Shielding nanoconjugates for use as delivery vehicles and molecular biological probes

Abstract

Novel nanoconjugate compositions, such as nanoparticle-oligomer arrays (NOAs), and related methods for precision therapeutics and molecular diagnostics. These compositions comprise a core, optionally a nanoparticle core, and a plurality of oligomeric strands forming a dense, highly oriented three-dimensional scaffold. This scaffold architecture is engineered to position functional molecules precisely at desired distances from the core and / or the external surface, thereby controlling their physical and chemical interactions with the core and / or microenvironment, enabling distinct functionalities not observed with the functional molecules in an unstructured formulation.

Description

[0001] Shielding Nanoconjugates for Use as Delivery Vehicles and Molecular Biological Probes

[0002] Related Application

[0003] This application claims the benefit of priority from U. S. Provisional Application No. 63 / 718,283 filed November 8, 2024, and U. S. Provisional Application No. 63 / 718,306 filed November 8, 2024, the contents of which are hereby incorporated by reference.

[0004] Reference To Sequence Listing

[0005] The Sequence Listing submitted herewith, as a text file named “1367.202WO.xml,” created on November 10, 2025, and having a size of 20,000 bytes is hereby incorporated by reference pursuant to 37 CFR § 1.52(e)(5).

[0006] Field of the Invention

[0007] The present invention relates generally to the fields of nanotechnology, biomolecular engineering, molecular diagnostics, and nanomedicine.

[0008] More specifically, the invention provides novel nanoconjugate compositions, such as nanoparticle-oligomer arrays (NOAs), and related methods for precision therapeutics and molecular diagnostics. These compositions comprise a core, optionally a nanoparticle core, and a plurality of oligomeric strands forming a dense, highly oriented three-dimensional scaffold. This scaffold architecture is engineered to position functional molecules precisely at desired distances from the core and / or the external surface, thereby controlling their physical and chemical interactions with the core and / or microenvironment, enabling distinct functionalities not observed with the functional molecules in an unstructured formulation.

[0009] First, the scaffold can position a functional molecule (e.g., a therapeutic agent or detectable moiety) at a desired distance from the external-facing surface, thereby shielding it sterically and / or electrostatically from interactions with the local microenvironment. In therapeutic applications, this shielding renders an agent pharmacologically dormant, minimizing off-target toxicity and creating a prodrug-like platform that activates upon a specific trigger. In diagnostic applications, shielding a detectable moiety (e.g., fluorophores) reduces nonspecific staining that leads to background noise, thereby significantly enhancing the signal-to-noise ratio upon target recognition. Second, and concurrently or alternatively, the scaffold is engineered to position a functional molecule at a precise distance from the core to exploit core-dependent physical phenomena. For example, a plasmonic nanoparticle core and a fluorophore can be fixed at an optimal separation to induce and maximize phenomena such as metal-enhanced fluorescence (MEF).

[0010] This precise, multi-functional spatial control mediated by the oligomeric scaffold, enabling both shielding from the exterior and specific interactions with the core, is a key aspect of the invention for creating advanced therapeutic and molecular detection nanomaterials.

[0011] Background of the Invention

[0012] The fields of molecular diagnostics and nanomedicine remain constrained by significant technological limitations that impede the realization of their full clinical and research potential. Specifically, advancements in the precise detection of biomolecules and the site-specific delivery of therapeutic agents are critically dependent upon the development of highly advanced and sophisticated nanoconstructs.

[0013] Deficiencies in Molecular Diagnostics

[0014] Current state-of-the-art molecular detection assays, including in situ hybridization (ISH), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC), are consistently challenged by insufficient signal-to-noise ratio (SnR). This limitation primarily arises from two fundamental problems in the prior art. First, traditional oligonucleotide or antibody probes, typically labeled with fluorophores, are highly susceptible to non-specific interactions with unintended cellular components or sequences due to charge, hydrophobic, hydrogen-bonding, or pi-pi stacking interactions. This non-specific binding is the predominant source of high background noise, which obscures the signal from the true target. Second, the innate fluorescent signal produced by individual fluorophore molecules is often insufficient, necessitating extensive, multi-step signal amplification processes (e.g., secondary antibodies or enzyme-mediated amplification cascades). These amplification protocols drastically increase the assay time and cost, introduce considerable sample-to-sample variability, and elevate the potential for error, thereby hindering rapid and reliable clinical diagnostics and large-scale biological research.

[0015] Deficiencies in Therapeutic Delivery In the critical realm of therapeutics, especially in cancer therapeutics, conventional drug-delivery systems suffer from severe systemic constraints. A major challenge of the prior art is achieving favorable biodistribution and pharmacokinetics that enable the therapeutic agent to accumulate selectively at the tumor site. Crucially, existing systems lack a reliable "prodrug-like" mechanism wherein the active compound is functionally inert until it encounters a specific pathological trigger.

[0016] Consequently, current treatments frequently lead to off-target effects and systemic toxicity, most notably affecting filtering organs such as the liver and kidneys, due to non-selective distribution of the active agent throughout the body. The need for a delivery platform that maintains a therapeutic agent in a pharmacologically quiescent state unless and until activation is triggered only by a disease-specific molecular marker remains a substantial, unmet clinical need for maximizing localized therapeutic efficacy while concurrently mitigating severe systemic organ damage.

[0017] Summary of the Invention

[0018] The present invention provides novel Nanoparticle-Oligomer Array (NOA) compositions and related methods, wherein said compositions utilize an oligomeric scaffold to create a highly versatile platform for advancements in molecular diagnostics and targeted therapeutics.

[0019] In a first aspect, the invention provides a Nanoparticle-Oligomer Array (NOA) composition comprising:

[0020] a core, optionally a nanoparticle core;

[0021] a plurality of oligomer strands, typically including nucleic acids, densely arrayed on and extending from the core to form a three-dimensional scaffold; and

[0022] at least one functional moiety (e.g., a detectable agent or a therapeutic agent),

[0023] wherein said scaffold is configured to position said functional moiety precisely at a desired location relative to the core and / or an external surface of the composition to thereby control the chemical and physical interactions of said moiety. A core principle of the NOA composition is the implementation of the highly oriented oligomer shell as a steric and / or electrostatic barrier. This barrier is engineered to maintain the functional moiety in a functionally quenched, shielded, or pharmacologically dormant state.

[0024] In one embodiment, the NOA functions as a molecular diagnostic probe. In this embodiment, the functional moiety is a detectable agent that is substantially shielded by the dense oligomer shell. This comprehensive steric hindrance is effective at preventing non-specific interactions of the detectable agent with the local microenvironment (a common issue in molecular diagnostic assays like ICC and FISH), thereby resulting in the significant reduction of background signal. Furthermore, the polyvalent nature of the array facilitates the inclusion of multiple detectable moieties in close proximity, thus affording opportunities to enhance signal intensity, while simultaneously reducing noise suffered by prior art probes.

[0025] In a further embodiment, the NOA functions as a prodrug-like therapeutic delivery platform. The functional moiety is a therapeutic agent or prodrug that is maintained in a pharmacologically inactive or reduced-activity state by virtue of its strategic placement within the sterically shielded core / shell structure. In some embodiments of the therapeutic, the presence of a specific pathological marker (e.g., a disease-associated messenger RNA) triggers a conformational change and results in the release or exposure of the therapeutic agent, thereby orchestrating the controlled, site-specific activation and delivery of the therapeutic agent exclusively at the disease locus. This target-activated release mechanism is critical for maximizing localized therapeutic efficacy in oncology while drastically reducing systemic toxicity.

[0026] The nanoparticle core primarily functions as an inert scaffold to support the high-density oligomer array. However, in certain preferred embodiments, the core material is specifically selected to augment the construct's functionality. For instance, the core may be chosen to enhance the detectable signal through proximity-dependent phenomena (e.g., metal-enhanced fluorescence by gold or silver cores) or may be comprised of an infinite coordination polymer (ICP) nanoparticle, which is a metal-organic particle with features such as biocompatibility and enhanced porosity for high loading capacity of small molecules.

