:85.
  • the molar ratio of lactic acid to glycolic acid monomers in the polymer of the particle ⁇ e.g., the PLGA block copolymer or PLGA-PEG block copolymer may be selected to optimize for various parameters such as water uptake, therapeutic agent release and/or polymer degradation kinetics can be optimized.
  • a disclosed particle can for example comprise a diblock copolymer of PEG and PL(G)A, wherein for example, the PEG portion may have a number average molecular weight of about 1 ,000-20,000, e.g., about 2,000-20,000, e.g., about 2 to about 10,000, and the PL(G)A portion may have a number average molecular weight of about 5,000 to about 20,000, or about 5,000-100,000, e.g., about 20,000-70,000, e.g., about 15,000-50,000.
  • an exemplary nanoparticle of the disclosure that includes from about 10 to about 99 weight percent poly(lactic) acid-poly(ethylene)glycol copolymer or poly(lactic)-co- poly(glycolic) acid-poly(ethylene)glycol copolymer, or from about 50 to about 99.75 weight percent, from about 20 to about 80 weight percent, from about 40 to about 80 weight percent, or from about 30 to about 50 weight percent, or from about 70 to about 90 weight percent, from about 70 to about 99.75 weight percent, from about 80 to about 99.75 weight percent, from about 70 to about 80 weight percent, or from about 85 to about 95 weight percent poly(lactic) acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly(glycolic) acid-poly(ethylene)glycol copolymer.
  • a therapeutic nanoparticle comprises about 50 weight percent, about 55 weight percent, about 60 weight percent, about 65 weight percent, about 70 weight percent, about 75 weight percent, about 80 weight percent, about 85 weight percent, about 90 weight percent or about 95 weight percent poly(lactic) acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly(glycolic) acid-poly(ethylene)glycol copolymer.
  • Exemplary poly(lactic) acid-poly(ethylene)glycol copolymers can include a number average molecular weight ranging from about 15 to about 20 kilodaltons (kDa), or from about 10 to about 25 kDa of poly(lactic) acid and a number average molecular weight from about 4 kDa to about 6 kDa, from about 4 kDa to about 10 kDa, from about 6 kDa to about 10 kDa, or from about 2 kDa to about 10 kDa of poly(ethylene)glycol.
  • kDa kilodaltons
  • an exemplary therapeutic nanoparticle that includes from 10 to 99 weight percent poly(lactic) acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly(glycolic) acid-poly(ethylene)glycol copolymer, or from 50 to 99.75 weight percent, from 20 to 80 weight percent, from 40 to 80 weight percent, or from 30 to 50 weight percent, or from 70 to 90 weight percent, from 70 to 99.75 weight percent, from 80 to 99.75 weight percent, from 70 to 80 weight percent, or from 85 to 95 weight percent poly(lactic) acid- poly(ethylene)glycol copolymer or poly(lactic)-co-poly(glycolic) acid-poly(ethylene)glycol copolymer.
  • a therapeutic nanoparticle comprises 50 weight percent, 55 weight percent, 60 weight percent, 65 weight percent, 70 weight percent, 75 weight percent, 80 weight percent, 85 weight percent, 90 weight percent or 95 weight percent poly(lactic) acid- poly(ethylene)glycol copolymer or poly(lactic)-co-poly(glycolic) acid-poly(ethylene)glycol copolymer.
  • Exemplary poly(lactic) acid-poly(ethylene)glycol copolymers can include a number average molecular weight ranging from 15 to 20 kDa, or from 10 to 25 kDa of poly(lactic) acid and a number average molecular weight from 4 kDa to 6 kDa, from 4 kDa to 10 kDa, from 6 kDa to 10 kDa, or from 2 kDa to 10 kDa of poly(ethylene)glycol.
  • a nanoparticle of the disclosure has an average molecular weight of from about 7 kDa to about 17 kDa. According to the present disclosure, a nanoparticle having an average molecular weight of greater than about 17 kDa is characterized as a "slow release” nanoparticle, whereas a nanoparticle having an average molecular weight of less than about 17 kDa is characterized as a "fast release” nanoparticle.
  • the present disclosure provides methods for regulating release kinetics in the context of a PLGA-SNA; the shell of oligonucleotides surrounding the PLGA nanoparticle (see, e.g., Figures 1 and 2) provides an entirely different construct relative to previously described PLGA nanoparticles.
  • a nanoparticle of the disclosure further comprises an agent that facilitates escape of the nanoparticle from an endosome and/or a therapeutic.
  • disclosed nanoparticles may include about 0.2 to about 35 weight percent, about 0.2 to about 25 weight percent, about 0.2 to about 20 weight percent, about 0.2 to about 10 weight percent, about 0.2 to about 5 weight percent, about 0.5 to about 5 weight percent, about 0.75 to about 5 weight percent, about 1 to about 5 weight percent, about 2 to about 5 weight percent, about 3 to about 5 weight percent, about 1 to about 20 weight percent, about 2 to about 20 weight percent, about 3 to about 20 weight percent, about 4 to about 20 weight percent, about 5 to about 20 weight percent, about 1 to about 15 weight percent, about 2 to about 15 weight percent, about 3 to about 15 weight percent, about 4 to about 15 weight percent, about 5 to about 15 weight percent, about 1 to about 10 weight percent, about 2 to about 10 weight percent, about 3 to about 10 weight percent, about 4 to about 10 weight percent, about 5 to about 10 weight percent, about 10 to about 30 weight percent, or about 15 to about 25 weight percent of the therapeutic.
