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WO2017044768A1 - Structures à triple hélice d'arn, compositions et méthodes - Google Patents

Structures à triple hélice d'arn, compositions et méthodes Download PDF

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WO2017044768A1
WO2017044768A1 PCT/US2016/050977 US2016050977W WO2017044768A1 WO 2017044768 A1 WO2017044768 A1 WO 2017044768A1 US 2016050977 W US2016050977 W US 2016050977W WO 2017044768 A1 WO2017044768 A1 WO 2017044768A1
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dendrimer
oligonucleotide
mirna
rna
triple helix
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Natalie Artzi
João Conde
Nuria OLIVA
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Massachusetts Institute of Technology
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Massachusetts Institute of Technology
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
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    • A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • 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
    • A61K47/6903—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 semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
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    • C12N2310/3517—Marker; Tag
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    • C12N2320/00—Applications; Uses
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    • C12N2320/31—Combination therapy
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • miRNA miRNA
  • PNA peptide nucleic acids
  • pRNA packaging RNA
  • compositions comprising an miRNA triple helix.
  • the miRNA triple helices comprise an miRNA sense oligonucleotide, an miRNA antisense oligonucleotide, and an antagomiRNA oligonucleotide.
  • the miRNA sense oligonucleotide and the miRNA antisense oligonucleotide may be associated with each other by a plurality of Watson-Crick hydrogen bonds, and the antagomiRNA oligonucleotide and the miRNA sense oligonucleotide may be associated with each other by a plurality of Hoogsteen hydrogen bonds.
  • compositions in some embodiments, further comprise a dendrimer having at least two branches with one or more surface groups, wherein the RNA triple helix is associated with the one or more surface groups of the dendrimer.
  • 100 % of the surface groups comprise at least one primary or secondary amine.
  • the dendrimer and RNA triple helix may form an RNA triple helix-dendrimer conjugate, which may be a particulate material.
  • the particulate material may aggregate to form a composition comprising a plurality of the aggregates.
  • therapeutic compositions comprising [1] a hydrogel comprising a polymer and a first dendrimer, and [2] at least one RNA triple helix associated with a second dendrimer.
  • the first dendrimer may have at least two branches with one or more surface groups, wherein at least a portion of the one or more surface groups comprise at least one primary or secondary amine. In embodiments, about 20 % to about 75 % of the surface groups of the first dendrimer include at least one primary or secondary amine. In a particular embodiment, about 25 % of the surface groups of the first dendrimer comprise at least one primary or secondary amine. In one embodiment, the second dendrimer comprises at least two branches with one or more surface groups, and 100 % of the surface groups of the second dendrimer comprise at least one primary or secondary amine.
  • kits for making a therapeutic composition comprise a first part which includes a first solution comprising a polymer component, wherein the polymer component comprises a polymer; and a second part which includes a second solution comprising a dendrimer component, wherein the dendrimer component comprises a first dendrimer having at least 2 branches with one or more surface groups, and a second dendrimer having a least 2 branches with one or more surface groups associated with an RNA triple helix.
  • 100 % of the surface groups of the second dendrimer comprise at least one primary or secondary amine.
  • about 20 % to about 75 % of the surface groups of the first dendrimer may include at least one primary or secondary amine.
  • about 25 % of the surface groups of the first dendrimer comprise at least one primary or secondary amine.
  • the methods comprise providing a first solution comprising a polymer component, wherein the polymer component comprises a polymer having three or more aldehyde groups; providing a second solution comprising a dendrimer component, wherein the dendrimer component comprises a first dendrimer having at least 2 branches with one or more surface groups, and a second dendrimer having a least 2 branches with one or more surface groups associated with an RNA triple helix; and combining the first and second solutions together to produce a therapeutic composition and contacting one or more biological tissues with the therapeutic composition.
  • 100 % of the surface groups of the second dendrimer comprise at least one primary or secondary amine.
  • about 20 % to about 75 % of the surface groups of the first dendrimer may include at least one primary or secondary amine.
  • about 25 % of the surface groups of the first dendrimer comprise at least one primary or secondary amine.
  • the adhesive hydrogel composition in one embodiment, comprises an RNA triple helix.
  • the RNA triple helix may comprise an miRNA mimic and an antagomiRNA.
  • the adhesive hydrogel composition comprises a dendrimer associated with the RNA triple helix.
  • FIG.1A is a schematic showing the self-assembly of one embodiment of an RNA triple helix.
  • FIG.1B depicts the secondary structure of the RNA triple helix of FIG.1A.
  • FIG.1C depicts one embodiment of an RNA triple helix-dendrimer conjugate, and one embodiment of a hydrogel in which the RNA triple helix-dendrimer conjugate is disposed.
  • FIG.2 depicts the effect of Mg 2+ and Na + on one embodiment of an RNA triple helix.
  • FIG.3 is a high-resolution scanning electron microscope (SEM) image of the nanoparticles of RNA triple helix-dendrimer conjugates depicted at FIG.1C.
  • FIG.4 is a high-resolution SEM image of an aggregate of the nanoparticles of FIG.3.
  • FIG.5A is a high-resolution SEM image of the aggregates of FIG.4 embedded in one embodiment of a dextran-dendrimer hydrogel.
  • FIG.5B is a high-resolution SEM image of the aggregates of FIG.4 embedded in one embodiment of a dextran-dendrimer hydrogel.
  • FIG.5C is a high-resolution SEM image of the aggregates of FIG.4 embedded in one embodiment of a dextran-dendrimer hydrogel.
  • FIG.6A is a table indicating the presence of certain embodiments of the RNA strands in the results depicted at FIG.6B, FIG.6C, and FIG.6D.
  • FIG.6B depicts the effect of temperature (25 °C) on the stability of the embodiments of the RNA strand combinations shown at FIG.6A.
  • FIG.6B depicts the effect of temperature (37 °C) on the stability of the embodiments of the RNA strand combinations shown at FIG.6A.
  • FIG.6B depicts the effect of temperature (65 °C) on the stability of the embodiments of the RNA strand combinations shown at FIG.6A.
  • FIG.7A depicts flow cytometry data which revealed the specific uptake of one embodiment of fluorescent RNA triple helix-dendrimer conjugate nanoparticles into MDA-MB- 231 cells.
  • FIG.7B depicts flow cytometry data which revealed the specific uptake of one embodiment of fluorescent RNA triple helix-dendrimer conjugate nanoparticles into MDA-MB- 231 cells.
  • FIG.7C depicts flow cytometry data which revealed the specific uptake of one embodiment of fluorescent RNA triple helix-dendrimer conjugate nanoparticles into MDA-MB- 231 cells.
  • FIG.7D depicts flow cytometry data which revealed the specific uptake of one embodiment of fluorescent RNA triple helix-dendrimer conjugate nanoparticles into MDA-MB- 231 cells.
  • FIG.8 depicts the expression of embodiments of RNA strands in breast cancer cells at different times.
  • FIG.9 depicts cell viability at certain time periods.
  • FIG.10 depicts the results of a wound closure assay.
  • FIG.11 depicts the results of a clonogenic survival assay with crystal violet following treatment with embodiments of the RNA triple helix-dendrimer conjugates.
  • RNA triple helix structures with improved stability, effectiveness, and/or tolerability.
  • Embodiments of the RNA triple helices provided herein can improve cancer cells co-transfection efficiency.
  • the RNA triple helices provided herein can, in certain embodiments, modulate the expression of endogenous miRs in cancer in vivo.
  • the miRNA triple helix is a self-assembled dual-color RNA-triple-helix structure comprising two miRNAs, an miR mimic (tumor suppressor miRNA), and an antagomiR (oncomiR inhibitor)(FIG.1A and FIG.1B), which can provide the ability to synergistically abrogate tumors through delivery of one or both of the miRNAs.
  • the miRNA triple helices provided herein can be associated with a dendrimer to form RNA triple helix-dendrimer conjugates, which, in embodiments, can be stable triplex particles, including nanoparticles, as depicted schematically at FIG.1C. The particles may aggregate to form aggregates.
  • the aggregates and/or particles may be contacted with a polymer to form a hydrogel, which may act as an adhesive scaffold (see FIG.1C).
  • a hydrogel may adhere to natural tissue amines in a number of biological tissues, including tumors.
  • a hydrogel and/or a dendrimer to which an RNA triple helix is conjugated can effect local and/or controlled release of the miRNA oligonucleotides of an RNA triple helix.
  • RNA triple helix-dendrimer conjugates are functional in vitro and in vivo, and certain embodiments have been demonstrated to achieve tumor shrinkage of up to about 90 % within two weeks after deployment in a triple-negative breast-cancer mouse model. Therefore, particular embodiments of the hydrogels containing RNA triple helix- dendrimer conjugates can be used as an efficient anticancer platform to locally modulate the expression of endogenous miRs in cancer.
  • compositions that include an RNA triple helix.
  • the RNA triple helix includes [1] an miRNA sense oligonucleotide, [2] an miRNA antisense oligonucleotide, and [3] an antagomiRNA oligonucleotide (oncomiR inhibitor).
  • the miRNA sense oligonucleotide and miRNA antisense oligonucleotide may be part of an miRNA mimic (a tumor suppressor miRNA).
  • the molar ratio of the miRNA antisense oligonucleotide, miRNA sense oligonucleotide, and the antagomiRNA oligonucleotide in the RNA triple helix compositions is about (0.5—1.5):(0.5—1.5):(0.5—1.5). In one embodiment, the molar ratio of the miRNA antisense oligonucleotide, miRNA sense oligonucleotide, and the antagomiRNA oligonucleotide in the RNA triple helix compositions is about (1):(0.5—1.5):(0.5—1.5). In another
  • the molar ratio of the miRNA antisense oligonucleotide, miRNA sense oligonucleotide, and the antagomiRNA oligonucleotide in the RNA triple helix compositions is about (0.5—1.5):(0.5—1.5):(1).
  • the molar ratio of the miRNA antisense oligonucleotide, miRNA sense oligonucleotide, and the antagomiRNA oligonucleotide in the RNA triple helix compositions is about (1):(1):(0.5—1.5). In yet another embodiment, the molar ratio of the miRNA antisense oligonucleotide, miRNA sense oligonucleotide, and the antagomiRNA oligonucleotide in the RNA triple helix compositions is about (0.5—1.5):(1):(1).