[0027] These compositions and methods overcome fundamental limitations of the prior art by providing a single, flexible, biocompatible architecture capable of achieving specificity and signal control in diagnostics, and profound site-specific activation of therapeutics. In a preferred embodiment, the invention provides a colloidal nanostructure, less than 200 nm in diameter, comprising:

[0028] a. A nanomaterial core; and

[0029] b. A scaffolding of synthetic oligomers, wherein the oligomers are connected to the core and arranged to form a three-dimensional shell around the core, providing a surface for further functionalization.

[0030] In yet another embodiment, the invention provides a nanostructure core, further comprising a flare oligonucleotide partially or fully hybridized to the scaffolding oligomers, wherein the flare oligonucleotide is covalently attached to a cargo molecule (therapeutic, diagnostic, or fluorogenic) at its 5’ or 3’ terminus or internally, positioned within the scaffolding or exposed on the external surface.

[0031] In another embodiment, the invention provides a nanostructure core, wherein the synthetic oligomers are selected from the group consisting of single-stranded oligonucleotides (DNA / RNA analogs), oligo(ethylene glycol), oligo(propylene glycol), oligo(acrylic acid), oligo(methacrylic acid), oligo(l-vinylpyrrolidone), and oligo(acrylamido(carboxybetaine)).

[0032] In yet another embodiment the invention provides a nanostructure core, wherein the singlestranded oligonucleotides are nuclease-resistant and selected from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), phosphorodiamidate morpholino oligonucleotides (PMOs), tricyclo-DNA (tcDNA), bicyclo-DNA (bcDNA), constrained ethyl LNA (cEt-LNA), trans-bridged nucleic acids (trans-BNAs), and spirocyclopropylene bridged nucleic acids (scpBNA) as well as derivatives and / or combinations of the above.

[0033] In another embodiment, the invention provides a nanostructure core, wherein the nanomaterial core is selected from the group consisting of a metal, polymer, silica, liposome, micelle, and metal-organic coordination polymer. In another embodiment the core is a metallic element coated with an azide-functionalized coating selected from the group consisting of:

[0034] (a) a binary mixture of o-thiol and co-azide terminated oligo(acrylic acid) with thiol- terminated oligo(ethylene glycol);

[0035] (b) oligo(ethylene glycol) possessing multiple thiol groups and one or more azido groups (c) a thiol or disulfide-terminated zwitterionic oligomer

[0036] (d) An oligonucleotide with one or more thiol or disulfide groups, and also containing an azide group.

[0037] In another embodiment, wherein the cargo molecule is an azide-containing therapeutic or other chemically active agent derivatized with A / -succinimidyl-(4-azidomethyl)benzoate (AMB-NHS) or an / V-hydroxyimide analog thereof.

[0038] In another embodiment, the invention provides a chemical compound N-succinimidyl-(4-azidomethyl)benzoate (AMB-NHS) and any A / -hydroxyimide analogs thereof, including but not limited to esters of A / -hydroxy-5-norbornene-2,3-dicarboximide, / V-hydroxyphthalimide, / V-hydroxynaphthalimide, AZ-hydroxyglutarimide, as well as esters of phenol derivatives.

[0039] In another embodiment, the invention provides a nanostructure core, wherein the therapeutic agent is an antineoplastic or immunomodulating agent (ATC codes L01-L04) or a cytotoxic agent, including prodrugs of said agents, including but not limited to lenalidomide, bortezomib, doxorubicin, paclitaxel, staurosporine, and cantharidin.

[0040] In another embodiment, the invention provides a method of preparing a flare oligonucleotide comprising reacting an oligonucleotide containing a strained alkyne moiety (e.g., BCN or DBCO) with an excess of the azide-containing cargo molecule in aqueous solution, wherein the cargo molecule is an azide-containing therapeutic or other chemically active agent derivatized with / V-succinimidyl-(4-azidomethyl)benzoate (AMB-NHS) or an A / -hydroxyimide analog thereof.

[0041] In another embodiment, the invention provides a method of synthesizing the nanostructure core, wherein the core is a metal-ligand coordination polymer, comprising introducing a labile salt of a metal ion to a basic solution of a ditopic 3-hydroxy-4-pyridinone ligand that contains one or more azide substituents.

[0042] In another embodiment, the invention provides a method for cross-linking the metal-ligand coordination polymer nanoparticles, wherein a molecule bearing two or more strained alkynes is introduced into a solution of coordination polymer nanoparticles and allowed to intramolecularly cross-link available azide groups, stabilizing the coordination polymer network with covalent bonds. In another embodiment, the invention provides a ditopic 3-hydroxy-4-pyridinone ligand, where two (3-hydroxy-4-pyridinone) substituents are connected to an aromatic group (including but not limited to benzene, naphthalene, pyridine, quinoline, fluorene, and anthracene) also containing one or more azide substituents. In particular, the compound claimed has the structural formula of the product in Scheme 9, wherein the 3-hydroxy-4-pyridinone groups are connected via their nitrogen atoms at the 3 and 5-positions of / \ / -(3-azidopropyl)benzamide.

[0043] DEFINITIONS

[0044] As used herein, unless otherwise indicated, the following terms have the meanings ascribed below. Singular forms include the plural unless the context clearly dictates otherwise.

[0045] “Functional moiety” refers to a cargo molecule, such as a therapeutic agent or fluorescent dye, that is capable of interacting with other molecules in the surrounding chemical or biological environment to generate a measurable effect or signal arising from the interaction.

[0046] “Oligomer” refers to a molecule with a repeating structure, typically containing 100 repeat units or less, which can comprise any combination of synthetic or naturally occurring monomers including nucleic acids and their analogs.

[0047] “Nanoparticle” refers to a material having an overall diameter less than 1000 nanometers in length, or preferentially less than 200 nanometers.

[0048] “Bonding” refers to any covalent, ionic, or dative interaction between two or more molecules.

[0049] “Derivative” refers to a chemical variant of a molecule in which a functional group or chemical backbone is modified.

[0050] BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1: Nanoconjugate examples with sterically shielded internal detectable molecules. A: Illustration of detectable molecules (fluorophores depicted) bound to an internally-located position of the nanoconjugate capture strands. B: Illustration of detection molecules bound terminally to non-capture oligomer, whereby the length of the detectable molecule-labeled oligomer renders the detectable molecule sterically and / or electrostatically shielded from the local microenvironment.

[0052] Figure 2. Illustration of NOA target binding and flare release. In this example the target is depicted as a complementary oligonucleotide which binds to the capture (recognition) sequence, displacing the flare and releasing the functional molecule (cargo)

[0053] Figure 3. Left: MDA-MB 231 “triple negative” breast cancer cells cultured without therapeutic intervention. Right: MDA-MB 231 GFP cells treated with GFP RNA-targeting NOAs that release cytotoxic (S)-IO-hydroxycamptothecin flares upon target binding. Cells cultured at 37° C in 5% CO2 were grown in DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin. Cells were treated with 5 nM (S)-IO-hydroxycapmptothecin-flare carrying NOAs. After 5 days, cells were imaged with a Zeiss Lattice SIM 3 structured illumination microscope, using a brightfield illumination source, highlighting the near complete cell death of cells treated with the target-triggered NOAs.

[0054] Figure 4. Optical imaging illustrating the impact of the external portion of oligomer shell on biological interactions and transport. Cells cultured at 37°C in 5% CO2 were grown in DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin. Cells were treated with 1nM nanoparticles functionalized with (left) LA-PEG5k, (middle) DNA SEQ ID NO: 13, or DNA amine-terminated SEQ ID NO: 14. Images taken with brightfield illumination on the Evos M7000 microscope show black dots that correspond to endocytosed nanoparticle clusters.