  • the disclosed nanoparticles include about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 1 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 weight percent of the agent.
  • disclosed nanoparticles may include 0.2 to 35 weight percent, 0.2 to 25 weight percent, 0.2 to 20 weight percent, 0.2 to 10 weight percent, 0.2 to 5 weight percent, 0.5 to 5 weight percent, 0.75 to 5 weight percent, 1 to 5 weight percent, 2 to 5 weight percent, 3 to 5 weight percent, 1 to 20 weight percent, 2 to 20 weight percent, 3 to 20 weight percent, 4 to 20 weight percent, 5 to 20 weight percent, 1 to 15 weight percent, 2 to 15 weight percent, 3 to 15 weight percent, 4 to 15 weight percent, 5 to 15 weight percent, 1 to 10 weight percent, 2 to 10 weight percent, 3 to 10 weight percent, 4 to 10 weight percent, 5 to 10 weight percent, 10 to 30 weight percent, or 15 to 25 weight percent of the therapeutic.
  • the disclosed nanoparticles include 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 weight percent of the agent.
  • a nanoparticle of the disclosure further comprises a therapeutic.
  • disclosed nanoparticles may include about 0.2 to about 35 weight percent, about 0.2 to about 25 weight percent, about 0.2 to about 20 weight percent, about 0.2 to about 10 weight percent, about 0.2 to about 5 weight percent, about 0.5 to about 5 weight percent, about 0.75 to about 5 weight percent, about 1 to about 5 weight percent, about 2 to about 5 weight percent, about 3 to about 5 weight percent, about 1 to about 20 weight percent, about 2 to about 20 weight percent, about 3 to about 20 weight percent, about 4 to about 20 weight percent, about 5 to about 20 weight percent, about 1 to about 15 weight percent, about 2 to about 15 weight percent, about 3 to about 15 weight percent, about 4 to about 15 weight percent, about 5 to about 15 weight percent, about 1 to about 10 weight percent, about 2 to about 10 weight percent, about 3 to about 10 weight percent, about 4 to about 10 weight percent, about 5 to about 10 weight percent, about 10 to about 30 weight percent, or
  • the disclosed nanoparticles include about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 1 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 weight percent of the therapeutic.
  • disclosed nanoparticles may include 0.2 to 35 weight percent, 0.2 to 25 weight percent, 0.2 to 20 weight percent, 0.2 to 10 weight percent, 0.2 to 5 weight percent, 0.5 to 5 weight percent, 0.75 to 5 weight percent, 1 to 5 weight percent, 2 to 5 weight percent, 3 to 5 weight percent, 1 to 20 weight percent, 2 to 20 weight percent, 3 to 20 weight percent, 4 to 20 weight percent, 5 to 20 weight percent, 1 to 15 weight percent, 2 to 15 weight percent, 3 to 15 weight percent, 4 to 15 weight percent, 5 to 15 weight percent, 1 to 10 weight percent, 2 to 10 weight percent, 3 to 10 weight percent, 4 to 10 weight percent, 5 to 10 weight percent, 10 to 30 weight percent, or 15 to 25 weight percent of the therapeutic.
  • the disclosed nanoparticles include 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 weight percent of the therapeutic.
  • the molar ratio of agent to therapeutic ⁇ e.g., initially during formulation of the nanoparticles and/or in the nanoparticles may range from about 0.25:1 to about 6:1 , in some embodiments from about 0.25:1 to about 5:1 , in some embodiments from about 0.25:1 to about 4:1 , in some embodiments, from about 0.25:1 to about 3:1 , in some embodiments from about 0.25:1 to about 2:1 , in some embodiments, from about 0.25:1 to about
  • 0.25:1 to about 0.5:1 in some embodiments from about 0.5:1 to about 6:1 , in some
  • the molar ratio of agent to therapeutic ⁇ e.g., initially during formulation of the nanoparticles and/or in the nanoparticles may range from 0.25:1 to 6:1 , in some embodiments from 0.25:1 to 5:1 , in some embodiments from 0.25:1 to 4:1 , in some embodiments, from 0.25:1 to 3:1 , in some embodiments from 0.25:1 to 2:1 , in some
  • from 0.25:1 to 1 .5:1 in some embodiments, from 0.25:1 to 1 :1 , in some embodiments, from 0.25:1 to 0.5:1 , in some embodiments from 0.5:1 to 6:1 , in some
  • from 1 :1 to 2:1 in some embodiments from 1 :1 to 1 .5:1 , in some embodiments, from 1 .5:1 to 6:1 , in some embodiments, from 1 .5:1 to 5:1 , in some embodiments from 1 .5:1 to 4:1 , in some embodiments from 1 .5:1 to 3:1 , in some embodiments from 2:1 to 6:1 , in some embodiments from 2:1 to 4:1 , in some embodiments, from 3:1 to 6:1 , in some embodiments, from 3:1 to 5:1 , and in some embodiments, from 4:1 to 6:1 .