  • the molar ratio of the miRNA antisense oligonucleotide, miRNA sense oligonucleotide, and the antagomiRNA oligonucleotide in the RNA triple helix compositions is about (1):(0.5—1.5):(1). In some embodiments, the molar ratio of the miRNA antisense oligonucleotide, miRNA sense oligonucleotide, and the antagomiRNA oligonucleotide in the RNA triple helix compositions is about (1):(1):(1).
  • the miRNA oligonucleotides of the RNA triple helix may be associated with one another by a plurality of hydrogen bonds.
  • the hydrogen bonds may include Watson-Crick hydrogen bonds, Hoogsteen hydrogen bonds, or a combination thereof. Not wishing to be bound by any particular theory, it is believed that the hydrogen bonds contribute, at least in part, to the formation of a stable RNA triple helix structure that is suitable for miR delivery.
  • the miRNA sense oligonucleotide and the miRNA antisense oligonucleotide are associated with each other by a plurality of Watson-Crick hydrogen bonds.
  • the antagomiRNA oligonucleotide and the miRNA sense are associated with each other by a plurality of Watson-Crick hydrogen bonds.
  • the miRNA sense oligonucleotide and the miRNA antisense oligonucleotide are associated with each other by a plurality of Watson-Crick hydrogen bonds
  • the antagomiRNA oligonucleotide and the miRNA sense oligonucleotide are associated with each other by a plurality of Hoogsteen hydrogen bonds.
  • the phrase“associated with each other by a plurality of Watson-Crick hydrogen bonds” refers to two miRNA oligonucleotides that are attracted to each other by Watson-Crick hydrogen bonds that form between two or more bases of the first miRNA oligonucleotide and two or more bases of the second miRNA oligonucleotide. Although possible, not all of the bases of an miRNA oligonucleotide must participate in the Watson-Crick hydrogen bonding to satisfy the foregoing definition.
  • the phrase“associated with each other by a plurality of Hoogsteen hydrogen bonds” refers to two miRNA oligonucleotides that are attracted to each other by Hoogsteen hydrogen bonds that form between two or more bases of the first miRNA oligonucleotide and two or more bases of the second miRNA oligonucleotide. Although possible, not all of the bases of an miRNA oligonucleotide must participate in the Hoogsteen hydrogen bonding to satisfy the foregoing definition.
  • the RNA triple helix is a self-assembled structure.
  • the RNA triple helix that may be formed by combining one or more miRNA oligonucleotides that form Watson-Crick and/or Hoogsteen hydrogen bonds.
  • the triplex-forming oligonucleotides (TFO) bind antiparallel to the purine-rich strand in RNA, which requires no base protonation and exhibits pH independent binding.
  • the RNA triple helix induces further stacking within the tertiary structure domain, which may further improve structural stability.
  • the RNA triple helix may include substituents capable of reporting or detecting the formation of the RNA triple helix and/or the release of one or more therapeutic miRNAs.
  • the RNA triple helix can include one or more dyes, and one or more quenchers.
  • the central miRNA oligonucleotide of the RNA triple helix is substituted with a quencher at its‘5 end, while the other two miRNA oligonucleotides are substituted with a dye at their 3’ ends.
  • the RNA triple helix includes stable two-pair fluorescence resonance energy transfer (FRET) donor/quencher RNA oligonucleotides that are able to report the in vivo release of the miRNA oligonucleotides, which may occur upon conjugation to complementary targets.
  • FRET fluorescence resonance energy transfer
  • the RNA triple helix is stable at a pH of about 4 to about 10. In some embodiments, the RNA triple helix is stable at a pH of about 5 to about 9. In one embodiment, the RNA triple helix is stable at a pH of about 5.5 to about 8.5. In another embodiment, the RNA triple helix is stable at a pH of about 6 to about 8. In a further embodiment, the RNA triple helix is stable at a pH of about 6.5 to about 7.5. In yet another embodiment, the RNA triple helix is stable at a pH of about 7.
  • the RNA triple helix is stable at a temperature of 70 °C or less. In another embodiment, the RNA triple helix is stable at a temperature of 65 °C or less. In a further embodiment, the RNA triple helix is stable at a temperature of 60 °C or less. In an additional embodiment, the RNA triple helix is stable at a temperature of 55 °C or less. In a still further embodiment, the RNA triple helix is stable at a temperature of 50 °C or less. The stability of the RNA triple helix at various temperatures may be determined by the methods of Example 2 herein.
  • the RNA triple helix is stable at a pH of about 4 to about 10, and a temperature of 70 °C or less. In further embodiment, the RNA triple helix is stable at a pH of about 4 to about 10, and a temperature of 65 °C or less. In additional embodiments, the RNA triple helix is stable at a pH of about 4 to about 10, and a temperature of 60 °C or less. In one embodiment, the RNA triple helix is stable at a pH of about 4 to about 10, and a temperature of 55 °C or less. In another embodiment, the RNA triple helix is stable at a pH of about 4 to about 10, and a temperature of 50 °C or less.
  • the RNA triple helix is stable at a pH of about 5 to about 9, and a temperature of 70 °C or less. In further embodiment, the RNA triple helix is stable at a pH of about 5 to about 9, and a temperature of 65 °C or less. In additional embodiments, the RNA triple helix is stable at a pH of about 5 to about 9, and a temperature of 60 ° C or less. In one embodiment, the RNA triple helix is stable at a pH of about 5 to about 9, and a temperature of 55 °C or less. In another embodiment, the RNA triple helix is stable at a pH of about 5 to about 9, and a temperature of 50 °C or less.
  • the RNA triple helix is stable at a pH of about 5.5 to about 8.5, and a temperature of 70 °C or less. In further embodiment, the RNA triple helix is stable at a pH of about 5.5 to about 8.5, and a temperature of 65 °C or less. In additional embodiments, the RNA triple helix is stable at a pH of about 5.5 to about 8.5, and a temperature of 60 ° C or less. In one embodiment, the RNA triple helix is stable at a pH of about 5.5 to about 8.5, and a temperature of 55 °C or less. In another embodiment, the RNA triple helix is stable at a pH of about 5.5 to about 8.5, and a temperature of 50 °C or less.
  • the RNA triple helix is stable at a pH of about 6 to about 8, and a temperature of 70 °C or less. In further embodiment, the RNA triple helix is stable at a pH of about 6 to about 8, and a temperature of 65 °C or less. In additional embodiments, the RNA triple helix is stable at a pH of about 6 to about 8, and a temperature of 60 ° C or less. In one embodiment, the RNA triple helix is stable at a pH of about 6 to about 8, and a temperature of 55 °C or less. In another embodiment, the RNA triple helix is stable at a pH of about 6 to about 8, and a temperature of 50 °C or less.
  • the miRNA sense oligonucleotide includes from 20 to 40 nucleotides (nt). In one embodiment, the miRNA sense oligonucleotide includes from 25 to 35 nt. In another embodiment, the miRNA sense oligonucleotide includes from 26 to 30 nt. In a particular embodiment, the miRNA sense oligonucleotide includes 28 nt.
  • the miRNA sense oligonucleotide may be modified to include a quencher.
  • the quencher may be used to verify triple helix formation when at least one of the other oligonucleotides of the triple helix is modified to include a dye.
  • the quencher may be a BLACK HOLE QUENCHER® (LGC Biosearch Technologies, USA).
  • the quencher may be placed at one end of the miRNA sense oligonucleotide, such as the 5’ end. The 5’ end may be appropriate when the 3’ end of the antisense and/or the 3’ end of the antagomiR oligonucleotides includes a dye.
  • the miRNA sense oligonucleotide may be modified to include a lipid.
  • the lipid may be cholesterol.
  • the miRNA sense oligonucleotide includes a cholesterol molecule at its 3’ end.
  • a lipid such as a neutral lipid, can facilitate the permeation of cell membranes, improve co-transfection efficiency in vivo, and/or protect oligonucleotides from degradation.
  • the miRNA sense oligonucleotide is an miR-205 oligonucleotide. In one embodiment, the miRNA sense oligonucleotide is an miR-205 oligonucleotide that includes a lipid, such as a cholesterol, which may be at its 3’ end. In a particular embodiment, the miRNA sense oligonucleotide is an miR-205 oligonucleotide that includes a quencher, such as a BLACK HOLE QUENCHER® (LGC Biosearch Technologies, USA), which may be at its 5’ end.
  • a quencher such as a BLACK HOLE QUENCHER® (LGC Biosearch Technologies, USA
  • the miRNA sense oligonucleotide is an miR-205 oligonucleotide that includes a cholesterol at its 3’ end, and a BLACK HOLE QUENCHER® (LGC Biosearch TECHNOLOGIES, USA) at its 5’ end.
  • the miRNA sense oligonucleotide is an miR-200 oligonucleotide, including, but not limited to miR-200a, miR-200b, miR-200c, miR-141, and miR-429.
  • miRNA sense oligonucleotides may be modified to include a lipid, such as a cholesterol, and/or a quencher, such as a BLACK HOLE QUENCHER® (LGC Biosearch Technologies, USA).
  • the lipid and/or quencher may be included at the ends of the miR-200 oligonucleotide.
  • the miRNA sense oligonucleotide may be an miR-200 oligonucleotide that is substituted at its 3’ end with a lipid, such as a cholesterol, and at its 5’ end with a quencher, such as a BLACK HOLE QUENCHER® (LGC Biosearch Technologies, USA).
  • a lipid such as a cholesterol
  • a quencher such as a BLACK HOLE QUENCHER® (LGC Biosearch Technologies, USA).
  • the miRNA antisense oligonucleotide and miRNA sense oligonucleotide are an miRNA mimic (tumor suppressor miRNA), such as a mature tumor suppressor miRNA- 205 (see FIG.1A and FIG.1B).
  • the miRNA mimic may be a mature miRNA duplex comprising both sense and antisense strands.
  • the miRNA antisense oligonucleotide includes from 20 to 40
  • the miRNA antisense oligonucleotide includes from 25 to 35 nt. In another embodiment, the miRNA antisense oligonucleotide includes from 26 to 30 nt. In a particular embodiment, the miRNA antisense oligonucleotide includes 28 nt.
  • the miRNA antisense oligonucleotide may be modified to include a dye.
  • the dye may be used to verify triple helix formation when at least one of the other
  • the dye may be a near infrared (NIR) dye, such as QUASAR® 705 (LGC Biosearch Technologies, USA).
  • NIR near infrared
  • the dye may be placed at one end of the miRNA antisense oligonucleotide, such as the 3’ end. The 3’ end may be appropriate when the 5’ end of the miRNA sense oligonucleotide and/or the 3’ end of the antagomiR oligonucleotide includes a quencher or a dye, respectively.