[0055] Figure 5. Effect of Survivin knockdown via morpholino-templated nanoparticles. NOAs functionalized with sequence SEQ ID NO 15 and Uni-2PEG23-N3 spacer were added to a solution of MDA “triple negative” breast cancer cells at a concentration of 5 nM, 500 pM, and 50 pM and incubated in DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin for 3 days. In parallel, cells were also treated with a standard transfection protocol from Gene Tools, Endo-Porter. After 3 days of incubation, cells incubated with Endo-Porter and 10,000 nM morpholino clearly exhibited a rounded apoptotic phenotype. Likewise, cells treated with 5 nM NOAs showed a similarly dramatic apoptotic phenotype, while untreated cells remained healthy and proliferative. Visualization of the SulfoCy5.5-labeled probes was performed using an Evos M7000 epifluorescent microscope, which revealed perinuclear localization of the nanoparticles, a well-documented and characteristic pattern after endocytosis of nucleotide-functionalized nanoparticles.

[0056] Figure 6: Fluorescence of nanoparticle-templated fluorophores. Left: quantification of the fluorescence value per particle observed relative to the corresponding fluorophore in solution. Nanoparticles with gold (Au) or silver (cores) were templated with fluorophore-conjugated DNA carrying Cy5, Cy3, or FITC, and the resulting fluorescence of the particles quantified. Right: Example of distance-dependent core- fluorophore interactions. Cy5-conjugated DNA strands in solution had nanoparticles added, upon which the fluorescence immediately decreases as a result of fluorophore surface adsorption and / or light scattering. After two hours of incubation, the DNA strands arrange radially, resulting in a favorable displacement of fluorophores from the NP surface to achieve MEF, resulting in boosted levels of fluorescence beyond that of fluorophores in solution.

[0057] Figure 7: Spectral shift of nanoparticle-conjugated fluorophores. Reference emission spectra (filled in plots) compared to FITC alone in solution (FITC control), FITC, Cy3, and Cy5 held near a gold nanoparticle surface via fluorophore-conjugated oligomers (line plots). Each fluorophore held near the gold surface exhibits a distinct spectral shift according to its interaction with the nanoparticle core, in this case the plasmon resonance of the gold core affecting emission strength, wavelength, and lifetime. Note that the excitation of the triple-fluorophore containing nanoparticle is excited at 514nm (FITC). As such, the emission of Cy3 and Cy5 arises due to FRET interactions between the fluorophores.

[0058] Figure 8: Example of color combination options on a NOA using common blue (B), green (G), yellow (Y), red (R), and far-red (FR) fluorophores. Table assumes detection filter sets defined by wavelength ranges corresponding to those colors, however more options are possible corresponding to the excitation and emission detection capabilities of the detection modality.

[0059] Figure 9: FRET-pairing from Nanoparticle-conjugated fluorophores. Gold nanoparticles with oligomers conjugated to Cy3 (FRET donor) and Cy5 (FRET acceptor). The close proximity of donor and acceptor fluorophores to one another due to their conjugation to the nanoparticle surface results in FRET. Shown: reference excitation spectra (dotted line plot) and emission spectra (filled in plot) of Cy3 and Cy5, overlaid with measured fluorescence values attained by exciting Cy3 conjugated to the nanoparticle surface with a 561 nm laser. Emission from the conjugated Cy3 and Cy5 (FRET) can be observed, along with the characteristic red-shifting of each fluorophore emission due to the nanoparticle plasmon resonance.

[0060] Figure 10: Immunocytochemistry using nanoparticle probes. U2OS cells treated with Left: Mouse anti-beta actin primary antibody followed by anti-mouse antibody conjugated to Alexa Fluor ® 647, and Right: Mouse anti-beta actin primary antibody functionalized with a DNA oligomer “target” sequence followed by nanoparticles functionalized with Alexa Fluor ® 647 and DNA “capture” oligomers that are complementary to the target sequence of the primary antibody.

[0061] Figure 11: Nanoparticle single molecule localization microscopy. Images taken with AF647-labeled NOAs (right) and the line profile (left, pixel intensities along the red line drawn on the image). With a 1 ms exposure enough light from blinking (illustrated in frame t= 1 ms) was collected to generate an SMLM image identifying the localization of particles (t = 1 ms overlay; white dots are the processed SMLM image from 10,000 x 1 ms frames).

[0062] Figure 12. Analytical acrylamide gel of in-house synthesized oligos. From left to right: unmodified GFP-flare, internal-BCN GFP flare, DNA impurities left from BCN-flare synthesis, GFP capture sequence (Integrated DNA Technologies), 5’DBCO GFP flare

[0063] Figure 13. UV-Vis spectrum of internal-Cy5.5-BCN flare showing DNA peak at 260 nm and Cy5.5 absorbance at 693 nm

[0064] Figure 14. Confocal microscope image of fluorescent AgNP- PAA- DNA- NOAs endocytosed into MDA-231 breast cancer cells.

[0065] Figure 15. DLS size distribution of AuNPs at each stage of functionalization (bare, intermediate, NO A)

[0066] Figure 16. ATR-FTIR spectrum of Uni-2PEG23-N3AuNPs with azide absorbance band at 2070 cm'1

[0067] Figure 17. Size distribution of CuNPs before and after reaction with Uni-2PEG23-N3 Figure 18. Size distribution of purified Fe-ICP-N3NPs (0.1pm / 50kDa filtered) compared to crude reaction mixture.

[0068] Figure 19. Size distribution of purified Fe-ICP-N3particles compared to Gd-ICP-N3particles

[0069] Figure 20. Size distribution of AuNPs before and after functionalization with azide-terminated zwitterionic polymer

[0070] Figure 21. Bis-cyclooctyne crosslinkers examined for Fe-ICP nanoparticles

[0071] Figure 22. “Cure shrinkage” of crude Fe-ICP particles with increasing concentration of crosslinker (DBCO-PEG5-DBCO). *lntensity size measurement is weighted differently

[0072] Figure 23. 1H-NMR spectrum of AMB-NHS (500 MHz, DMSO-d6)

[0073] Figure 24. UV-Vis spectrum of bare vs oligomer-functionalized Fe-ICP particles

[0074] Figure 25. DLS size (by scattering intensity) of MOR / PEG NOAs vs DNA-NOAs exposed to 100% DMEM for 24h.

[0075] Figure 26. FRET from ICP cores containing FRET pairs BoDIPY and AF568 (Left chart) and AF568 and AF647 (Right chart). Quantified is the fold increase in fluorescence of each nanoparticle relative to fluorophore in solution, or fluorophore mixture in solution for the purposes of FRET quantification. In each case, the FRET pair show strong coupling, resulting in high fluorescence levels of the FRET acceptor upon excitation of the FRET donor. Fluorescence measurements were acquired on the GloMax plate reader with the following settings: BoDIPY Excitation (BoDIPY Ex): 475nm, AF 568 Excitation (568 Ex): 520nm, AF647 Excitation (647 Exc.) 627nm, BoDIPY Emission (BoDIPY Em): 500-550nm, AF 568 Emission (568 Em):580-640nm, AF647 Emission (647 Em.) 660-720nm.

[0076] Figure 27. NOA-held fluorophores reduce nonspecific binding in biomolecular assays. MDA-MB-231 cells were fixed and permeabilized with a paraformaldehyde / methanol mixture for 10 minutes. Subsequently, cells were treated with (row 1) SulfoCy5.5-DBCO dye, (row 2) SulfoCy5.5-DBCO clicked to LA-PEG5K-azide, (row 3) SulfoCy5.5-DBCO clicked to LA-PEG5K-azide templated on a 13 nm gold nanoparticle, and (row 4) NOAs functionalized with SEQ ID NO: 8 and carrying SulfoCy5-functionalized flares (SEQ ID NO 2). Mitochondrial localization was observed in all exposed-dye forms, while NOAs with shielded fluorophores demonstrated no observable colocalization.