  • the particle size of a PLGA-SNA created by a method of the disclosure is less than or equal to about 50 nanometers.
  • a plurality of PLGA-SNAs is produced and the PLGA-SNAs in the plurality have a mean diameter of less than or equal to about 50 nanometers ⁇ e.g., about 5 nanometers to about 50 nanometers, or about 5 nanometers to about 40 nanometers, or about 5 nanometers to about 30 nanometers, or about 5 nanometers to about 20 nanometers, or about 10 nanometers to about 50 nanometers, or about 10
  • the PLGA-SNAs in the plurality have a mean diameter of less than or equal to about 20 nanometers, or less than or equal to about 25 nanometers, or less than or equal to about 30 nanometers, or less than or equal to about 35 nanometers, or less than or equal to about 40 nanometers, or less than or equal to about 45 nanometers.
  • disclosed nanoparticles substantially immediately release the agent and/or the therapeutic ⁇ e.g., from about 1 minute to about 30 minutes, about 1 minute to about 25 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 1 hour, about
  • the release profile is slower: about 2% or less; about
  • FBS fetal bovine serum
  • nanoparticles comprising the agent and/or the therapeutic may release the agent and/or the therapeutic when placed in a solution ⁇ e.g., 10% FBS) e.g., at 25°
  • nanoparticles comprising the agent and/or the therapeutic may release the agent and/or the therapeutic when placed in a solution ⁇ e.g., 10% FBS), e.g., at 25° C. and/or at 37° C, at a rate substantially corresponding to about 10 to about 70%, in some embodiments about 10 to about
  • nanoparticles comprising the agent and/or the therapeutic may release the agent and/or the therapeutic when placed in a solution ⁇ e.g., 10% FBS) e.g., at 25° C. and/or at 37° C, at a rate substantially corresponding to about 0.01 to about 50%, in some embodiments about 0.01 to about 25%, in some embodiments about 0.01 to about 1 5%, in some embodiments about 0.01 to about 10%, in some embodiments about 0.01 to about 5%, and in some embodiments about 0.01 to about 3% of the agent and/or the therapeutic released by weight over about 4 hours.
  • nanoparticles comprising the agent and/or the therapeutic may release the agent and/or the therapeutic when placed in a solution (e.g., 1 0% FBS) e.g., at 25° C.
  • nanoparticles comprising the agent and/or the therapeutic may release the agent and/or the therapeutic when placed in serum (e.g., 1 0% FBS) e.g., at 25° C.
  • nanoparticles comprising the agent and/or the therapeutic may release the agent and/or the therapeutic when placed in serum (e.g., 10% FBS) e.g., at 25° C.
  • nanoparticles comprising the agent and/or the therapeutic may release the agent and/or the therapeutic when placed in serum (e.g., a phosphate buffer solution) e.g., at 25° C.
  • serum e.g., a phosphate buffer solution
  • disclosed nanoparticles substantially immediately release the agent and/or therapeutic (e.g., from 1 minute to 30 minutes, 1 minute to 25 minutes, 5 minutes to 30 minutes, 5 minutes to 1 hour, 1 hour, or 24 hours).
  • the release profile is slower: 2% or less; 5% or less; 10% or less; 15% or less; 20% or less; 25% or, 30% or less 40% or less of the agent and/or the therapeutic, by weight is released for example, when placed in 10% FBS, at room temperature ⁇ e.g., 25° C.) and/or at 37° C.
  • nanoparticles comprising the agent and/or the therapeutic may release the agent and/or the therapeutic when placed in serum (e.g., 10% FBS) e.g., at 25° C. and/or at 37° C, at a rate substantially corresponding to 0.01 to 50%, in some embodiments 0.01 to 25%, in some embodiments 0.01 to 15%, in some embodiments 0.01 to 10%, in some embodiments 1 to 40%, in some embodiments 5 to 40%, and in some embodiments 10 to 40% of the agent and/or the therapeutic released by weight over 1 hour.
  • nanoparticles comprising the agent and/or the therapeutic may release the agent and/or the therapeutic when placed in serum (e.g., 10% FBS), e.g., at 25° C.
  • nanoparticles comprising the agent and/or the therapeutic may release the agent and/or the therapeutic when placed in serum (e.g., 10% FBS) e.g., at 25° C.
  • nanoparticles comprising the agent and/or the therapeutic may release the agent and/or the therapeutic when placed in serum (e.g., 10% FBS) e.g., at 25° C.
  • nanoparticles comprising the agent and/or the therapeutic may release the agent and/or the therapeutic when placed in serum (e.g., a phosphate buffer solution) e.g., at 25° C.
  • serum e.g., a phosphate buffer solution
  • nanoparticles comprising the agent and/or the therapeutic may release the agent and/or the therapeutic when placed in serum (e.g., 10% FBS) e.g., at 25° C.
  • nanoparticles comprising the agent and/or the therapeutic may release the agent and/or the therapeutic when placed in serum (e.g., 10% FBS) e.g., at 25° C.
  • disclosed nanoparticles may substantially retain the agent and/or the therapeutic, e.g., for at least about 1 minute, at least about 1 hour, or more, when placed in 10% FBS at 37° C.