  • the antagomiRNA oligonucleotide may be a synthetic single-stranded RNA (ssRNA) used to inhibit an omcomiRNA.
  • ssRNA synthetic single-stranded RNA
  • the antagomiRNA oligonucleotide includes from 20 to 40 nucleotides (nt). In one embodiment, the antagomiRNA oligonucleotide includes from 25 to 35 nt. In another embodiment, the antagomiRNA oligonucleotide includes from 28 to 32 nt. In a particular embodiment, the antagomiRNA oligonucleotide includes 30 nt.
  • the antagomiRNA oligonucleotide may be modified to include a dye.
  • the dye may be used to verify triple helix formation when at least one of the other
  • the dye may be a near infrared (NIR) dye, such as QUASAR® 570 (LGC Biosearch Technologies, USA).
  • NIR near infrared
  • the dye may be placed at one end of the antagomiRNA oligonucleotide, such as the 3’ end. The 3’ end may be appropriate when the 5’ end of the miRNA sense oligonucleotide and/or the 3’ end of the miRNA antisense oligonucleotide includes a quencher or a dye, respectively.
  • the antagomiRNA oligonucleotide is an antagomiR-221
  • the RNA triple helix is formed by contacting the miRNA
  • the medium may be an incubation buffer.
  • the incubation buffer is a tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7).
  • the Tris buffer may be supplemented with one or more supplements, such as MgCl 2 , spermine, CuSO 4 , or a combination thereof.
  • the Tris buffer is a 10 mM Tris buffer supplemented with MgCl 2 , such as 10 mM MgCl 2 .
  • the Tris buffer is a 10 mM Tris buffer supplemented with spermine, such as 1 mM spermine.
  • the Tris buffer is a 10 mM Tris buffer supplemented with CuSO 4 , such as 0.8 mM CuSO 4 .
  • the Tris buffer is a 10 mM Tris buffer supplemented with MgCl 2 , such as 10 mM MgCl 2 , and spermine, such as 1 mM spermine.
  • the Tris buffer is a 10 mM Tris buffer supplemented with MgCl 2 , such as 10 mM MgCl 2 , and CuSO 4 , such as 0.8 mM CuSO 4 .
  • the Tris buffer is a 10 mM Tris buffer supplemented with CuSO 4 , such as 0.8 mM CuSO 4 , and spermine, such as 1 mM spermine.
  • the Tris buffer is a 10 mM Tris buffer supplemented with 10 mM MgCl 2 , 1 mM spermine, and 0.8 mM CuSO 4 .
  • the supplemental copper ions may favor the intercalation of the nitrogen atoms in the minor groove of the RNA triple helix or triplex where copper binding occurs (see, e.g., Francois, J.C. et al. Proceedings of the National Academy of Sciences of the United States of America, 86, 9702-9706 (1989)).
  • the spermine as a natural polyamine, may improve triplex formation by reducing the electrostatic repulsive forces between the negatively charged phosphate backbones of the RNA strands (see, e.g., Tung, C.H. et al. Nucleic Acids Research 215489-5494 (1993)).
  • Mg 2+ may promote RNA triple helix formation due to charge neutralization.
  • Mg 2+ is believed to bind to the phosphate groups of the RNA triple helix, which may reduce the repulsion between the three phosphate frameworks, thereby increasing the efficiency of the triple-helix formation.
  • Na + is believed to be a possible inhibitor of DNA triple helix formation (see FIG.2).
  • the hindering effect of Na + may be explained by the polyelectrolyte effect: a high concentration of Na + is believed to lower the population of Mg 2+ in the vicinity of DNA or RNA, thereby decreasing the probability of triple helix formation. It also is believed that Na + had the ability to form undesirable dimers and tetramers, thereby decreasing the efficiency of triple helix formation.
  • the RNA triple helix is formed by [1] contacting the miRNA
  • oligonucleotides in an appropriate medium such as a Tris buffer, which may be supplemented as provided herein, and [2] heating the medium to a temperature of about 60 to about 100 °C, about 70 to about 90 °C, or about 80 °C.
  • the medium may be heated for about 1 to about 10 minutes, from about 3 to about 7 minutes, or about 5 minutes.
  • the medium may then be cooled to a temperature of about 1 to about 10 °C, from about 2 to about 6 ° C, or about 4 °C.
  • the RNA triple helix may be associated with a dendrimer to form an RNA triple helix- dendrimer conjugate.
  • the dendrimer of the RNA triple helix-dendrimer conjugate may allow the release of the RNA triple helix and/or one or more miRNAs of the RNA triple helix from the RNA triple helix-dendrimer conjugates to be controlled.
  • the RNA triple helix-dendrimer conjugate may be a particulate material.
  • the particulate material may be substantially spherical.
  • the particulate material comprises nanoparticles.
  • the particulate material comprises substantially spherical nanoparticles.
  • the nanoparticles may have an average largest dimension of about 30 to about 80 nm, from about 40 to about 70 nm, or from about 50 to about 60 nm. The average largest dimension may be determined by SEM images or X-ray diffraction, and is the diameter when the particulate material is substantially spherical.
  • the RNA triple helix-dendrimer conjugates may be a particulate material that forms aggregates.
  • the aggregates of the particulate material may be substantially spherical.
  • the aggregates of the particulate material may be a microscale material.
  • the microscale material may be substantially spherical.
  • the aggregates may have an average largest dimension of about 1 to about 10 ⁇ m, from about 2 to about 8 ⁇ m, from about 3 to about 5 ⁇ m, or from about 3 to about 4 ⁇ m.
  • the average largest dimension may be determined by SEM images or X-ray diffraction, and is the diameter when the aggregates are substantially spherical.
  • the RNA triple helix-dendrimer conjugates are in the form of a particulate material, aggregates of a particulate material, or a combination thereof. In another embodiment, the RNA triple helix-dendrimer conjugates are in the form of aggregates of a particulate material. In yet another embodiment, the RNA triple helix-dendrimer conjugates are in the form of a particulate material. In a still further embodiment, a first portion of the RNA triple helix-dendrimer conjugates are in the form of a particulate material, and a second portion of the RNA triple helix-dendrimer conjugates are in the form of aggregates of a particulate material.
  • RNA triple helices provided herein can be provided at various scales, including nanoscale, microscale, and macroscale.
  • an RNA triple helix- dendrimer conjugates can be provided as nanoparticles.
  • FIG.3 is a high-resolution SEM image of the RNA triple helix-dendrimer conjugate nanoparticles of FIG.1C, which have an average largest dimension of about 50 nm to about 60 nm.
  • RNA triple helix-dendrimer conjugates can be provided as microparticles, which may be aggregates of the RNA triple helix-dendrimer conjugate nanoparticles.
  • FIG.4 is a high-resolution SEM image of an aggregate of the nanoparticles of RNA triple helix-dendrimer conjugates of FIG.3, and the aggregate has a largest dimension of about 3 ⁇ m to about 4 ⁇ m.
  • RNA triple helix-dendrimer conjugates can be provided at the macroscale (> 1 mm) when embedded in a hydrogel, as shown at FIG.5A, FIG.5B, and FIG.5C, which are high-resolution SEM images of the aggregates of FIG.4 embedded in one embodiment of a dextran-dendrimer hydrogel.
  • the RNA triple helix may be associated with the dendrimer through one or more attractive forces.
  • the attractive forces may include an interaction between the phosphates of the miRNA oligonucleotides and the surface groups of the dendrimer.
  • the surface groups may have a cationic character.
  • a cationic surface of the dendrimer provides an easy platform for conjugating the dendrimer with miRNA oligonucleotides via electrostatic interactions between the cationic surface groups, such as positively charged terminal amines from PAMAM dendrimers, and the negatively charged RNA phosphate of the miRNA oligonucleotides.
  • the dendrimer of the RNA triple helix-dendrimer conjugates comprises a dendrimer having amines on at least a portion of its surface groups, which are commonly referred to as“terminal groups” or“end groups.”
  • the dendrimer may have amines on from about 75 % to 100 % of its surface groups, from about 80 % to about 100 % of its surface groups, from about 85 % to about 100 % of its surface groups, from about 90 % to about 100 % of its surface groups, from about 95 % to about 100 % of its surface groups, or from about 98 % to about 100 % of its surface groups.
  • the dendrimer has amines on 100 % of its surface groups.
  • the term“dendrimer” refers to any compound with a polyvalent core covalently bonded to two or more dendritic branches.
  • the polyvalent core is covalently bonded to three or more dendritic branches.
  • the amines are primary amines.
  • the amines are secondary amines.
  • one or more surface groups have at least one primary and at least one secondary amine.
  • the dendrimer of the RNA triple helix-dendrimer conjugates extends through at least 2 generations. In another embodiment, the dendrimer of the RNA triple helix- dendrimer conjugates extends through at least 3 generations. In yet another embodiment, the dendrimer of the RNA triple helix-dendrimer conjugates extends through at least 4 generations. In still another embodiment, the dendrimer of the RNA triple helix-dendrimer conjugates extends through at least 5 generations. In a further embodiment, the dendrimer of the RNA triple helix- dendrimer conjugates extends through at least 6 generations. In still a further embodiment, the dendrimer of the RNA triple helix-dendrimer conjugates extends through at least 7 generations.
  • the dendrimer of the RNA triple helix-dendrimer conjugates has a molecular weight of from about 1,000 to about 1,000,000 Daltons. In a further embodiment, the dendrimer of the RNA triple helix-dendrimer conjugates has a molecular weight of from about 3,000 to about 120,000 Daltons. In another embodiment, the dendrimer of the RNA triple helix- dendrimer conjugates has a molecular weight of from about 10,000 to about 100,000 Daltons. In yet another embodiment, the dendrimer of the RNA triple helix-dendrimer conjugates has a molecular weight of from about 20,000 to about 40,000 Daltons. Unless specified otherwise, the “molecular weight” of the dendrimer of the RNA triple helix-dendrimer conjugates refers to the number average molecular weight.
  • the dendrimer of the RNA triple helix-dendrimer conjugates may be made using any known methods.
  • the dendrimer of the RNA triple helix- dendrimer conjugates is made by oxidizing a starting dendrimer having surface groups comprising at least one hydroxyl group so that at least a portion of the surface groups comprise at least one amine.
  • the dendrimer of the RNA triple helix-dendrimer conjugates is made by oxidizing a starting generation 5 (G5) dendrimer having surface groups comprising at least one hydroxyl group so that at least about 75 % to about 100 % of the surface groups comprise at least one amine.