[0077] Figure 28. Target-triggered flare release from novel NOA formulations. A solution of 1 nM NOAs of compositions indicated, each carrying Cy5-labeled flares held by the oligomer matrix near the nanoparticle surface in order to achieve quenching of the fluorophores, were mixed with 1 pM of DNA oligomers fully complementary to the NOA capture strands, which then competed with and released flares from the nanoparticle surface, resulting in measurable fluorescence. Fluorescence levels were measured on a GloMax plate reader for 30 minutes following target addition. Importantly, NOAs made with a combination of negatively charged spacers (LNA-Tio) and neutral capture strands (morpholino) were able to perform target capture and flare release at comparable levels to pure DNA strands, while having the added benefits of presenting neutral, nuclease resistant morpholinos to the external environment.

[0078] Figure 29. Target capture from novel NOA formulations. NOAs were tested for their ability to capture targets in physiological conditions were diluted to a concentration of 10 nM, and the fluorescence measured. Then, complementary fluorescent flares were added to the nanoparticle solution at a concentration of 100 nM, and the fluorescence measured on a GloMax plate reader for the following 30 minutes. The inclusion of a condition using nanoparticle functionalized with a noncomplementary sequence (SEQ ID NO: 11) showed a measured increase in fluorescence due to the free, unbound fluorescent flares in solution. However, all complementary NOAs showed markedly decreased fluorescence, as the complementary nanoconjugates bound the fluorescent flares and sequestered them within the oligomer matrix, holding them near the gold core and quenching fluorescence.

[0079] DETAILED DESCRIPTION OF THE INVENTION

[0080] The NOA construct, in a most preferred embodiment, comprises a plurality of specifically oriented oligomers which form a shell-like configuration. These oligomers may be oriented around a nanoparticle core, or by coordination of a different means. The present invention overcomes the drawbacks of existing functional moiety-carrying nanoconjugates by utilizing a shell of oligomers which provide steric and / or electrostatic shielding for internally held functional moieties with non-random placement. Non-random placement of the functional moiety may be accomplished by bonding the functional moiety to one or more oligomers within the oligomer shell. In other embodiments placement of the functional moiety is accomplished by bonding the functional moiety to the core, which is also sterically and / or electrostatically shielded by the shell of oligomers.

[0081] Optionally, the NOA construct further comprises a second oligomer, termed a “flare,” which is bound to the plurality of specifically oriented oligomers in such a way that the shell of oligomers keeps all or a portion of the flare shielded via steric hindrance and / or electrostatics from the local microenvironment while bound.

[0082] When this NOA construct binds with a targeted moiety, it results in a change in the conformation of the bound oligomer, which displaces or exposes the flare in such a way that it becomes more available to interact with the local environment. Optionally, where the flare contains a therapeutic, this displacement results in a therapeutic effect

[0083] In some alternative embodiments, the NOA construct may target a nucleotide or nucleotide analogue sequence. In other embodiments, it may target a protein, peptide sequence, sugar, or other molecules or macromolecule.

[0084] In further embodiments, the NOA construct comprises two or more types and / or sequences of oligomer in the shell. Additional types of oligomers may contain flares, or may be designed to perform other functions, such as affecting the construct’s chemical or biological function.

[0085] In yet other embodiments, individual oligomers may be a sequence of the same type of molecule (such as DNA oligonucleotides); in other optional embodiments, individual oligomers may further comprise mixtures of different molecules (for example LNA “gapmers” which contain mixtures of DNA and LNA) in order to alter the interaction with target molecules, or to alter the interaction with the local microenvironment.

[0086] In certain alternative embodiments, two or more types of flares are included on one particle. These flares may have identical or distinct therapeutic effects.

[0087] Alternatively, molecules other than DNA or nucleotides may be included within the shell of oligonucleotides in order to alter the NOAs. In some circumstances, these other molecules may be used instead of DNA entirely. Such materials may be phosphorodiamidate morpholino oligonucleotides (“morpholinos”) and / or or polyethylene glycol) (PEG) or their derivatives, which present neutrally charged moieties to the external microenvironment, and may reduce opsonization and increase circulation time.

[0088] In some embodiments the flare may be displaced entirely after the binding of a target; in others the flare may be moved but remain attached, as in the case of hairpin structures or other methods of linking the flare.

[0089] In some embodiments the core is made of gold. In other embodiments the core is made of lipid, polymer, infinite coordination polymer, other materials, or there is no core and the 3D oligomeric shell structure is formed by intermolecular coordination of the oligomers via hydrogen-bonding, hydrophobic association, or similar supramolecular assembly method. In further embodiments the core is made from a combination of multiple materials, some of which may be present for a specific biological, chemical, magnetic, or thermal effect, as in the case of a therapeutic contained in a lipid or polymer nanoparticle, or gold shell useful for photothermal therapy.

[0090] Examples

[0091] General

[0092] NMR & mass spectrometry were performed by NuMega Resonance Labs (San Diego, CA). Mass spectra were collected on a Perkin Elmer PE-SCIEX API-150 mass spectrometer equipped with an electrospray ionization source. NMR spectra were collected on a 500 MHz Bruker Avance II NMR spectrometer. FTIR spectra were collected on a Bruker Alpha II ATR-FTIR spectrophotometer. Solvents were acquired from TCI America (Portland, OR) and Oakwood Chemical (Estill, SC) and stored over 3A molecular sieves unless otherwise indicated.

[0093] 4-(chloromethyl)benzoic acid, / V-hydroxysuccinimide, doxorubicin HCI, and 10-hydroxycamptothecin were obtained from Ambeed (Arlington Heights, IL). Phosphorodiamidate morpholino oligonucleotides were purchased from Gene Tools, LLC (Philomath, OR). All oligonucleotide synthesis was carried out on an Applied Biosystems ABI 391 A DNA synthesizer using 4,5-dicyanoimidiazole as the activator (0.25M in acetonitrile) and 3% dichloroacetic acid in dichloromethane as the deblock solution. The manufacturer’s standard 1 pmol synthesis cycle was used for all syntheses, except the coupling time for regular phosphoramidites was set to 90 seconds, and 180 seconds for LNA phosphoramidites. All syntheses were performed with the 5’ DMT group left intact (where applicable). All DNA, LNA & modifier phosphoramidites were purchased from Glen Research (Sterling, VA) and dissolved in anhydrous acetonitrile before use.

[0094] Section A. Example of bifunctional azido-NHS ester linker

[0095] The bifunctional azido-NHS ester, / V-hydroxysuccinimidyl-(4-azidomethyl)benzoate (AMB-NHS) was synthesized (Scheme 1) as a versatile molecule for introducing azides into biomolecules, macromolecules, cancer drugs, and other compounds of interest. This linker has been described in the I iterature[i] but not explicitly claimed as a distinct entity in the patent literature nor has the method of manufacture. The low cost of the precursors and ease of synthesis (no column chromatography or inert atmosphere required) make this a particularly attractive modifier from a process chemistry standpoint.

[0096]

[0097] Scheme 1. Synthesis of NHS-AMB. No chromatography or inert atmosphere is required.

[0098] Example 1. Synthesis of 4-(azidomethyl)benzoic acid. To a 100-mL round-bottomed flask with a magnetic stirrer was added 5.00 grams (29.31 mmol) of 4-(chloromethyl)benzoic acid and 2.10 grams (32.30 mmol) of sodium azide, followed by 25 ml_ of anhydrous dimethyl sulfoxide. The vessel was stoppered and the reaction mixture was allowed to stir for 24 hours at room temperature, during which time sodium chloride precipitated from the solution. The reaction mixture was poured into 100 ml_ distilled H2O, causing a white precipitate to form. The solids were collected by vacuum filtration on a Buchner funnel, washed with an additional 100 mL of H2O, and allowed to dry on the filter. Recovered: 4.57g (88.0%).1H NMR (500 MHz, DMSO-d6) δ 13.03 (s, 1H), 7.98 (d, 2H), 7.46 (d, 2H), 4.52 (s, 2H).