  • disclosed nanoparticles may substantially retain the agent and/or the therapeutic, e.g., for at least 1 minute, at least 1 hour, or more, when placed in 10% FBS at 37° C.
  • the oligonucleotide shell of disclosed nanoparticles substantially immediately dissociate ⁇ e.g., from about 1 minute to about 30 minutes, about 1 minute to about 25 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 1 hour, about 1 hour, or about 24 hours).
  • the dissociation profile is slower: about 2% or less; about 5% or less; about 10% or less; about 15% or less; about 20% or less; about 25% or, about 30% or less about 40% or less of the oligonucleotides, by weight is dissociated for example, when placed in, e.g., 10% fetal bovine serum (FBS), at room temperature ⁇ e.g., 25° C.) and/or at 37° C.
  • FBS fetal bovine serum
  • the PLGA-SNA constructs of the disclosure further comprise an agent that enhances endosomal escape.
  • the agent is an endosome escaping peptide that is encapsulated in the PLGA matrix.
  • the endosomal release agents include include imidazoles, poly or oligoimidazoles, PEIs, peptides, fusogenic peptides, polycaboxylates, polyacations, masked oligo or poly cations or anions, acetals, polyacetals, ketals/polyketyals, orthoesters, polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges.
  • a PLGA-SNA construct of the disclosure further comprises an endosomal escape agent as disclosed in U.S. Patent Application Publication No. 2017/0275650 or U.S. Patent Application Publication No. 2017/0260274, incorporated by reference herein in their entireties.
  • PLGA-SNAs of the disclosure comprise one or more oligonucleotides conjugated to the surface.
  • the one or more oligonucleotides are not encapsulated in or otherwise inside the PLGA-SNA.
  • Oligonucleotides contemplated for use according to the disclosure are from about 5 to about 100 nucleotides in length. Methods and compositions are also contemplated wherein the oligonucleotide is about 5 to about 90 nucleotides in length, about 5 to about 80 nucleotides in length, about 5 to about 70 nucleotides in length, about 5 to about 60 nucleotides in length, about 5 to about 50 nucleotides in length about 5 to about 45 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 35 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to about 15 nucleotides in length, about 5 to about 10 nucleotides in length, and all oligonucleotides intermediate in length of the sizes specifically disclosed to the extent that the oligonucleot
  • oligonucleotides of 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24,25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, and 100 nucleotides in length are contemplated.
  • Modified Oligonucleotides include those containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are considered to be within the meaning of "oligonucleotide.”
  • Modified oligonucleotide backbones containing a phosphorus atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates,
  • phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters,
  • selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5', or 2' to 2' linkage.
  • oligonucleotides having inverted polarity comprising a single 3' to 3' linkage at the 3'-most internucleotide linkage, i.e. a single inverted nucleoside residue which may be abasic (the nucleotide is missing or has a hydroxyl group in place thereof). Salts, mixed salts and free acid forms are also contemplated.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • oligonucleotide mimetics wherein both one or more sugar and/or one or more internucleotide linkage of the nucleotide units are replaced with "non- naturally occurring” groups.
  • this embodiment contemplates a peptide nucleic acid (PNA).
  • PNA compounds the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone. See, for example US Patent Nos. 5,539,082; 5,714,331 ; and 5,719,262, and Nielsen et al., 1991 , Science, , 254: 1497-1500, the disclosures of which are herein incorporated by reference.
  • oligonucleotides are provided with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and including— CH 2 — NH— O— CH 2 — ,— CH 2 — N(CH 3 )— O— CH 2 — classroom— CH 2 — O— N(CH 3 )— CH 2 — ,— CH 2 — N(CH 3 )— N(CH 3 )— CH 2 — and—0—N(CH 3 )—CH 2 —CH 2 — described in US Patent Nos. 5,489,677, and 5,602,240.
  • oligonucleotides with morpholino backbone structures described in US Patent No. 5,034,506.
  • RH is selected form hydrogen and Ci- 4 -alkyl
  • R" is selected from Ci- 6 -alkyl and phenyl
  • Modified oligonucleotides may also contain one or more substituted sugar moieties.
  • oligonucleotides comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to C10 alkyl or C 2 to C10 alkenyl and alkynyl.
  • oligonucleotides comprise one of the following at the 2' position: Ci to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN,
  • a modification includes 2'- methoxyethoxy (2 , -0-CH2CH 2 OCI-l3, also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al., 1995, Helv. Chim. Acta, 78: 486-504) i.e., an alkoxyalkoxy group.
  • 2'-dimethylaminooxyethoxy i.e., a 0(CH 2 )20N(CH 3 )2 group, also known as 2'-DMAOE, as described in examples herein below
  • 2'-dimethylaminoethoxyethoxy also known in the art as 2'-0-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE
  • 2'-0— CH 2 — O— CH 2 — N(CH 3 ) 2 i.e., 2'-0— CH 2 — O— CH 2 — N(CH 3 ) 2 .
  • the 2'-modification may be in the arabino (up) position or ribo (down) position.
  • a 2'-arabino modification is 2'-F.
  • oligonucleotide Similar modifications may also be made at other positions on the oligonucleotide, for example, at the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. See, for example, U.S. Pat. Nos. 4,981 ,957; 5,1 18,800; 5,319,080;
  • a modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.