  • G5 starting generation 5
  • the dendrimer of the RNA triple helix-dendrimer conjugates is a poly(amidoamine)-derived (PAMAM) dendrimer.
  • the dendrimer of the RNA triple helix-dendrimer conjugates is a G5 PAMAM-derived dendrimer.
  • the dendrimer of the RNA triple helix-dendrimer conjugates is a G5 PAMAM- derived dendrimer having primary amines on about 75 to about 100 % of the dendrimer’s surface groups.
  • the dendrimer of the RNA triple helix-dendrimer conjugates is a G5 PAMAM-derived dendrimer having primary amines on about 80 to about 100 % of the dendrimer’s surface groups.
  • the dendrimer of the RNA triple helix-dendrimer conjugates is a G5 PAMAM-derived dendrimer having primary amines on about 90 to about 100 % of the dendrimer’s surface groups.
  • the dendrimer of the RNA triple helix-dendrimer conjugates is a G5 PAMAM-derived dendrimer having primary amines on 100 % of the dendrimer’s surface groups.
  • the dendrimer of the RNA triple helix-dendrimer conjugates is a poly(propyleneimine)-derived dendrimer.
  • an RNA triple helix and/or an RNA triple helix-dendrimer conjugate is disposed in a hydrogel.
  • the hydrogel may comprise a polymer component and a dendrimer component.
  • an RNA triple helix is disposed in the hydrogel.
  • the RNA triple helix may be substantially evenly dispersed in the hydrogel.
  • an RNA triple helix-dendrimer conjugate is disposed in a hydrogel.
  • the RNA triple helix- dendrimer conjugate may be substantially evenly dispersed in the hydrogel.
  • an RNA triple helix and an RNA triple helix-dendrimer conjugate is disposed in a hydrogel. At least one of the RNA triple helix and RNA triple helix-dendrimer conjugate may be substantially evenly dispersed in the hydrogel.
  • the dendrimer component of the hydrogel comprises a dendrimer that includes one or more surface groups capable of reacting with a polymer group.
  • the dendrimer component of the hydrogel comprises a first dendrimer that includes one or more surface groups capable of reacting with a polymer group, and a second dendrimer that is associated with an RNA triple helix.
  • the first dendrimer and the second dendrimer that is associated with an RNA triple helix in a particular embodiment, have substantially identical structures.
  • the first dendrimer and the second dendrimer that is associated with an RNA triple have different structures.
  • the first dendrimer is referred to herein as the“hydrogel-forming dendrimer.”
  • the first dendrimer is referred to as a“hydrogel- forming dendrimer,” the use of this phrase is not intended to convey that the second dendrimer does not participate, at least to some degree, in hydrogel formation, although it is possible that the second dendrimer may not participate in hydrogel formation, at least in some embodiments.
  • both the first dendrimer and the second dendrimer contribute to the formation of a hydrogel.
  • the first dendrimer contributes to hydrogel formation, and the second dendrimer does not substantially contribute to hydrogel formation.
  • the hydrogel-forming dendrimer has amines on at least a portion of its surface groups, which are commonly referred to as“terminal groups” or“end groups.”
  • the hydrogel-forming dendrimer may have amines on from about 20 % to 100 % of its surface groups. In some embodiments, the hydrogel-forming dendrimer has amines on about 20 % to about 80 % of its surface groups. In one embodiment, the hydrogel-forming dendrimer has amines on less than 75 % of its surface groups. In a particular embodiment, the hydrogel- forming dendrimer has amines of about 20 % to about 75 % of its surface groups.
  • the hydrogel-forming dendrimer has amines of about 20 % to about 60 % of its surface groups. In a certain embodiment, the hydrogel-forming dendrimer has amines of about 20 % to about 50 % of its surface groups. In another embodiment, the hydrogel-forming dendrimer has amines of about 20 % to about 50 % of its surface groups. In a further embodiment, the hydrogel-forming dendrimer has amines of about 20 % to about 40 % of its surface groups. In yet another embodiment, the hydrogel-forming dendrimer has amines of about 20 % to about 30 % of its surface groups. In a still further embodiment, the hydrogel- forming dendrimer has amines on about 25 % of its surface groups.
  • the hydrogel-forming dendrimer extends through at least 2 generations. In another embodiment, the hydrogel-forming dendrimer extends through at least 3 generations. In yet another embodiment, the hydrogel-forming dendrimer extends through at least 4 generations. In still another embodiment, the hydrogel-forming dendrimer extends through at least 5 generations. In a further embodiment, the hydrogel-forming dendrimer extends through at least 6 generations. In still a further embodiment, the hydrogel-forming dendrimer extends through at least 7 generations.
  • the hydrogel-forming dendrimer has a molecular weight of from about 1,000 to about 1,000,000 Daltons. In a further embodiment, the hydrogel-forming dendrimer has a molecular weight of from about 3,000 to about 120,000 Daltons. In another embodiment, the hydrogel-forming dendrimer has a molecular weight of from about 10,000 to about 100,000 Daltons. In yet another embodiment, the hydrogel-forming dendrimer has a molecular weight of from about 20,000 to about 40,000 Daltons. Unless specified otherwise, the “molecular weight” of the hydrogel-forming dendrimer refers to the number average molecular weight.
  • the hydrogel-forming dendrimer may be made using any known methods.
  • the hydrogel-forming dendrimer is made by oxidizing a starting dendrimer having surface groups comprising at least one hydroxyl group so that at least a portion of the surface groups comprise at least one amine.
  • the hydrogel-forming dendrimer is made by oxidizing a starting generation 5 (G5) dendrimer having surface groups comprising at least one hydroxyl group so that at least a portion of the surface groups comprise at least one amine.
  • the hydrogel-forming dendrimer is made by oxidizing a starting G5 dendrimer having surface groups comprising at least one hydroxyl group so that about 25 % of the surface groups comprise at least one amine.
  • the hydrogel-forming dendrimer is a G5 dendrimer having primary amines on about 25 % of the dendrimer’s surface groups.
  • the hydrogel-forming dendrimer is a poly(amidoamine)-derived (PAMAM) dendrimer.
  • the hydrogel-forming dendrimer is a G5 PAMAM-derived dendrimer.
  • the hydrogel-forming dendrimer is a G5 PAMAM-derived dendrimer having primary amines on about 25 % of the dendrimer’s surface groups.
  • the hydrogel-forming dendrimer is a poly(propyleneimine)-derived dendrimer.
  • the dendrimer component is combined with a liquid to form a dendrimer component solution.
  • the dendrimer component solution is an aqueous solution.
  • the solution comprises water, phosphate buffer saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), or any combination thereof.
  • PBS phosphate buffer saline
  • DMEM Dulbecco’s Modified Eagle’s Medium
  • the dendrimer component concentration in the dendrimer component solution is about 5 % to about 25 % by weight.
  • the dendrimer component concentration in the dendrimer component solution is about 10 % to about 20 % by weight.
  • the dendrimer component concentration in the dendrimer component solution is about 11 % to about 15 % by weight.
  • the dendrimer component comprises an RNA triple helix, and the concentration of the RNA triple helix in the dendrimer component solution is about 0.1 to about 1.0 micromolar.
  • the dendrimer component comprises a first dendrimer that includes one or more surface groups capable of reacting with a polymer group, and a second dendrimer that is associated with an RNA triple helix, and the concentration of the second dendrimer in the dendrimer component solution is about 0.01 to about 5 mg/mL, and the concentration of the RNA triple helix is about 0.1 to about 1.0 micromolar.
  • the dendrimer component comprises a first dendrimer that includes one or more surface groups capable of reacting with a polymer group, and a second dendrimer that is associated with an RNA triple helix, and the concentration of the second dendrimer in the dendrimer component solution is about 0.05 mg/mL, and the concentration of the RNA triple helix is about 1.0 micromolar.
  • the dendrimer component or dendrimer component solution further includes one or more additives.
  • the amount of additive may vary depending on the application, tissue type, concentration of the dendrimer component solution, the type of dendrimer component, concentration of the polymer component solutions, and/or the type of polymer component.
  • suitable additives include, but are not limited to, pH modifiers, thickeners, antimicrobial agents, colorants, surfactants, and radio-opaque compounds. Specific examples of these types of additives are described herein.
  • the dendrimer component solution comprises a foaming additive.
  • the dendrimer component or dendrimer component solution includes one or more drugs.
  • the polymer component or polymer component solution includes one or more drugs.
  • the hydrogel may serve as a matrix material for controlled release of the one or more drugs.
  • the drug may be essentially any drug suitable for local, regional, or systemic administration from a quantity of the hydrogel that has been applied to one or more tissue sites in a patient.
  • the dendrimer component or dendrimer component solution includes one or more cells.
  • the polymer component or polymer component solution includes one or more cells.
  • the pH modifier is an acidic compound.
  • acidic pH modifiers include, but are not limited to, carboxylic acids, inorganic acids, and sulfonic acids.
  • the pH modifier is a basic compound.
  • basic pH modifiers include, but are not limited to, hydroxides, alkoxides, nitrogen-containing compounds other than primary and secondary amines, basic carbonates, and basic phosphates.
  • the thickener may be selected from any known viscosity-modifying compounds, including, but not limited to, polysaccharides and derivatives thereof, such as starch or hydroxyethyl cellulose.
  • the surfactant may be any compound that lowers the surface tension of water.
  • the surfactant is an ionic surfactant—for example, sodium lauryl sulfate.
  • the surfactant is a neutral surfactant. Examples of neutral surfactants include, but are not limited to, polyoxyethylene ethers, polyoxyethylene esters, and
  • the radio-opaque compound is barium sulfate, gold particles, or a combination thereof.
  • the polymer component includes a polymer with one or more functional groups capable of reacting with one or more functional groups on a biological tissue and/or one or more functional groups on a dendrimer of the dendrimer component.
  • the polymer is at least one polysaccharide.
  • the at least one polysaccharide may be linear, branched, or have both linear and branched sections within its structure.
  • the at least one polysaccharide may be natural, synthetic, or modified—for example, by cross-linking, altering the polysaccharide’s substituents, or both.
  • the at least one polysaccharide is plant-based. In another embodiment, the at least one polysaccharide is animal-based.
  • the at least one polysaccharide is a combination of plant-based and animal-based polysaccharides.
  • Non-limiting examples of polysaccharides include, but are not limited to, dextran, chitin, starch, agar, cellulose, hyaluronic acid, or a combination thereof.