[0099] Example 2. Synthesis of A / -hydroxysuccinimidyl-(4-azidomethyl)benzoate (AMB-NHS). To a 200-mL round- bottomed flask with a magnetic stirrer was added (4-azidomethyl)benzoic acid (1.00 g, 5.64 mmol) and HATU (2.15g, 5.64 mmol) in 25 mL of anhydrous DMSO with stirring. Once the solids dissolved, A / , / V-diisopropylethylamine (2.45 mL, 14.11 mmol) was added and the color of the solution changed from clear to bright yellow. Subsequently, A / -hydroxysuccinimide (0.682g, 5.93 mmol) dissolved in DMSO (5 mL) was added to the HATU-activated carboxylic acid. The reaction vessel was stoppered, and stirring continued overnight for 16 hours. The next day, the reaction mixture was slowly added to 150 mL of dilute hydrochloric acid (0.1M). A large amount of precipitate appeared which was quickly collected by vacuum filtration on a Buchner funnel, washing with 100 mL of H2O. After drying on the filter, the product was isolated as a fluffy white powder, freely soluble in DMSO and acetone. Recovered: 1.22g (79.0%).1H NMR (500 MHz, DMSO-d6) δ 8.14 (d, 2H), 7.63 (d, 2H) 4.67 (s, 2H), 2.90 (s, 4H). (Figure)

[0100] The active ester moiety is not limited to / V-hydroxysuccinimide, other suitable compounds include A / -hydroxy-5-norbornene-2,3-dicarboximide (HONB), AAhydroxynaphthalimide, / V-hydroxyphthalimide, A / -Hydroxy-A / -phenylbenzamide, / V-hydroxyglutarimide, or any compound containing a / V-hydroxyimide group. The A / -hydroximide group may be created by reacting a molecule containing an anhydride functional group in the presence of hydroxylamine hydrochloride and pyridine under reflux conditions (Scheme 2). Other suitable reaction partners include: 3-oxabicyclo[3.1.0]hexane-2, 4-dione, caronic anhydride, 2,2-dimethylsuccinic anhydride, 3,3-dimethylglutaric anhydride, 2,3-pyridinedicarboxylic anhydride, tetrahydrophthalic anhydride, hexahydrophthalic anhydride, and other cyclic anhydrides. The active ester moiety may also be a substituted phenol, particularly those substituted with electron-withdrawing groups, such as pentafluorophenol, p-nitrophenol, and related compounds. The coupling agent may be HATU or another uronium salt including but not limited to HBTU, TBTU, and COMU.

[0101]

[0102] Scheme 2. Synthesis of alternative activated ester version of AMB starting from caronic anhydride.

[0103]

[0104]

[0105]

[0106] Table 1. Oligonucleotide sequences in Examples. J = morpholino oligonucleotide. NHS = modifier installed via NHS chemistry, CPG = 3’ modifier included on solid support. T = LNA-deoxythymidine. All oligonucleotides were made synthetically by Integrated DNA Technologies or in-house.

[0107]

[0108] Table 2. Zeta potential measurements of NOA constructs described in examples

[0109] Section B. Examples of oligonucleotide-cargo “flares” made using NHS-AMB

[0110] Example 3. Synthesis of Doxorubicin-(azidomethyl)benzamide (DOX-BA) (Scheme 3).

[0111] In one embodiment, a 50 ml_ round-bottomed flask with magnetic stirrer was charged with 0.2g (.345 mmol) of doxorubicin hydrochloride suspended in 10 mL anhydrous tetrahydrofuran, followed by 50 pl_ (.362 mmol) of triethylamine to liberate the hydrochloride salt of doxorubicin. Then, / V-hydroxysuccinimidyl-(4-azidomethyl)benzoate was added and the reaction mixture blanketed under argon while stirring. The vessel was sealed with a rubber septum and left at room temperature overnight protected from light. The following day, the reaction mixture was poured into 100 ml_ of 1X PBS resulting in the precipitation of a red solid, which was collected by vacuum filtration and washed with H2O to afford 0.23g (94.7%) of doxorubicin-(azidomethyl)benzamide (DOX-BA). ESI-MS (m / z) [M+Na]+Calc’d for C35H34N4NaO12725.2, found 725.3.

[0112]

[0113] Scheme 3. Synthesis of DOX-BA from doxorubicin hydrochloride.

[0114] Example 4. synthesis of Doxorubicin-flare.

[0115] In one embodiment, a 1mg / ml_ stock solution of DOX-BA was prepared in anhydrous DMSO (equivalent to 1.14 mM). 10 pL of 300 pM BC22 Target 3’DBCO-DNA solution (SEQ ID NO: 4) (Integrated DNA Technologies) was mixed with 10 pL of DOX-BA solution (~5x excess) in a 1.5-mL centrifuge tube, diluted to 150 pL in H2O, and allowed to shake for 17 h at 37°C. The mixture was diluted to 500 pL in 1X PBS and purified by gravity filtration using a NAP-5 gel column (Cytiva) equilibrated in 1X PBS. Fractions were collected and analyzed by UV-Vis. The main fraction was found to have [DNA] = 6.1 pM and [DOX] = 4.8 pM, using Amax=480 nm for doxorubicin (£48o = 11,500 L • M'1• cm’1) and Amax=260 nm for DNA (E26O = 206,700 L • M'1• cm’1) suggesting a successful nearly quantitative conjugation, with the extinction coefficient of the modified doxorubicin being estimated from the literature.

[0116] Example 5. Synthesis of 10-hvdroxycamptothecin-(azidomethyl)benzoate (CPT-BA) (Scheme 4L In one embodiment, a 50 mL round- bottomed flask with magnetic stirrer was charged with 97.3 mg (.549 mmol) of 4-(azidomethyl)benzoic acid dissolved in 10 mL dry DMSO along with,209g HATU (.549 mmol). / , A / -diisopropylethylamine (0.238 mL, 1.37 mmol) was subsequently added and the reaction mixture stirred under argon for several minutes to allow activation of the carboxylic acid. Then, (S)-10-hydroxycamptothecin pre-dissolved in DMSO (0.200g) was added to the reaction mixture with stirring and the esterification was allowed to commence under inert atmosphere. Due to the large difference in pKa between the phenolic and aliphatic hydroxy groups, the esterification preferentially occurs at the 10-position. [ii] After stirring overnight, the product was recovered by pouring the reaction mixture into dilute HOI (100 mL, 0.1M) causing an orange powder to precipitate, which was isolated by vacuum filtration and washed with H2O. Recovered: 0.17g (59%) of camptothecin 10-azidomethylbenzoate (CPT-BA). ESI-MS (m / z) Calc’d for C28H2iN5O6[M+H]+524.2, Found 524.1.

[0117]

[0118] Scheme 4. Synthesis of CPT-BA from (S)-10-hydroxycamptothecin.

[0119] Example 6. Synthesis of 10-QH-camptothecin-flare (Scheme 5).

[0120] In one embodiment, a 1mg / ml_ stock solution of CPT-BA was prepared in anhydrous DMSO (equivalent to 1.91 mM). 10 pl_ of BC22 Target 3’DBCO-DNA (SEQ ID. NO: 4) (300 pM, Integrated DNA Technologies) was added to a 1.5-mL centrifuge tube along with 10 pL CPT-BA stock solution and 80 pL H2O. The mixture, which was initially turbid due to the low solubility of CPT-BA, turned clear after shaking at 37°C for 17 h. The mixture was diluted to 500 pL in 1X PBS and purified by gravity filtration using a NAP-5 gel column (Cytiva) equilibrated in 1X PBS. Fractions were collected and analyzed by UV-Vis. The main fraction was found to have [DNA] = 4.16 pM and [CPT] = 5.03 pM, using Amax=378 nm for 10-OH-CPT (£378= 19,900 L • M'1• cm’1) and Amax=260 nm for DNA (E26O = 206,700 L • M'1• cm’1) suggesting a successful conjugation, with the extinction coefficient of the modified 10-OH-CPT being estimated from the literature.

[0121]

[0122] Scheme 5. Synthesis of CPT-BA chemoflare from DBCO-DNA.