  • the linkage is in certain aspects is a methylene (— CH 2 — ) n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2.
  • LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.
  • Oligonucleotides may also include base modifications or substitutions.
  • "unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified bases include other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoro
  • Further modified bases include tricyclic pyrimidines such as phenoxazine cytidine(1 H-pyrimido[5 ,4-b][1 ,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1 H-pyrimido[5 ,4-b][1 ,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine ⁇ e.g.
  • Modified bases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further bases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859,
  • 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1 .2°C. and are, in certain aspects combined with 2'-0-methoxyethyl sugar modifications. See, U.S. Pat. Nos. 3,687,808, U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273;
  • a "modified base” or other similar term refers to a composition which can pair with a natural base ⁇ e.g., adenine, guanine, cytosine, uracil, and/or thymine) and/or can pair with a non-naturally occurring base.
  • the modified base provides a T m differential of 15, 12, 10, 8, 6, 4, or 2°C. or less.
  • Exemplary modified bases are described in EP 1 072 679 and WO 97/12896.
  • nucleobase is meant the naturally occurring nucleobases adenine (A), guanine
  • G cytosine
  • C cytosine
  • T thymine
  • U uracil
  • nucleobases such as xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7-deazaxanthine, 7-deazaguanine, N 4 ,N 4 -ethanocytosin, N',N'-ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C 3 — C 6 )- alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-tr- iazolopyridin, isocytosine, isoguanine, inosine and the "non-naturally occurring" nucleobases described in Benner et al., U.S. Pat. No. 5,432,272 and Susan M. Freier and Karl-Hein
  • nucleobase thus includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Further naturally and non-naturally occurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan, et al.), in Chapter 15 by Sanghvi, in Antisense Research and Application, Ed. S. T. Crooke and B.
  • nucleosidic base or “base unit” is further intended to include compounds such as heterocyclic compounds that can serve like nucleobases including certain "universal bases” that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases.
  • universal bases are 3-nitropyrrole, optionally substituted indoles ⁇ e.g., 5-nitroindole), and optionally substituted hypoxanthine.
  • Other desirable universal bases include, pyrrole, diazole or triazole derivatives, including those universal bases known in the art.
  • RNA can be an inhibitory RNA (RNAi) that performs a regulatory function, and in various embodiments is selected from the group consisting of a small inhibitory RNA (siRNA), an RNA that forms a triplex with double stranded DNA, and a ribozyme.
  • RNAi inhibitory RNA
  • the RNA is microRNA that performs a regulatory function.
  • the DNA is, in some embodiments, an antisense-DNA.
  • the methods of the disclosure allow for the production of PLGA-SNA nanoparticles having a surface density of nucleic acid that is at least about 2 pmol/cm 2 . .
  • the surface density of nucleic acid on the surface of the PLGA-SNA nanoparticle is approximately 10 pmol/cm 2 ,1 1 pmol/cm 2 , 12 pmol/cm 2 , 13 pmol/cm 2 , 14 pmol/cm 2 , 15 pmol/cm 2 , 16 pmol/cm 2 , 17 pmol/cm 2 , 18 pmol/cm 2 , 19 pmol/cm 2 , 20 pmol/cm 2 , or higher.
  • the surface density of oligonucleotide on the surface of the PLGA-SNA nanoparticle is at least 2 pmol/cm 2 , at least 3 pmol/cm 2 , at least 4 pmol/cm 2 , at least 5 pmol/cm 2 , at least 6 pmol/cm 2 , at least 7 pmol/cm 2 , at least 8 pmol/cm 2 , at least 9 pmol/cm 2 , at least 10 pmol/cm 2 , at least about 15 pmol/cm2, at least about 19 pmol/cm 2 , at least about 20 pmol/cm 2 , at least about 25 pmol/cm 2 , at least about 30 pmol/cm 2 , at least about 35 pmol/cm 2 , at least about 40 pmol/cm 2 , at least about 45 pmol/cm 2 , at least about 50 pmol/cm 2 ,
  • the density of oligonucleotide on the surface of the PLGA-SNA is measured by the number of oligonucleotides on the surface of a PLGA-SNA.
  • a PLGA-SNA as described herein comprises from about 1 to about 100 oligonucleotides on its surface.
  • a PLGA-SNA comprises from about 10 to about 100, or from 10 to about 90, or from about 10 to about 80, or from about 10 to about 70, or from about 10 to about 60, or from about 10 to about 50, or from about 10 to about 40, or from about 10 to about 30, or from about 10 to about 20
  • a PLGA-SNA comprises at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 oligonucleotides on its surface.
  • Methods for inhibiting gene product expression include those wherein expression of the target gene product is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% compared to gene product expression in the absence of a PLGA-SNA.
  • the degree of inhibition is determined in vivo from a body fluid sample or from a biopsy sample or by imaging techniques well known in the art. Alternatively, the degree of inhibition is determined in a cell culture assay, generally as a predictable measure of a degree of inhibition that can be expected in vivo resulting from use of a specific type of PLGA-SNA and a specific oligonucleotide.
  • a PLGA-SNA performs both a gene inhibitory function as well as a therapeutic agent delivery function.