  • the at least one polymer has a molecular weight of from about 1,000 to about 1,000,000 Daltons. In one embodiment, the at least one polymer has a molecular weight of from about 5,000 to about 15,000 Daltons. Unless specified otherwise, the“molecular weight” of the polymer refers to the number average molecular weight.
  • the polymer is functionalized so that its structure includes one or more functional groups that will react with one or more functional groups on a biological tissue and/or one or more functional groups on a dendrimer of the dendrimer component. In other embodiments, the polymer is functionalized so that its structure includes three or more functional groups that will react with one or more functional groups on a biological tissue and/or one or more functional groups on a dendrimer of the dendrimer component. In one embodiment, the functional group incorporated into the polymer’s structure is aldehyde.
  • the polymer’s degree of functionalization is adjustable.
  • the “degree of functionalization” generally refers to the number or percentage of groups on the polymer that are replaced or converted to the desired one or more functional groups.
  • the one or more functional groups include aldehydes.
  • the degree of functionalization is adjusted based on the type of tissue to which the hydrogel is applied, the concentration(s) of the components, and/or the type of polymer or dendrimer used in the hydrogel.
  • the degree of functionalization is from about 10 % to about 75 %.
  • the degree of functionalization is from about 15 % to about 50 %.
  • the degree of functionalization is from about 20 % to about 30 %.
  • the polymer is a polysaccharide having from about 10 % to about 75 % of its hydroxyl groups converted to aldehydes. In another embodiment, the polymer is a polysaccharide having from about 20 % to about 50 % of its hydroxyl groups converted to aldehydes. In yet another embodiment, the polymer is a polysaccharide having from about 10 % to about 30 % of its hydroxyl groups converted to aldehydes.
  • the polymer is dextran with a molecular weight of about 10 kDa. In another embodiment, the polymer is dextran having about 50 % of its hydroxyl group converted to aldehydes. In a further embodiment, the polymer is dextran with a molecular weight of about 10 kDa and about 50 % of its hydroxyl groups converted to aldehydes.
  • a polysaccharide is oxidized to include a desired percentage of one or more aldehyde functional groups.
  • this oxidation may be conducted using any known means.
  • suitable oxidizing agents include, but are not limited to, periodates, hypochlorites, ozone, peroxides, hydroperoxides, persulfates, and percarbonates.
  • the oxidation is performed using sodium periodate.
  • different amounts of oxidizing agents may be used to alter the degree of functionalization.
  • the polymer component is combined with a liquid to form a polymer component solution.
  • the polymer component solution is an aqueous solution.
  • the solution comprises water, PBS, DMEM, or any combination thereof.
  • the polymer component solution may have any suitable concentration of polymer component.
  • the polymer component concentration in the polymer component solution is about 5 % to about 40 % by weight.
  • the polymer component concentration in the polymer component solution is about 5 % to about 30 % by weight.
  • the polymer component concentration in the polymer component solution is about 5 % to about 25 % by weight.
  • the concentration may be tailored and/or adjusted based on the particular application, tissue type, and/or the type and concentration of dendrimer component used.
  • the polymer component or polymer component solution may also include one or more additives.
  • the additive is compatible with the polymer component.
  • the additive does not contain primary or secondary amines.
  • the amount of additive varies depending on the application, tissue type, concentration of the polymer component solution, the type of polymer component and/or dendrimer component.
  • suitable additives include, but are not limited to, pH modifiers, thickeners, antimicrobial agents, colorants, surfactants, radio-opaque compounds, and the other additives described herein.
  • the polymer component solution comprises a foaming agent.
  • the pH modifier is an acidic compound.
  • acidic pH modifiers include, but are not limited to, carboxylic acids, inorganic acids, and sulfonic acids.
  • the pH modifier is a basic compound.
  • basic pH modifiers include, but are not limited to, hydroxides, alkoxides, nitrogen-containing compounds other than primary and secondary amines, basic carbonates, and basic phosphates.
  • the thickener may be selected from any known viscosity-modifying compounds, including, but not limited to, polysaccharides and derivatives thereof, such as starch or hydroxyethyl cellulose.
  • the surfactant may be any compound that lowers the surface tension of water.
  • the surfactant is an ionic surfactant—for example, sodium lauryl sulfate.
  • the surfactant is a neutral surfactant. Examples of neutral surfactants include, but are not limited to, polyoxyethylene ethers, polyoxyethylene esters, and
  • the radio-opaque compound is barium sulfate, gold particles, or a combination thereof.
  • the polymer component or polymer component solution includes one or more drugs.
  • the hydrogel may serve as a matrix material for controlled release of drug.
  • the drug may be essentially any drug suitable for local, regional, or systemic administration from a quantity of the hydrogel that has been applied to one or more tissue sites in a patient.
  • the polymer component or polymer component solution includes one or more cells.
  • the hydrogel may serve as a matrix material for delivering cells to a tissue site at which the hydrogel has been applied.
  • the hydrogels described herein may be formed by combining the polymer component or polymer component solution, and the dendrimer component or dendrimer component solution in any manner.
  • the polymer component or polymer component solution, and the dendrimer component or dendrimer component solution are combined before contacting a biological tissue with the hydrogel.
  • the polymer component or polymer component solution, and the dendrimer component or dendrimer component solution are combined, in any order, on a biological tissue. Therefore, the combining and contacting steps may be performed simultaneously or in any order.
  • the polymer component or polymer component solution is applied to a first biological tissue, the dendrimer component or dendrimer component solution is applied to a second biological tissue, and the first and second biological tissues are contacted.
  • the polymer component or polymer component solution is applied to a first region a biological tissue, the dendrimer component or dendrimer component solution is applied to a second region of a biological tissue, and the first and second regions are contacted.
  • the hydrogel comprises an RNA triple helix and/or one or more RNA triple helix-dendrimer conjugates.
  • the RNA triple helix and/or one or more RNA triple helix-dendrimer conjugates may be combined with or added to a component and/or component solution prior to hydrogel formation, or, alternatively or additionally, the RNA triple helix and/or one or more RNA triple helix-dendrimer conjugates may be added to a hydrogel during or after hydrogel formation and/or curing.
  • the RNA triple helix and/or one or more RNA triple-helix dendrimer conjugates is combined with or added to the dendrimer component or the dendrimer component solution prior to hydrogel formation. In one embodiment, the RNA triple helix and /or one or more RNA triple-helix dendrimer conjugates is disposed in a hydrogel during and/or after formation and/or curing of the hydrogel.
  • the hydrogels may be applied to one or more biological tissues as an adhesive, sealant, and/or treatment.
  • the biological tissue includes a tumor.
  • the biological tissue includes a tumor, tissue adjacent to a tumor, or a combination thereof.
  • the phrase“applied to,” as used herein, refers to the placement of a hydrogel on, in, and/or adjacent to a biological tissue.
  • the one or more biological tissues may be diseased, damaged (e.g., dissected), healthy, or some combination thereof.
  • the hydrogel is applied to one or more biological tissues as an adhesive.
  • the hydrogel is applied to one or more biological tissues as a sealant.
  • the hydrogel is applied to one or more biological tissues as a treatment.
  • an additional biological tissue includes a tumor.
  • the biological tissue includes a tumor, tissue adjacent to a tumor, or a combination thereof.
  • the phrase“applied to,” as used herein, refers to the placement of a hydrogel on, in, and/or adjacent to a biological tissue.
  • the hydrogel is applied to one or more biological tissues as an adhesive and sealant. In still another embodiment, the hydrogel is applied to one or more biological tissues as an adhesive and treatment. In yet another embodiment, the hydrogel is applied to one or more biological tissues as a sealant and treatment. In a still further embodiment, the hydrogel is applied to one or more biological tissues as an adhesive, sealant, and treatment.
  • the hydrogel may be applied to the biological tissue using any suitable tool and methods.
  • suitable tool and methods include the use of syringes or spatulas. Double barrel syringes with rigid or flexible discharge tips, and optional extension tubes, known in the art are envisioned.
  • the hydrogel is a“treatment” when it improves the response of at least one biological tissue to which it is applied.
  • the improved response is lessening tumor growth, lessening tumor size, lessening overall inflammation, improving the specific response at the wound site/ interface of the tissue and hydrogel, enhancing healing, or a combination thereof.
  • the phrase“lessening tumor growth” refers to a decrease in the rate of tumor growth or the temporary or permanent cessation of tumor growth.
  • the phrase“lessening tumor size” refers to a decrease in one or more dimensions of a tumor.
  • the phrase“lessening overall inflammation” refers to an improvement of histology scores that reflect the severity of inflammation.
  • the phrase“improving the specific response at the wound site/interface of the tissue and hydrogel” refers to an improvement of histology scores that reflect the severity of serosal neutrophils.
  • the phrase“enhancing healing” refers to an improvement of histology scores that reflect the severity of serosal fibrosis.
  • the formulation may be allowed adequate time to cure or gel.
  • hydrogel“cures” or“gels,” as those terms are used herein it means that the reactive groups on the polymer component, dendrimer component, and one or more biological tissues have undergone one or more reactions.
  • the hydrogels described herein are effective because the polymer component reacts with both the dendrimer component and the surface of the biological tissues.
  • the polymer component s aldehyde functional groups react with the amines on a dendrimer of the dendrimer component and the biological tissues to form imine bonds.
  • the amines on the dendrimer component react with a high percentage of the aldehydes on the polymer component, thereby reducing toxicity and increasing biocompatibility of the hydrogels.
  • the time needed to cure or gel the hydrogels will vary based on a number of factors, including, but not limited to, the characteristics of the polymer component and/or dendrimer component, the concentrations of the polymer component solution and/or the dendrimer component solution, and the characteristics of the one or more biological tissues.
  • the hydrogel will cure sufficiently to provide bonding or sealing shortly after the components are combined.
  • the gelation or cure time should provide that a mixture of the components can be delivered in fluid form to a target area before becoming too viscous or solidified and then once applied to the target area sets up rapidly thereafter.
  • the gelation or cure time is less than 120 seconds.
  • the gelation or cure time is between 3 and 60 seconds.
  • the gelation or cure time is between 5 and 30 seconds.
  • one or more foaming agents are added to the polymer component solution and/or the dendrimer component solution before the solutions are combined.
  • the foaming agents comprise a two part liquid system comprising Part 1 and Part 2, wherein Part 1 comprises a bicarbonate and Part 2 comprises an aqueous solution of di- or polyaldehydes and a titrant.
  • Part 1 comprises a bicarbonate
  • Part 2 comprises an aqueous solution of di- or polyaldehydes and a titrant.