[0123] Example 7. Synthesis of internal bicyclo[6.1.01non-4-yne (BCN) flares with improved purity

[0124] In one embodiment, the GFP-Flare DNA sequence was synthesized but with the innermost thymine position replaced with Fmoc Amino-Modifier C6-dT (Glen Research) (SEQ ID. NO: 6). Upon completion of DNA synthesis, the solid supports were left in the column and first washed with acetonitrile (1 ml_). The cyanoethyl protecting groups on the phosphate backbone were removed by treatment with 10% A / -ethylisopropylamine in acetonitrile for 5 minutes. The supports were washed with acetonitrile (3 ml_) and then treated with 20% 4-methylpiperidine in dimethylformamide for 5 minutes. This step was repeated three times, then the supports were washed with DMF (3 mL) followed by acetonitrile (6 mL). Finally, the supports were treated with a solution of ~3mg (10.3 pmol, >10x molar excess) encto-BCN-NHS carbonate in DMSO / triethylamine (99:1) for 3-4h. This step was repeated again to ensure complete conversion of the internal amino modifier into the desired carbamate. Importantly, the carbamate linkage between BCN and the oligonucleotide is stable to ammonium hydroxide deprotection.

[0125] The supports were washed with acetonitrile (5 mL) to remove unbound BCN modifier, then the CPGs were removed from the column and treated with concentrated ammonia solution (30% in H2O) for 17h at room temperature. The ammonia was evaporated on a hot plate under low heat, and the resulting crude DNA solution was purified using a 150 mg Glen-Pak™ cartridge (Glen Research) according to the manufacturer’s protocol. After purification, the DNA solution was concentrated on a SpeedVac to remove acetonitrile, then the oligo solution was diluted and loaded on an acrylamide-bis electrophoresis gel and compared with the analogous strand bearing a 5’DBCO modifier. The DBCO-flare shows a smeared band with multiple species corresponding to DBCO-oligos clustered with similar molecular weights (Figure 12). The BCN-flare showed a single strong band with faint bands corresponding to co-eluted failure sequences and dimer(s), respectively, suggesting that BCN-flares are more easily purified and thus a more preferable platform for chemoflare synthesis. Therefore, any examples of flares described in this invention are also produced analogously with BCN.

[0126] Example 8. Synthesis of internal SulfoCvanine5.5 / Cvanine5.5 GFP Flares

[0127] In one embodiment, a solution of internal-BCN GFP-flare DNA (300 pL) (SEQ ID. NO: 6) was prepared (500 pM DNA) and was mixed with 2 molar equivalents of either SulfoCyanine5.5 azide or Cyanine5.5 azide DMSO stock solution (Lumiprobe) and allowed to react at 37 °C for 17 h. The flares were purified by diluting the reaction mixture in PBS and applying it to a Glen Research Gel-Pak™ column equilibrated with 1X PBS. Fractions were collected and analyzed by UV-Vis to determine the success of the conjugation and purification (Figure 13), then stored protected from light at 4° C.

[0128] Example 9. 3’Cvanine5.5-GT-NonTarqet synthesis (purchased DNA oligonucleotide)

[0129] In another embodiment, a 100 pM stock solution of Cyanine5.5-azide (Lumiprobe) was prepared in anhydrous DMSO. 10 pL of 300 pM 3’DBCO-GT-NonTarget DNA solution (SEQ ID. NO: 1) (Integrated DNA Technologies) was mixed with 30 pL of Cyanine5.5-azide solution (1:1 molar ratio) in a 1.5-mL centrifuge tube, diluted to 150 pL in H2O, and allowed to shake protected from light for 17 h at 37° C. The mixture was diluted to 500 pL in 1X PBS and purified by gravity filtration using a column of Sepharose CL-4B equilibrated in 1X PBS. Fractions were collected and analyzed by UV-Vis. The main fraction was found to have [DNA] = 3.2 pM and [Cy5.5] = 2.9 pM, using Amax=680 nm for Cyanine5.5-azide (E48O ~ 198,000 L • M'1• cm-1) and Amax=260 nm for GT-NonTarget-DNA (E26O - 235,500 L • M’1• cm’1) suggesting a successful nearly quantitative conjugation, with the extinction coefficient of the Cyanine5.5-azide provided by the

[0130] manufacturer. Example 10. 5’Cvanine5-GFP-Flare synthesis (in-house synthesized DNA oligonucleotide)

[0131] In another embodiment, Glen UnySupport™ 500 (Glen Research) were used as the solid support for DNA synthesis. The sequence was 5’DBCO GFP Flare (SEQ ID. NO: 2). The final base in the synthesis was DBCO-TEG phosphoramidite (Glen Research). Upon completion of DNA synthesis, the solid supports were separated from the column and immersed in 2 mL of deprotection solution (30% ammonia in water) for 17 hours at room temperature. Then, the ammonia was gently evaporated under low heat on a hot plate, and the resulting crude DNA solution purified using a 150-mg Glen-Pak cartridge (Glen Research) according to the manufacturer’s protocol, except that the 2% trifluoroacetic acid detritylation step was omitted due to the lack of a 5’ dimethoxytrityl group. The DBCO-DNA was eluted with 50:50 acetonitrile:water and the acetonitrile removed in vacuo via SpeedVac. The concentration of DNA was measured by the absorbance at 260 nm and DBCO conjugation efficiency was measured by the absorbance at 310 nm. The 5’DBCO-Flare DNA (€260 = 136,900 L • M’1• cm’1) stock concentration was calculated (935 pM) and 53.5 pL of 5’DBCO-GFP Flare DNA (SEQ ID. NO: 6) was mixed with 60 pL of Cyanine5-azide stock solution (1.66 mM, Lumiprobe) and allowed to shake protected from light for 17 h at 37°C. Afterwards, the Cy5-conjugated DNA was purified using a NAP-5 Sephadex G-25 column (Cytiva) equilibrated in 1X PBS. The main fraction was found to have a DNA concentration of 64.3 pM and a Cy5 concentration of 76.0 pM, using Amax=649 nm for Cyanine5-azide (estimated €649 = 250,000 L • M’1• cm-1), suggesting a quantitative conjugation. ESI-TOF (m / z): [M+H]+Calc’d 5196.007, Found: 5198.760.

[0132] Example 11. 3’Cyanine5-BC22-Target morpholino synthesis (purchased morpholino oligonucleotide)

[0133] In another embodiment, morpholino oligonucleotides were purchased from Gene Tools, LLC with a BCN moiety incorporated at the 3’ terminus. Briefly, 20 pL of 3’BCN-BC22-Target morpholino (300 pM in H2O) (SEQ ID. NO: 9) was mixed with 5 pL of Cyanine5-azide stock solution (1.66 mM in DMSO) and diluted to 100 pL in 1X PBS. The mixture was allowed to shake protected from light for 17 h at 37° C. Afterwards, the Cy5-conjugated morpholino was diluted to 0.5 mL in 1X PBS and purified using a NAP-5 Sephadex G-25 column (Cytiva) equilibrated in 1X PBS. The main fraction was found to have a morpholino concentration of 3.8 pM and a Cy5 concentration of 3.4 pM, using Amax=265 nm for 3’BCN-BC22-Target morpholino (€265 = 279,740 L • M'1• cm’1), suggesting a quantitative conjugation. Example 12. Functional testing of chemoflare-loaded NOAs (capture-and-release)

[0134] In one embodiment, the AuNP-NOA containing oligomers with the compositions shown in Table 3

[0135]

[0136] Table 3. Composition of AuNP-NOAs described in Example land where each capture strand had the nucleotide sequence 3’cyclooctyne-GFP-Capture (SEQ ID NO: 8 or 3) were synthesized by incubating 1000 molar equivalents of oligomer (50% of each in embodiments with co-adsorbed oligomers) in a solution of 13 nm citrate-capped gold nanoparticles (50 nM) and.01% Triton X-100 (w / v). The solution was shaken at 50°C for 1 hour, then sodium chloride was slowly added overnight to a final concentration of 300 mlVI. In one embodiment, a bifunctional 3’-lipoamide and 5’ azide-modified LNA-Tw oligonucleotide was synthesized as a versatile linker for preparing azide-functionalized metal nanoparticles (Ag, Au) for further elaboration with oligomers, fluorescent dyes, etc. (Scheme 6)