  • a therapeutic agent is encapsulated in and/or conjugated to a PLGA-SNA of the disclosure and the particle is additionally functionalized with one or more oligonucleotides designed to effect inhibition of target gene expression or perform some other regulatory function (e.g., target cell recognition).
  • the methods include use of an oligonucleotide which is 100% complementary to the target polynucleotide, i.e., a perfect match, while in other aspects, the oligonucleotide is at least (meaning greater than or equal to) about 95% complementary to the polynucleotide over the length of the oligonucleotide, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20% complementary to the polynucleotide over the length of the oligonucleotide to the extent that the oligonucleotide is able to achieve the desired degree of inhibition of a target gene product.
  • sequence of an antisense compound is 100%
  • an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
  • the sequence of an antisense compound may be about 75%, about 80%, about 85%, about 90%, or about 95% complementary to that of its target nucleic acid.
  • an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event ⁇ e.g., a loop structure or hairpin structure). The percent complementarity is determined over the length of the oligonucleotide.
  • the oligonucleotide would be 90 percent complementary.
  • the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleotides.
  • Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
  • This method comprises the step of hybridizing a polynucleotide encoding said gene product with one or more oligonucleotides complementary to all or a portion of said polynucleotide, said oligonucleotide being attached to a PLGA-SNA, wherein hybridizing between said polynucleotide and said oligonucleotide occurs over a length of said
  • polynucleotide with a degree of complementarity sufficient to inhibit expression of said gene product.
  • the inhibition of gene expression may occur in vivo or in vitro.
  • the oligonucleotide utilized in the methods of the disclosure is either RNA or DNA.
  • the RNA can be an inhibitory RNA (RNAi) that performs a regulatory function, and in various embodiments is selected from the group consisting of a small inhibitory RNA (siRNA), an RNA that forms a triplex with double stranded DNA, and a ribozyme.
  • RNAi inhibitory RNA
  • the RNA is microRNA that performs a regulatory function.
  • the DNA is, in some embodiments, an antisense-DNA.
  • TLRs Toll-like receptors
  • the mammalian immune system uses two general strategies to combat infectious diseases. Pathogen exposure rapidly triggers an innate immune response that is characterized by the production of immunostimulatory cytokines, chemokines and polyreactive IgM antibodies.
  • the innate immune system is activated by exposure to Pathogen Associated Molecular Patterns (PAMPs) that are expressed by a diverse group of infectious microorganisms. The recognition of PAMPs is mediated by members of the Toll-like family of receptors.
  • PAMPs Pathogen Associated Molecular Patterns
  • TLR receptors such as TLR 4, TLR 8 and TLR 9 that response to specific oligonucleotide are located inside special intracellular compartments, called endosomes.
  • endosomes special intracellular compartments, called endosomes.
  • the mechanism of modulation of TLR 4, TLR 8 and TLR9 receptors is based on DNA-protein interactions.
  • immunomodulatory oligonucleotides that contain CpG motifs that are similar to those found in bacterial DNA stimulate a similar response of the TLR receptors. Therefore immunomodulatory oligonucleotides have various potential therapeutic uses, including treatment of immune deficiency and cancer. Employment of PLGA-SNAs conjugated to immunomodulatory oligonucleotides will allow for increased preferential uptake and therefore increased therapeutic efficacy.
  • Down regulation of the immune system would involve knocking down the gene responsible for the expression of the Toll-like receptor.
  • This antisense approach involves use of PLGA-SNAs conjugated to specific antisense oligonucleotide sequences to knock down the expression of any toll-like protein.
  • the method either up-regulates or down-regulates the Toll-like-receptor through the use of a TLR agonist or a TLR antagonist, respectively.
  • the method comprises contacting a cell having a toll-like receptor with a PLGA-SNA of the disclosure.
  • the toll-like receptors modulated include toll-like receptor 1 , toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 6, toll-like receptor 7, toll-like receptor 8, toll-like receptor 9, toll-like receptor 10, toll-like receptor 1 1 , toll-like receptor 12, and toll-like receptor 13.
  • a PLGA-SNA nanoparticle of the disclosure further comprises a therapeutic.
  • the therapeutic in various embodiments, is encapsulated in the PLGA-SNA nanoparticle, is conjugated to the surface of the PLGA-SNA nanoparticle, or both.
  • a "therapeutic” as used herein means any compound useful for therapeutic or diagnostic purposes.
  • the term as used herein is understood to mean any compound that is administered to a patient for the treatment or diagnosis of a condition.
  • Therapeutics that may be used in methods of the disclosure include oligonucleotides ⁇ e.g., siRNA as disclosed herein) and chemotherapeutics.
  • chemotherapeutics include, but are not limited to, an anti-PD-1 antibody, alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferatives, antivirals, aurora kinase inhibitors, apoptosis promoters (for example, Bcl-2 family inhibitors), activators of death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody drug conjugates, biologic response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth
  • therapeutics include small molecules.
  • small molecule refers to a chemical compound, for instance a peptidometic that may optionally be derivatized, or any other low molecular weight organic compound, either natural or synthetic. Such small molecules may be a therapeutically deliverable substance or may be further derivatized to facilitate delivery.