  • aqueous glyoxal ethanedial
  • aqueous glutaraldehyde penentadial
  • Water soluble mixtures of di- and polyaldehydes prepared by oxidative cleavage of appropriate carbohydrates with periodate, ozone or the like may also be useful.
  • a titrant is most preferably employed in the liquid solution of Part 2. More specifically, the titrant is an organic or inorganic acid, buffer, salt, or salt solution which is capable of reacting with the bicarbonate component of Part 1 to generate carbon dioxide and water as reaction by- products.
  • the carbon dioxide gas that is generated creates a foam-like structure of the hydrogel and also causes the volume of the hydrogel to expand.
  • the titrant is an inorganic or organic acid that is present in an amount to impart an acidic pH to the resulting mixture of the Part 1 and Part 2 components.
  • Preferred acids that may be employed in the practice of the present invention include phosphoric acid, sulfuric acid, hydrochloric acid, acetic acid, and citric acid.
  • a dual-color RNA triple helix was constructed using three pieces of RNA
  • RNA oligomer sequences of this example are depicted in the following table:
  • the miR-205 sense of this example was a 28 nucleotide (nt) RNA oligo double-modified with a Black-Hole dark quencher (BHQ2) at 5’ and a cholesterol molecule at 3’.
  • the miR-205 antisense was a 28 nt RNA oligo modified with a near infrared (NIR) dye, QUASAR® 705 (LGC Biosearch Technologies, USA), at 3’.
  • NIR near infrared
  • the antagomiR-221 was a 30 nt RNA oligo modified with a QUASAR® 570 at 3’. Scrambled miRs (Biosearch Technologies, USA) synthesized with the same
  • RNA strands were mixed in an equal molar ratio (1:1:1, final concentration 1 ⁇ M each) or several combination ratios at room temperature in an incubation buffer including 10 mM Tris buffer pH 7 (Bio-Rad) supplemented with 10 mM MgCl 2 (Sigma), 1 mM spermine (Sigma), and 0.8 mM of CuSO 4 (Sigma), and then heated to 80 °C for 5 minutes before being rapidly cooled to 4 °C.
  • Tris buffer pH 7 Bio-Rad
  • RNA triple-helix formation In order to verify the RNA triple-helix formation and determine an optimal molar ratio between the three oligos of this example, fluorescence intensity of the RNA oligos (two doubles or triple helix forming structures) with graded molar ratios was measured at room temperature using a live imaging system. A nearly 100% quenching effect (no fluorescence emission) induced by the proximity of the chromophores with the quencher BHQ2 was observed at a 1:1:1 molar ratio or higher.
  • the emission spectra of QUASAR® 705 and 570 oligos were collected after incubation with different ratios of the oligo containing the quencher BHQ2.
  • the fluorescence intensity of the RNA oligos at several molar ratios measured at room temperature was evaluated in order to compare quenching efficiencies, which were induced by the proximity of the chromophores with the quencher BHQ2.
  • the region of interest (ROI) quantification of the Q570 and Q705 channels also was evaluated for several molar ratios of the components, and in the presence and absence of the quencher BHQ2.
  • RNA strands were mixed in an equal molar ratio (1:1:1, final concentration 1 ⁇ M each) or at several combination ratios at room temperature in an incubation buffer and then heated to 80 oC for 5 minutes and rapidly cooled to 4oC. Fluorescence spectra were then taken using a microplate reader (Varioskan Flash Multimode Reader, Thermo Scientific) and fluorescence images using the IVIS Spectrum-bioluminescent and fluorescent imaging system (Xenogen XPM-2 Corporation).
  • the Quasar570 has an Exc/Emi at 548/570 nm and the Quasar705 at 690/705 nm.
  • RNA triple helix Successful self-assembly of an RNA triple helix was also confirmed using gel electrophoresis at 25 oC, which revealed slower migration rates for the RNA triple helix compared with two double-helices (including two of the three components of the RNA triple helix) and with the single RNA oligos of each component of the RNA triple helix.
  • RNA oligos alone and conjugated to form the double and triple helices were collected.
  • the triplex design afforded quenching effects between the dye/quencher pair indicating the specific spatial proximity of the three RNA oligonucleotides composing the 3D structure, and consequently, the formation of the triple-helix assembly.
  • Example 1 The self-assembled RNA triple helix of Example 1 was subjected to the following characterization and competition and specificity assays.
  • Ionic strength In the ionic strength test, the effect of Mg 2+ and Na + on the formation of the RNA triple helix on TBE gel was measured. It was revealed that Na+ decreased the probability of triple helix formation, and likely caused the formation of undesirable dimers and tetramers.
  • FIG.2 depicts the effect of Mg 2+ and Na + on the formation of the RNA triple helix on TBE gel. Increasing amounts of NaCl and MgCl 2 were incubated with 1 ⁇ M of the RNA triple- helix. Na + decreased the probability of triple helix formation and resulted in undesirable dimers and tetramers.
  • Presence of urea The effect of urea concentration on the stability of the double- and triple helices were evaluated in the absence of urea, under physiological concentration (0.007 M), and at super-physiological urea concentration (7 M). Normal human adult blood typically contains between 0.004 and 0.0071 M urea, and the triple helix structures remained stable, without significant dissociation, under physiological urea concentration, and with declined dissociation in super-physiological conditions.
  • RNA triple-helix oligos concentration of single RNA and the RNA triple-helix oligos (equal molar ratio 1:1:1, final concentration 1 ⁇ M each) was fixed and the samples were loaded on 20% TBE PAGE gel without and with urea (0.007 and 7M) (Invitrogen).
  • Tm melting temperature
  • RNA triple helix oligos The concentration of single RNA and the RNA triple helix oligos at a particular molar ratio (1:1:1, final concentration 1 ⁇ M each) was fixed and the samples were incubated with different pH solutions (from 3 to 12) for 30 minutes and then loaded on 20% TBE gel (Invitrogen).
  • the triple helix had remarkable stability over a pH range of 5 to 9, which corroborated the pH-independent binding of the triplex, as purine motifs of triplex were believed to present significant stability under physiological pH.
  • RNA triple helix and control helix (scrambled miRs) structure was measured by gel electrophoresis, and on the emission of QUASAR® 705 and 570 on the oligos that formed the RNA triple helix.
  • the presence of quenching effects between the dye/quencher pair was believed to indicate the specific spatial proximity of the three RNA oligonucleotides composing the 3D structure, and, consequently, the formation of the triple-helix assembly. All experiments were done in triplicate.
  • RNA triple helix structure and single RNA oligos were measured by gel electrophoresis after incubation in 50 % serum solution for a pre-determined period of time, and on the emission of Quasar 705 and 570 on the oligos that formed the RNA triple helix and control triplex.
  • the single RNA and the RNA triple helix oligos were incubated in 50% fetal bovine serum (Gibco). A sample of RNA was taken at 0, 5, 10, 24, 48 and 72 hr time points after incubation at 37 °C, followed by analysis using 20% TBE gel (Invitrogen).All of these experiments were done in triplicate.
  • the triple helix structure was mostly intact (even after 72 hours of incubation) when compared to the single RNA oligos, which were rapidly degraded after 24 hours.
  • RNA triple-helix oligos were recognized and cleaved into smaller RNA products only in the presence of recombinant AGO2, when compared to the RNA triple-helices without AGO2 treatment. Therefore, the strong RNA triple-helix recognition by AGO2 depicted the self-assembled RNA triple helix as a highly specific structure.
  • the fluorescence signal of the dyes present in the triple helix was activated only at high AGO2 concentrations.
  • AGO2 was the only enzyme able to recognized and cleave the ⁇ 20-30 nt RNA oligos (only dsRNAs larger than 300 nt), as confirmed by the absence of RNA cleaved products, and by the absence of fluorescence signal from the triple helix dyes. These results confirmed the key role of AGO2 in the recognition of the RNA triple helix.
  • RNA triple-helix was previously incubated in equal molar ratio 1:1:1, final concentration 1 ⁇ M each oligo to form the structure.
  • DICER specificity assay increasing amounts of recombinant human Dicer (Genlantis) from 0.5 to 2 units were incubated with 1 ⁇ M of the triple-helix oligos during 16 hours at 37oC and then collected and inhibited by adding Dicer stop solution (Genlantis). The samples were then run on a 20% TBE gel (Invitrogen) to evaluate the by-products of the reaction.
  • Ago2 assay For Ago2 assay, increasing amounts of recombinant human AGO2/EIF2C2 (Sino Biological Inc.) from 5 to 160 ng were incubated with 1 ⁇ M of the triple-helix oligos during 60 minutes at 37 oC. Reactions were stopped by incubation at 65 oC for 30 minutes. The samples were then run on a 20% TBE gel (Invitrogen) to evaluate the by-products of the reaction.
  • RNA triplex nanoparticles were formed by complexation of the triple helix strands of Example 1 with polyamidoamine (PAMAM) G5 dendrimer creating a branched sponge-like nanoscopic structure readily visible using cryo-electron microscopy (cryo-EM, FIG.1C) and in the high-resolution scanning electron microscope (SEM, FIG.4, FIG.3, FIG.5A, FIG.5B, FIG.5C) images.
  • PAMAM polyamidoamine
  • RNA aggregates (3.4 ⁇ 1.1 ⁇ m as measured by SEM, FIG.4) were formed by interactions between the RNA triplex nanoparticles (56.6 ⁇ 3.9 nm as measured by SEM, FIG. 3) and the naked PAMAM G5 dendrimer (5.3 ⁇ 0.7 nm as measured by cryo-EM). These nanoparticles exhibited a densely packed molecular structure approximately 50 nm in diameter, which was also confirmed by Dynamic Light Scattering (DLS) analysis, which revealed a hydrodynamic diameter of 48.4 ⁇ 5.7 nm. Also confirmed was the fact that the branched sponge- like dendritic structures contained complexed RNA. This test was performed with SYBR green II staining, a sensitive dye used to detect RNA. Green fluorescence emitted from the RNA triplex nanoparticles confirmed that this structure was composed of dendrimer containing RNA.
  • DLS Dynamic Light Scattering
  • RNA triplex assembly at the nanoscale (small dendrimer triplex nanoparticles, ⁇ 50-60 nm, FIG.
  • an electrophoretic mobility shift assay was performed on agarose gel using increasing concentrations of PAMAM G5 (0.01 to 5 mg/mL), pre-incubated with 1 ⁇ M of the RNA triple-helix. Substantial change in the electrophoretic mobility of RNA triple-helix complexed with PAMAM G5 was evident for dendrimer concentrations higher than 0.025 mg/mL, indicating successful complexation. While PAMAM G5 alone imparted cytotoxicity in a dose dependent manner (0.1 to 5 mg/mL), its complex with RNA triplex or control triplex was cyto-compatible even at high dendrimer concentrations.