[0137]

[0138] Scheme 6. Structure of 5’ terminus of LA-LNA-T10-N3 before cleavage from CPGs and detritylation

[0139] These functionalized nanoparticles were hybridized with complementary oligomers of the sequence Cy5-ATGGTGAGCAAGG (SEQ ID NO: 12). Flares were annealed onto the NOA scaffold by adding a stoichiometric ratio of 10 flares per nanoparticle, heating to 70°C for 10 minutes, and cooling slowly to room temperature overnight, after which the nanoparticles were spun at 21,000 x ref for 20 minutes, and the supernatant removed. This process was repeated 3x to remove unbound flares. Flare-containing NOAs were then diluted in 1X PBS to a concentration of 10 nM, and the fluorescence of each nanoparticle solution measured on a GloMax plate reader. Then, each well was mixed with 1 pM of DNA oligonucleotides fully complementary to the capture strands, to compete with and release flares from the nanoparticle surface. Importantly, NOAs made with a combination of negatively charged spacers (LNA-T10) and neutral capture strands (morpholino) were able to perform target capture and flare release at comparable levels to pure DNA strands, while having the added benefits of presenting neutral, nuclease-resistant morpholines to the external environment. However, similar morpholino oligonucleotides having a neutral spacer (PEG 1K) in place of LNA performed extremely poorly, illustrating the nontrivial and nonobvious functional differences that can arise on self-assembled surfaces with seemingly minor changes, such as the inclusion of nuclease-resistant oligonucleotides in the scaffolding.

[0140] DNA and morpholino-functionalized NOAs were also tested for their ability to capture targets in physiological conditions (Figure 29). In this test, the same nanoparticle constructs (without flares) were diluted to a concentration of 10 nM, and the fluorescence measured. Then, fluorescent flares were added to the nanoparticle solution at a concentration of 100 nM, and the fluorescence measured for the following 30 minutes. The inclusion of a condition using NOAs functionalized with a noncomplementary sequence (SEQ ID NO: 11) showed a measured increase in fluorescence due to the free, unbound fluorescent flares in solution. However, all complementary NOAs showed markedly decreased fluorescence, as the complementary nanoconjugates bound the fluorescent flares and sequestered them within the oligomer scaffold, holding them near the gold core and quenching fluorescence. Once again, the PEG1K-morpholino particle showed the worst performance, sequestering fewer complementary strands than any other construct of the same sequence, while the LNA-morpholino chimera performed similarly to DNA-NOAs.

[0141] The capture and release of flares has similarly demonstrated remarkable utility in therapeutic applications. MDA-MB 231 GFP “triple negative” breast cancer cells, which also express green fluorescent protein (GFP), were incubated with 5 nM NOAs that carried cytotoxic (S)-10-hydroxycamptothecin-conjugated flares. After 5 days of incubation, cells treated with NOAs targeting GFP mRNA for target-triggered flare release showed significant, nearly-complete cell death (Figure 3).

[0142] Section C. Bifunctional LA-LNA-T10-N3 spacer for modification of metal nanoparticles & target capture / release

[0143] Example 13. Synthesis of LA-LNA-T10-N3 spacer oligonucleotide (SEQ ID NO: 7)

[0144] 3’ Dithiol Serinol CPGs (Glen Research) were used as the solid support. The final base in the synthesis was Fmoc Amino Modifier C6-dT (Glen Research). Upon completion of DNA synthesis, the solid supports were left in the column and first washed with acetonitrile (1 ml_). The cyanoethyl protecting groups on the phosphate backbone were removed by treatment with 10% A / -ethyl isopropylamine in acetonitrile for 5 minutes. The supports were washed with acetonitrile (3 mL) and then treated with 20% 4-methylpiperidine in dimethylformamide for 5 minutes. This step was repeated three times, then the supports were washed with DMF (3 mL) followed by acetonitrile (6 mL). Finally, the supports were treated with a solution of 3mg (>10 pmol, 10x molar excess) A / -hydroxysuccinimidyl-(4-azidomethyl)benzoate (AMB-NHS) in DMSO / triethylamine (99:1) for 3-4h. This step was repeated again to ensure complete conversion of the 5’ amino modifier into the desired amide. The supports were washed with acetonitrile (5 mL) to remove unbound azide modifier, then the CPGs were removed from the column and treated with concentrated ammonia solution (30% in H2O) for 2h at room temperature. The ammonia was evaporated on a hot plate under low heat, and the resulting crude DNA solution was purified using a 150 mg Glen-Pak™ cartridge (Glen Research) according to the manufacturer’s protocol. After purification, the DNA solution was concentrated on a SpeedVac to remove acetonitrile and the concentration calculated by UV-Vis absorbance at 260 nm before moving to the next step (vide infra).

[0145] Example 14. Conjugation of LA-LNA-T10-N3 to morpholino oligonucleotides & functionalization of nanoparticles for capture / release experiments

[0146] In one embodiment employing the disulfide-bearing-LNA-Tio spacer described in the previous Example, a solution of 3’ BCN-conjugated morpholino strands were mixed in solution with lipoamide-LNA-Tio-Na (1:2 stoichiometric ratio of morpholino: T10) and allowed to shake at 37° C for 24 hours. The “clicked” strands were then purified using 10k MWCO centrifuge filters to elute and remove all unreacted strands. Upon purification, the chimeric DNA-morpholino oligonucleotide was used for particle functionalization as mentioned in example 12.

[0147] Section D. Iron “infinite coordination polymer” (ICP) nanoparticle synthesis & functionalization with morpholino oligonucleotides

[0148] Metal-ligand coordination polymer nanoparticles were first developed by Calabrese and Briley at Northwestern University and described in a seminal Angewandte Chemie paper.[iii] Derived from the FDA-approved iron chelator deferiprone (itself derived from the natural flavor compound maltol), Fe-ICP particles are an attractive biocompatible scaffold for therapeutic applications. In their original form, the Fe-ICP nanoparticles were conjugated with 100% DNA oligonucleotides on the surface, limiting their clinical utility. Since then, their capabilities have been greatly expanded, with new versions featuring a neutral surface coating of oligomers, enabling their use in clinical medicine. The synthesis of the particles has also been improved to give lower polydispersity. The Fe-ICP nanoparticles are formed from a rigid ditopic 3,4-hydroxypyridinone ligand (bearing an azide functional handle) and iron(lll) nitrate nonahydrate in dilute NaOH, affording colloidally-stable iron coordination-polymer nanoparticles. To our knowledge, this is the first material in its class that has been deployed in vivo and represents a significant advance in biocompatible nanomaterials synthesis. Distinct from MOFs, which are crystalline and well-ordered metal-organic structures prepared by solvothermal synthesis in organic solvents such as DMF (and often incompatible with aqueous conditions), Nanotrace Fe-ICPs are amorphous and form nearly instantaneously in water upon mixing of the two components, greatly simplifying their manufacturing.

[0149] Example 15. Synthesis of diaminobenzoic acid mono-HOPO intermediate (D-MHP) (Scheme 7)

[0150] To a 200 mL round-bottomed flask with a magnetic stirrer was added 10 grams of 3,5-diaminobenzoic acid (65.72 mmol) and 8.28 grams of maltol (65.72 mmol). 50 mL of acidic n-propanol was added (49:1 n-propanol:12M HCI) and the reaction vessel fitted with a Vigreux condenser and heated to reflux overnight exposed to ambient atmosphere. A solid precipitate appeared, and the suspension was vacuum-filtered while still hot and the solids collected on a Buchner funnel. The filter cake was washed repeatedly with acetone to afford 6.65g of D-MHP (38.9% isolated yield) as a beige solid, insoluble in acetone and MTBE, sparingly soluble in water, soluble in acetonitrile, DMF and DMSO. Acetone washing is the preferred method to isolate the pure mono product and no suitable substitutes have been found.1H NMR (500 MHz, DMSO-d6) 07.65 (d, 1H), 7.31 (t, 1H) 6.97 (t, 1H), 6.73 (t, 1H), 6.36 (d, 1H) 6.27 (t, 1H), 2.02 (s, 1H).