  • low molecular weight is meant compounds having a molecular weight of less than 1000 Daltons, typically between 300 and 700 Daltons.
  • Low molecular weight compounds are about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 1000 Daltons.
  • Lipid-modified PLGA constructs Lipid-modified nucleic acid is conjugated to the surface of poly (lactic-co-glycolic acid) (PLGA) nanoparticles via hydrophobic-hydrophobic interaction.
  • PLGA-SNA construct with a high nucleic acid coverage ⁇ i.e., high surface density) enters cells as early as 30 minutes without the use of transfection agent.
  • TEM microscopy
  • Figures 5 and 7 show the sharp melting transition of the PLGA-SNAs.
  • Figure 5 additionally shows the ability of the PLGA-SNAs to freely enter cells.
  • 250 nM PLGA-SNAs labeled with Cy5 was incubated with a SKOV-3 cell line.
  • Cellular uptake of PLGA-SNAs was evaluated by confocal microscopy at four different time points. Note that Figure 5 corresponds to DBCO-DNA melting while
  • Figure 7 corresponds to lipid-DNA melting.
  • Figure 6 shows the size distribution of PLGA-SNAs in water, 1 X PBS, and 1 X PBS containing 0.1 % Tween 20.
  • the PLGA-SNAs were shown to exhibit cooperative melting transition characteristic of a high surface nucleic acid density.
  • the construct was also shown to enter human cells ⁇ e.g., SKOV-3) effectively without the use of a transfection agent that is toxic to cells.
  • the PLGA-SNA constructs of the disclosure are modified to further comprise an agent that enhances or facilitates endosomal escape ⁇ e.g., Endo-porter, Gene Tools LLC [Summerton,
  • the PLGA-SNAs were utilized to encapsulate a hydrophobic model drug, coumarin 6, which can then be released in a polymer composition-dependent tunable manner, while the dissociation rate of the nucleic acid shell remains relatively constant, regardless of core composition.
  • a hydrophobic model drug coumarin 6
  • NP gold nanoparticle conjugate SNAs
  • PLGA-SNAs freely enter Raw-Blue cells and can be used to activate toll-like receptor 9 (TLR9) in a sequence- and dose-dependent manner.
  • Oligonucleotide Synthesis The nucleic acids used in this work (Table 2) were synthesized according to recommendation from Glen Research with standard solid-phase phosphonamidites synthesis on an Applied Biosystems 394 DNA/RNA synthesizers. The oligonucleotides were cleaved from the CPG support with a fast deprotection method (50% methylamine: 50% Ammonia, v/v) for two hours. Ultramild deprotection method (0.05 M K2C03 in MeOH) was utilized when Cy5 was used to label the oligonucleotide.
  • oligonucleotides were purified with a C4 column on a reverse-phase high performance liquid chromatography system using a gradient from 10% Acetonitrile to 100% Acetonitrile in 45 minutes. Fractions from HPLC were collected and lyophilized for MALDI-TOF analysis.
  • the reaction mixture was then incubated at 25 °C for 48 hours.
  • the purified SNA product was collected from the filter and analyzed by 1 % agarose gel electrophoresis ( Figure 10A).
  • the linear nucleic acids and PLGA-SNAs exhibit different electrophoretic mobility shifts due to size and charge.
  • the PLGA-PEG- N 3 NPs and PLGA-SNAs were imaged by atomic force microscopy (AFM) in the liquid phase, allowing us to visualize the solution-phase NPs and to obtain quantitative size distributions ( Figures 11 A and 11 C).
  • NPs deposited on a modified Si substrate show that PLGA-SNAs retain their spherical shape upon surface functionalization ( Figure 11 A, 11 C, and 10B).
  • NanoSight NS300 Series (Malvern Instruments, United Kingdom). A sample of PLGA-PEG-N 3 NPs or PLGA-SNAs was injected manually with a syringe. Each nanoparticle tracking analysis was conducted three times in duplicate (1 :10000 and 1 :20000 dilution, v/v) using a default script. Nanoparticle concentration was calculated based on the average of the duplicate.
  • AFM atomic force microscope
  • Agarose gel electrophoresis A horizontal agarose gel electrophoresis (1 %, w/v) was utilized to resolve linear DNA and PLGA-SNAs. In a typical gel electrophoresis, the horizontal gel chamber was filled with 1 X Tris/Borate/EDTA buffer. Linear DNA (12 ⁇ ) and PLGA-SNAs were loaded with 5% glycerol. The gel analysis was performed at 100 V for 30 minutes. The Cy5 signal was detected by a gel scanner equipped with a Cy5 filter (Fuji Film, Japan).
  • PAGE Polyacrylamide gel electrophoresis
  • a vertical denaturing polyacrylamide gel electrophoresis gel was utilized to analyze linear DNA degradation product for the stability assay.
  • the denaturing polyacrylamide gel electrophoresis (12 ⁇ sample including 6 ⁇ loading dye and 6 ⁇ DNA) was performed at 350 V for 90 minutes before the DNA was stained with SYBR gold.
  • the reactions were quenched at each desirable time point by adding 2.2 ⁇ of 10% SDS.