  • RNA triple-helix hydrogel nanoconjugates would be achieved by coating the breast tumor with the adhesive hydrogel scaffold that has been shown to enhance the stability of embedded nanoparticles used for local gene delivery (Conde, J. et al. Proceedings of the National Academy of Sciences of the United States of America 112, E1278-E1287 (2015); and Segovia, N., et al. Adv.Healthc.Mater.4, 271-280 (2015)).
  • a fluorescent image of the dual color RNA triple-helix in the form of a hydrogel scaffold was collected. Epi-fluorescence images following hydrogel cryo-sectioning showed a distinct and punctuate signal from the RNA triple-helix nanoparticles throughout the hydrogel network.
  • RNA triple helix hydrogel nanoconjugates that were pre-incubated with the complementary target for both strands show a positive fluorescent signal, whereas the ones without target did not show any fluorescent signal from the triple helix. This confirmed that individual RNA triple- helix modules complexed with the hydrogel network retained their original folding.
  • Triple helix nanoparticles (1 ⁇ M of RNA oligos complexed with 5% PAMAM G5 dendrimer) were mixed with 5% dextran aldehyde to form a hydrogel to a total of 12.5% dendrimer amine.
  • RNA triple- helix hydrogel scaffolds showed high triplex stability with a complete discharge release within 24 to 48 hours.
  • RNA triple-helix hydrogel scaffold fluorescence images Pre-cured fluorescently labeled scaffolds alone (control) or doped with triple-helix nanoparticles with or without target pre- incubation were snap-frozen in liquid nitrogen and kept at -80oC for 24 hours. Then, 12 ⁇ m-thick cryosections (Cryostat Leica CM1850) were analyzed by fluorescence microscopy (NIS- Elements NIKON®). Controls for this experiment included empty scaffold (without
  • nanoparticles and scaffold with non-hybridized triple-helices (quenched fluorescence from the oligos).
  • RNA triple-helix release from hydrogel scaffold in vitro Pre-cured disks of fluorescently labeled hydrogel scaffold alone or doped with triple-helix nanoparticles with or without target pre-hybridization were incubated in phosphate buffer saline (PBS) at 37oC. At different time points, samples were collected from the PBS and fluorescence of released products was quantified (Varioskan Flash Multimode Reader, Thermo Scientific). Data was plotted as percent of total hybridized oligo or dextran aldehyde released for each time point. Controls for this experiment included empty scaffold (without nanoparticles) and scaffold with non-hybridized triple-helices (quenched fluorescence from the oligos).
  • PBS phosphate buffer saline
  • RNA dendrimer nanoconjugates The cellular uptake of a complex PAMAM G5 dendrimer (Mr.28 kDa) with the RNA oligos was analyzed using the MDA-MB-231 TNBC cell line.
  • PAMAM G5 dendrimer Mr.28 kDa
  • LYSOTRACKER® Blue was used to label these structures. Briefly, cells were seeded at a density of 2 ⁇ 10 5 cells/well in 24-well plates and grown for 24 hours prior to incubation of RNA dendrimer nanoconjugates (0.05 mg/mL of dendrimer complexed with 1 ⁇ M of triplex oligos).
  • the lysosomal dye LYSOTRACKER® Blue DND-22 (Invitrogen) was included at a final concentration of 500 nM. Then, cells were fixed with 4% paraformaldehyde in 1 ⁇ PBS for 15 min at 37 oC. Finally, cells were mounted in PROLONG® Gold Antifade Reagent (Invitrogen). Images of cells were taken with a NIKON®A1R Ultra-Fast Spectral Scanning Confocal Microscope.
  • MDA-MB-231 triple negative breast cancer cells (ATCC® Cat. No. HTB-26TM, tested for mycoplasma contamination by the Division of Comparative Medicine Diagnostic Lab at MIT via IMPACT PCR, which was negative) were grown in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 4 mM glutamine, 10 % heat inactivated fetal bovine serum (Gibco, Life Technologies), 100 U/ml penicillin and 100 ⁇ g/ml streptomycin (Invitrogen) and maintained at 37 oC in 5% CO2.
  • DMEM Dulbecco's modified Eagle's medium
  • Gibco 10 % heat inactivated fetal bovine serum
  • Penicillin 100 ⁇ g/ml streptomycin
  • endocytosis can be divided into macropinocytosis, clathrin-dependent endocytosis and caveolae-mediated endocytosis (Conner, S.D. et al. Nature 422, 37-44 (2003)).
  • macropinocytosis clathrin-dependent endocytosis
  • caveolae-mediated endocytosis Conner, S.D. et al. Nature 422, 37-44 (2003).
  • the endocytosis inhibitors used were chlorpromazine, filipin, rottlerin, brefeldin A, colchicine and chloroquine.
  • RNA triple-helix dendrimer nanoconjugates cells were treated with several internalization inhibitors. Briefly, cells were seeded at a density of 2 ⁇ 10 5 cells/well in 24-well plates and grown for 24 hours prior to incubation of inhibitors and RNA dendrimer nanoconjugates. Cells were then treated with chlorpromazine (10 ⁇ g/ml), or filipin (5 ⁇ g/ml), or rottlerin (10 ⁇ M), or brefeldin A (5 ⁇ M), or colchicine (10 ⁇ M) or chloroquine (10 ⁇ M) (all from Sigma) in normal culture medium for 60 min at 37 oC.
  • RNA triple-helix dendrimer nanoconjugates 0.05 mg/mL of dendrimer complexed with 1 ⁇ M of triplex oligos
  • the naked PAMAM dendrimer labelled with ALEXA-FLUOR® 594
  • the dendrimer complexed with triplex oligos 0.05 mg/mL of dendrimer complexed with 1 ⁇ M of oligos
  • co-localization within the lysosomes at 0, 3, 6, 24 and 48h after exposure was studied.
  • the co-localization of naked dendrimer with the lysosomes was more evident than the dendrimer complexed with triplex oligos and decreased with time, which was consistent with the accumulation of PAMAM dendrimer in lysosomes and with the dendrimers’ capability to escape the endosome via the“proton-sponge effect”.
  • PAMAM dendrimers utilized endocytosis to enter cells and bypass the lysosomes
  • the triplex dendrimer nanoconjugates were believed to utilize micropinocytosis, probably, though not wishing to be bound by any particular theory, by triggering actin-mediated membrane ruffling. This difference in uptake mechanisms was believed to be dependent on the particles' physicochemical characteristics. Properties such as size and charge were ascertained to influence the endocytosis of these kinds of nanomaterials.
  • the dendrimer- triplex assembly structures size was 48.4 ⁇ 5.7 nm in diameter (as measured by DLS) and their aggregation occurred at the microscale (3.4 ⁇ 1.1 ⁇ m as measured by SEM, FIG.4) while the naked dendrimer size was only 6.7 ⁇ 0.4 nm in diameter as measured by DLS and 5.3 ⁇ 0.7 nm as measured by cryo-EM.
  • nanoparticles larger than 1 ⁇ m were most likely to be engulfed via micropinocytosis in this example, while the size involved in caveolae mediated endocytosis was about 60-80 nm (Conner, S.D. et al. Nature 422, 37-44 (2003)), all of which were consistent with the dendrimer-triplex microstructure and the naked dendrimer sizes.
  • RNA triplex nanoparticles (0.05 mg/mL of dendrimer complexed with 1 ⁇ M of triplex oligos) were able to transfect nearly 100% (99.8 ⁇ 2.5%) of the cancer cells with a strong and uniform signal from both dyes (Q705 and Q570) (FIG.7A, FIG.7B, FIG.7C, and FIG.7D).
  • the RNA-dendrimer complexes accumulated in MDA-MB-231 cells as verified by confocal microscopy at 24 h. Confocal imaging showed efficient uptake of the RNA-dendrimer into targeted cells, as demonstrated by the outstanding co-localization and overlap of the dual color RNA triple-helix at 24 h.
  • FIG.7A, FIG.7B, FIG.7C, and FIG.7D the specific uptake of fluorescent RNA-dendrimer nanoparticles into MDA-MB-231 cells.
  • Negative and positive controls were cells only and cells treated with the RNA double-helices
  • the flow cytometry was collected by the following procedure: MDA-MB-231 cells incubated with naked PAMAM G5 dendrimer alone or RNA oligos complexed with PAMAM G5 dendrimers were washed with PBS and detached with 0.25% trypsin-EDTA (Life Technologies).
  • FACS running buffer 500 ⁇ l, consisting of 98% PBS and 2% heat inactivated fetal bovine serum (Gibco, Life Technologies), was added to each well. Cells were mixed thoroughly and then transferred to FACS tube with filter lid, and the Alexa-Fluor 594 (for naked dendrimer) and Q705 and Q570 (for triplex) signals were acquired on FACS LSR Fortessa HTS- 1 (BD Biosciences) flow cytometer.
  • Alexa-Fluor 594 for naked dendrimer
  • Q705 and Q570 for triplex
  • a luciferase reporter was constructed to assess RNA triplex nanoparticles activity, which proved the functional role of altering the expression of the studied miRNAs in abrogating cancer.
  • the miRNA sensor was generated by inserting the antisense sequence to miR-205 or the sense sequence to miR-221 into the 3’ untranslated region of Luciferase on vector.
  • These cloning vectors were produced by Cyagen Biosciences Inc.
  • the vectors were constructed in E. coli Stbl3 host to ensure sequence stability with ampicillin as the antibiotic resistance marker.
  • the E.coli transformed with the vectors were grown in Luria-Bertani (LB) broth (Invitrogen) supplemented with 100 ⁇ g/mL of ampicillin (Invitrogen) at 37oC overnight. After this, the vectors were extracted and purified from the cultured E. coli using a Plasmid Midi Kit (Qiagen).
  • MDA-MB-231 cells not expressing luciferase were stably transfected with miR- Luc-vectors (1 ⁇ g per well) using LIPOFECTAMINE® 3000 (Invitrogen) according to the manufacturer’s protocol.
  • Cell lysates were measured with 150 ⁇ g/mL of D-luciferin (Caliper LifeSciences) for luciferase activity after 48 hours of luciferase vectors transfection.