  • the reaction products were loaded onto gel electrophoresis (Amersham Typhoon) and the band intensity were quantified by the
  • TLRs Toll-like receptors
  • solubilizing buffer 100 ⁇ ; SDS 10% in 0.01 M HCI
  • 100 ⁇ of solubilizing buffer 100 ⁇ ; SDS 10% in 0.01 M HCI
  • the absorbance was measured at 570 nm using Biotek Synergy H4 Hybrid Reader.
  • Cell viability was normalized to the untreated control, i.e., (A S ampie A U ntreated control) * 100 and plotted as a percentage of cell viability. The experiment was performed in triplicates and the error was calculated as standard error of the mean.
  • Raw-Blue cells were seeded on a 96-well plate at a seeding density of -100,000/each well. Seeded cells were incubated with PLGA-SNAs bearing TLR9-activating oligonucleotide or control sequence or linear TLR9-activating oligonucleotide overnight. The level of TLR9 activation was then evaluated by a Quanti-Blue assay (InvivoGen, USA) according to manufacturer's recommendation. TLR9 activation was recorded in triplicate and is normalized to the untreated cells.
  • oligonucleotides for SNAs affects many of their biochemical properties [Cutler et al., J. Am. Chem. Soc. 2012, 134, 1376], the average number of strands per SNA conjugate was determined by making PLGA-SNAs with Cy5-tagged T 20 oligonucleotides and measuring both particle size and absolute number of DNA strands by using fluorescence spectroscopy methods. After NP isolation and purification, the NPs were redispersed in buffer, diluted in water, and the concentration was measured with a Nanoparticle Tracking Analysis (NTA) system ( Figure 12).
  • NTA Nanoparticle Tracking Analysis
  • the oligonucleotide concentration was measured by first dissolving the PLGA core of the SNA, which results in release of the oligonucleotides that define the shell, and quantifying and comparing the Cy5 fluorescence against a standard curve ( Figure 12).
  • Each approximately 65 nm (50 nm core) PLGA-SNA has an average of 199 ⁇ 16 strands or a surface density of 5.2 pmol/cm 2 . It was noted that this surface density is lower than a typical 13 nm Au SNA, where the surface density is approximately 30 pmol/cm 2 [Hurst et al, Abstr. Pap. Am. Chem. Soc.
  • NPs made of soft materials have been utilized to encapsulate a wide range of drugs, such as chemotherapy agents and nucleic acids. Furthermore, co-delivery of such agents within one nanoscale entity has been shown to enhance therapeutic efficacy in certain cases [Chen et al., Small 2009, 5, 2673].
  • a challenge with such combination therapy strategies pertains to how one can precisely control the temporal release of both drugs independently so that therapeutic effects can be maximized.
  • encapsulating both hydrophobic and hydrophilic drugs in one entity often results in poor drug loading efficiencies [Barichello et al., Drug Dev. Ind. Pharm. 1999, 25, 471 ], involves complicated preparation processes [Govender et al., J. Control.
  • the PLGA-SNA construct can spatially compartmentalize a chemotherapy drug and therapeutic nucleic acids in a single entity, so the loading and release of surface-conjugated nucleic acids and encapsulated drugs can be independently controlled.
  • the release kinetics of the encapsulated drugs can be tuned while the release rates of the nucleic acid shell remain relatively constant.
  • the increased stability of the PLGA SNAs is likely due to the covalent bond utilized to immobilize the nucleic acids on the NPs and the intrinsically higher stability of polymer NPs (as compared with liposomes). Additionally, nucleic acids conjugated to the PLGA NP also exhibited increased stability against DNase I degradation, as compared to their linear counterparts ( Figure 18). The enhanced stability of these SNAs will likely make them last longer under physiological conditions, leading to increased therapeutic efficacies in certain settings ⁇ e.g., systemic use).
  • nucleic acids are immobilized on the surface of the SNAs using the same attachment chemistry for each formulation and the dissociation rate of nucleic acids remains relatively constant for all formulations studied, which suggested that the governing mechanism of nucleic acid shell dissociation is the hydrolysis of the ester backbone defining the PLGA polymer.
  • PLGA-SNA construct As a therapeutic platform, PLGA-SNAs with Cy5- tagged T 20 oligonucleotides were synthesized and quantified their cellular uptake in a Raw-Blue macrophage reporter cell line.

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Abstract

La présente invention concerne de manière générale des acides nucléiques sphériques (SNA) de poly(acide lactique-co-glycolique) (PLGA) ayant une stabilité améliorée, des méthodes de fabrication de ceux-ci, et leurs utilisations. Les PLGA-SNA sont utiles dans la régulation génique et l'administration de médicament.
PCT/US2018/023367 2017-03-20 2018-03-20 Acides nucléiques sphériques de poly(acide lactique-co-glycolique) (plga) WO2018175445A1 (fr)

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RU2734710C1 (ru) * 2019-06-14 2020-10-22 Федеральное государственное бюджетное учреждение науки "Научный центр биомедицинских технологий Федерального медико-биологического агентства" (ФГБУН НЦБТМ ФМБА России) Наночастицы на основе биодеградирующих полимеров с инкапсулированными в них противоопухолевыми препаратами
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WO2021211734A1 (fr) * 2020-04-15 2021-10-21 The Regents Of The University Of California Compositions et procédés de traitement

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