  • RNA triple-helix dendrimer nanoconjugates were incubated with increasing amounts of the RNA triple-helix dendrimer nanoconjugates and controls for 24, 48 and 72 hours and the optimal sensor activity was evaluated by fluorescent imaging system (Xenogen XPM-2 Corporation) and quantification of cell lysates luminescence using a microplate reader (Varioskan Flash Multimode Reader, Thermo Scientific).
  • RNA triple-helices significantly inhibited the growth of MDA-MB-231 cells compared with the two double-helices (Q570 and Q705), and with a control triplex (FIG.9).
  • FIG.10 RNA triple-helix nanoparticles restricted MDA-MB-231 cell migration.
  • FIG.11 the number of colonies in the cells transfected with the RNA triple-helix was compared with control triplex at 24, 48 and 72 hours of incubation.
  • RNA migration and proliferation was studied using a wound closure assay where cells were allowed to grow in a 24-well plate until confluency and a wound was created using a sterile pipet tip. The cells were then incubated with the RNA-dendrimer complexes and wound closure was monitored using light microscopy. Cells treated with a control triplex were able to close the wound almost completely ( ⁇ 90%) within 72 h (FIG.10). Cells treated with the two double-helix miRs separately showed a partial closure of the wound over this time frame (75% for Q570 and 35% for Q705 oligos), while cells treated with the RNA triple-helix were not able to close the wound at all.
  • RNA oligos were seeded at a density of 1 ⁇ 10 5 cells per well in 24-well culture plates in complete DMEM (500 ⁇ l) with serum. After 24 hours of exposure to dendrimer with and without RNA oligos, the medium was removed and the cells were washed twice with sterile PBS and 300 ⁇ l of fresh medium with serum was added. Then 16.7 ⁇ l of sterile MTT stock solution (5 mg/mL in PBS) was added to each well.
  • the medium was removed and the formazan crystals were resuspended in 300 ⁇ l of dimethyl sulfoxide (Sigma).
  • the solution was mixed and its absorbance was measured at 540 nm as a working wavelength and 630 nm as reference using a microplate reader (Varioskan Flash Multimode Reader, Thermo Scientific).
  • the cell viability was normalized to that of cells cultured in the culture medium with PBS treatment.
  • MDA-MB-231 cells were grown to >95% confluency in a 24-well plate and a wound was made using a sterile pipet tip. The cells were then incubated with the RNA-dendrimer complexes and the wound closure was monitored using light microscopy.
  • MDA-MB-231 cells were harvested and resuspended in the culture medium.
  • 24-well plates 1 ⁇ 10 5 cells/well were seeded and allowed to grow until visible colonies formed (6 days).
  • Cell colonies were fixed using 4% paraformaldehyde (Sigma) for 5 minutes at 37 oC and stained with 0.05% crystal violet (Sigma) in Mili-Q water filtered at room temperature for 30 minutes. Cell colonies were then washed three times with water and incubated in a half of the total well volume of methanol (Sigma) to solubilize the dye. Then, the absorbance at 540 nm was measured using a microplate reader (Varioskan Flash Multimode Reader, Thermo Scientific). Each sample had three replicates and the mean ⁇ SD.
  • the in vivo nanoconjugates pharmacokinetics and platform therapeutic efficacy in an orthotopic breast cancer mouse model was studied.
  • the efficacy of the gene-therapy based therapeutic platform was compared to chemotherapy drugs that are part of the current standard of care for human breast cancer including doxorubicin (DOX), paclitaxel (PTX), and the monoclonal antibody bevacizumab (AVASTIN®).
  • DOX doxorubicin
  • PTX paclitaxel
  • AVASTIN® monoclonal antibody bevacizumab
  • TNBC triple-negative breast cancer
  • TNBC could benefit from gene therapy approaches, including endogenous miR modulation. It was believed that when a miRNA that was up-regulated intimately contributed to breast cancer progression (e.g. miR-221 0 ), an oncogenic miR inhibition therapy could be used to sterically hinder the miR expression via an antagomiR. Additionally, if a miR was downregulated in breast cancer (e.g. miR-205), the delivery of mature miRs (miR mimic oligonucleotide with the same sequence as the endogenous mature miR) would restore balanced miR expression levels.
  • miR miR mimic oligonucleotide with the same sequence as the endogenous mature miR
  • Avastin 0.07 ⁇ M) were implanted adjacent to the tumor in the mammary fat pad of SCID hairless congenic mice when tumors reached a desired volume of ⁇ 100 mm 3 . Inhibition of tumor progression was measured by luciferase expression while each RNA oligo release was tracked fluorescently via live imaging system for two weeks post-hydrogel implantation. No signs of inflammation were observed at the surgical site and no changes in body weight were observed before or after breast tumor induction or hydrogel implantation, which was believed to suggest that the hydrogels were biocompatible with no associated toxicity or side effects.
  • 50% tumor reduction was attained following each miR administration separately, while hydrogel only, control triplex (scrambled miRs) and Avastin-loaded hydrogel showed no tumor size reduction.
  • Dox and PTX-loaded hydrogels showed a 25% and 35% reduction in tumor size, respectively.
  • RNA nanoconjugates uptake and biodistribution were examined by quantifying fluorescence images of mice organs (liver, kidneys, spleen, heart, lungs and intestines) 13 days post-implantation. At 13 days, 20-30% of the miRs persisted in the tumoral tissue exclusively, when compared to major organs. No fluorescent signal was found in any of the major organs.
  • a time curve documenting the triplex signal (Q705 and Q570 dyes) in organs harvested from mice treated with the RNA triple-helix hydrogel scaffolds for 2, 6, 24, 48, 72 h and days 5, 8, 11 and 13 was collected. The triplex nanoparticles accumulated exclusively in the tumor tissue as demonstrated by ex vivo images of the organs and time curves for each organ. There appeared to be no accumulation of the triplex nanoparticles in any major organ at all time points during the 13 days, except some background signal in the intestine (also observed at 0h of incubation).
  • mice treated with hydrogel scaffolds loaded with the triplex and control triplex were treated with the triplex and control triplex, as well as for the chemotherapeutic drugs (DOX, PTX and Avastin).
  • miR-205 directly targeted LAMC1, a protein altered in human breast cancer that has been implicated in a wide variety of biological processes including cell adhesion, differentiation, proliferation, migration, signaling, and metastasis.
  • LAMC1 a protein altered in human breast cancer that has been implicated in a wide variety of biological processes including cell adhesion, differentiation, proliferation, migration, signaling, and metastasis.
  • miR-205 expression was enhanced by the triple-helix scaffold, the expression of LAMC1 decreased considerably, altering cancer cells proliferation as corroborated by the foregoing in vitro and in vivo data.
  • miR-205 also directly targeted E2F1, a gene that was overexpressed in triple-negative breast tumors (Piovan, C., et al. Mol Oncol 6, 458-472 (2012)), in which the level of miR-205 was low.
  • E2F1 was a target of miR-205 in triple negative breast cancer tissues.
  • p53 acted like an enhancer of miR-205 expression as the increase in the expression of this miR was followed by the up-regulation of p53.
  • miR-221 expression which was found to be up- regulated in triple-negative breast tumors and in a panoply of cancer types (Nassirpour, R. et al. Plos One 8(2013)), has been shown to directly target proliferation and adhesion genes, such as E-cadherin, Snail1 and Slug (Snail2).
  • RNA from MDA-MB-231 cells and breast tumors from SCID xenografted mice was extracted using RNeasy Plus Mini Kit (Qiagen) according to the manufacture’s protocol.
  • cDNA was produced using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) using 500 ng of total RNA.
  • qRT-PCR was performed with Taqman miRNA assays and Taqman probes FAM-MGB for miR-205, miR-221, VEGF, LAMC1, E2F1, p53, E-cadherin, Snail1 and Slug (Snail2) (Applied Biosystems).
  • RNU6B was used as a reference gene.
  • the reactions were processed using Light Cycler 480 II Real-time PCR machine (Roche) using TaqMan® Gene Expression Master Mix (Applied Biosystems) under the following cycling steps: 2 min at 50 °C for UNG activation; 10 min at 95 °C; 40 cycles at 95 °C for 15 s; 60 °C for 60 s. At least three independent repeats for each experiment were carried out. Gene expression was determined as a difference in fold after normalizing to the housekeeping gene RNU6B.
  • RNA triple-helix hydrogel scaffold synthesis and in vivo implantation Tagged hydrogel scaffolds were developed as previously described (Oliva, N., et al. Langmuir 28, 15402-15409 (2012)).
  • RNA nanoparticles (1 ⁇ M of RNA oligos doped in 5% PAMAM dendrimer generation 5 with 100% amines) or the free drugs: doxorubicin (DOX, from LC Laboratories), paclitaxel (PTX, from LC Laboratories) and the monoclonal antibody
  • bevacizumab (Avastin® from Roche) were added to the dendrimer solution prior to hydrogel formation. All solutions were filtered through a 0.22 ⁇ m filter prior to hydrogel formation for in vivo implantation. Pre-cured disks of fluorescently labeled scaffold with or without RNA nanoparticles were formed and implanted subcutaneously on top of the mammary tumor in mice.
  • Dextran aldehyde tagging reaction Dextran aldehyde (Mr 10,000 Da, 50% oxidation; 10 mg) was tagged by reaction with 2 mg of Alexa-Fluor® 405 Cadaverine (Invitrogen) in 20 mL of 50 mM carbonate buffer (pH 8.5) for 1 hour at room temperature. Then, the reaction crude was cooled down in an ice-water bath and imine bonds were reduced with 20 mL of 30 mM sodium cyanoborohydrate in PBS for 4 hours. Then, tagged dextran aldehyde was dialyzed four times through a 3,000 Da MWCO Centrifugal Filter (Millipore) for 20 min each time at room temperature and 4,000 RCFs. The purified product was lyophilized.

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Abstract

La présente invention concerne des structures à triple hélice d'ARN. La triple hélice d'ARN peut être associée à un dendrimère afin de former un conjugué dendrimère à triple hélice d'ARN, qui peut être appliqué sur un tissu biologique, tel qu'une tumeur. Le conjugué dendrimère à triple hélice d'ARN peut être disposé dans un hydrogel. L'hydrogel peut être appliqué sur un tissu biologique, tel qu'une tumeur. L'hydrogel et/ou le dendrimère peuvent réguler la libération de la triple hélice d'ARN. L'invention concerne également des méthodes de traitement d'un tissu biologique et des kits.
PCT/US2016/050977 2015-09-10 2016-09-09 Structures à triple hélice d'arn, compositions et méthodes Ceased WO2017044768A1 (fr)

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