[0151]

[0152] Scheme 7. Synthesis of D-MHP

[0153] Example 16. Synthesis of diaminobenzoic acid HQPO intermediate (D-BHP) (Scheme 8)

[0154]

[0155] To a 100 mL round-bottomed flask with a magnetic stirrer was added 6.50 grams of D-MHP (24.98 mmol), 3.94 grams of maltol (31.22 mmol) and 30 mL of acidic 2-ethoxyethanol (49:1 ethoxyethanol:12M HCI). The reaction vessel was fitted with a Vigreux condenser and the mixture heated to reflux temperature (135 °C) for 72 hours under argon atmosphere. The reaction mixture was allowed to cool to room temperature, causing the precipitation of a dark brown crude material (~6.0g) which was collected by vacuum filtration. The crude material was stirred in 100 mL of boiling pyridine until fully dissolved, then cooled to room temperature to allow precipitation of the pyridinium salt of the desired product. The pyridine was liberated by dissolving the material in 100 mL of boiling dimethylformamide and allowing the solution to cool, causing the desired product to precipitate as a grey powder. Isolated: 1.62 g (17.5% yield) of D-BHP. Soluble in hot DMSO and DMF, practically insoluble in H2O and other organic solvents. ESI-MS (m / z) Calc’d for CI9HI6N2O6[M+H]+369.1, Found 369.1.

[0156]

[0157] Scheme 8. Synthesis of D-BHP

[0158] Example 17. Synthesis of diaminobenzoic acid HOPO azide (D-BHP-C3-N3) (Scheme 9)

[0159]

[0160] D-BHP (0.500g, 1.35 mmol) and DMSO (20 mL) were added to a 100 mL round-bottomed flask with a magnetic stirrer. The mixture was heated in a glycerol bath to 60°C until all the solids dissolved, then allowed to cool to room temperature with continuous stirring. Then, 0.513g (1.35 mmol) of HATU was added, followed by 0.59 mL of A / , A / -diisopropylethylamine. The reaction mixture was capped with a septum and rendered inert under a blanket of argon. Then, 0.132 mL of 3-azidopropylamine (1.35 mmol) was injected to react with the HATU-activated carboxylic acid. After stirring for 6 hours at room temperature, the reaction was quenched by the addition of 1 volume of water and allowed to stand for several hours, after which a solid material precipitated. This material was collected by vacuum filtration and washed with H2O to afford 0.25 g (41.1% yield) D-BHP-C3-N3 as a grey solid.1H NMR (500 MHz, DMSO-d6) 58.77 (t, 1H), 8.02 (t, 2H), 7.94 (t, 1H), 7.65 (d, 2H) 6.25 (d, 2H), 3.42 (m, 2H), 3.32 (m, 2H) 2.04 (s, 6H) 1.79 (m, 2H). FTIR (ATR): vmax2097 cm1(N=N=N stretch)

[0161]

[0162] Scheme 9. Synthesis of D-BHP-C3-N3

[0163] Notably, this ligand employs a shorter spacer between the iron-coordinating moieties and the azide functional handle (3 carbons instead of 4 carbons) compared to the original synthesis described in Angewandte Chemie. The high conjugation efficiency of the ICP particles is maintained despite shortening the linker length, while the 3-azido-propan-1-amine precursor is far less expensive to make, starting from the cheap and abundant 3-chloropropylamine hydrochloride (<$0.10 / g, Ambeed), enabling future scale-up.

[0164] Example 18. Synthesis of Fe-ICP-N3nanoparticles from D-BHP-N3 (Scheme 10)

[0165] A stock solution was prepared consisting of 2.28 mM D-BHP-N3 by dissolving.052 grams of the ligand in 50 mL of 24.5 mM NaOH. A stock solution of Fe(NO3)3*9H2O (10.8 mM) in dilute HCI (4 mM) was also freshly prepared. The shelf-life of the D-BHP-N3 / NaOH stock solution is indefinite under ambient conditions; the Fe(lll) stock solution must be made fresh every 1-2 weeks and stored protected from light in a refrigerator.

[0166]

[0167] te

[0168] Scheme 10. Synthesis of ICP nanoparticles from metal ions and organic ligands 44 mL of D-BHP-N3 stock solution was mixed with 6 mL of Fe(lll) stock solution (slight molar excess of ligand, Fe2L3stoichiometry) and vortexed immediately, causing the color of the solution to turn deep red (indicating iron complex formation from its characteristic absorbance band at 460 nm). This crude reaction mixture was stored in a 50-mL polypropylene centrifuge tube; it is indefinitely stable, and aliquots can be drawn and purified when a batch of Fe-ICPs is needed. Importantly, the size distribution of the Fe-ICP-N3particles can be controlled by sizeexclusion filtration of the crude reaction mixture (vide infra).

[0169] 10 mL of the crude Fe-ICP-N3stock solution was back-loaded into a 20-mL syringe fitted with a 32mm diameter PALL Acrodisc™ 0.1 pm Supor™ polyethersulfone (PES) membrane filter. The solution was pushed through the filter and A460 of the solution was measured before and after, indicating ~86% pass-through of particles post-filtration. The azide concentration of the filtered stock solution was calculated using the known extinction coefficient of the Fe(lll) complex at 460 nm (£460 = 2870 L’M'1cnr1) and multiplying by 1.5 to account for the Fe2L3stoichiometry of the particles. The Fe-ICP-N3particle solution was then purified by ultrafiltration using an Amicon Ultra-15 centrifugal filter with a 50 kDa MWCO membrane. The size of the ICP particles was measured by DLS and found to have an average hydrodynamic radius of 13 nm (Figure 18). The final size distribution of the purified Fe-ICPs is dependent on the filter type(s) and MWCO used; larger or smaller distributions may be obtained based on filter selection. The stock solution of purified Fe-ICP-N3particles in distilled water is stable indefinitely and no sedimentation is observed upon standing even after >6 months.

[0170] Example 19. Functionalization of Fe-ICP-N3nanoparticles to generate Fe-ICP-NOAs

[0171] Fe-ICP-N3nanoparticles are employed as the core material; they can react with a wide range of strained alkyne-bearing molecules while maintaining colloidal stability. For initial biodistribution experiments, a mixture of phosphorodiamidate morpholino oligonucleotides, oligo(ethylene glycol) (MW 5,000) and SulfoCy5.5-DBCO were chosen for optimal blood circulation and in-vivo fluorescent imaging (I VI S).

[0172] To a 2.0-mL Eppendorf LoBind® centrifuge tube was added the following:

[0173] 1) 255 pL Fe-ICP-N3stock solution (2.2 mM in N3)

[0174] 2) 50 pL Triton X-100 (1% w / v) 3) 80 pL DBCO-PEG5K (500 pM, Vector Labs)

[0175] 4) 80 pL 3’BCN-GFP Capture Morpholino (SEQ ID NO: 8) (500 pM, Gene Tools)

[0176] 5) 130 pL DBCO-SulfoCyanine5.5 (100 pM, Lumiprobe)

[0177] 6) Dilute to final volume of 2 mL in H2O

[0178] The mixture was shaken at 37 °C for 17h to allow the DBCO-bearing molecules to click to the nanoparticle surface. Afterwards, the particles were purified by ultrafiltration using an Amicon Ultra-4 centrifugal filter with 50 kDa MWCO. The buffer was exchanged to 0.005% Tween-20 in PBS (w / v) and the particles were stored at 4°C until ready for use.

[0179] Morpholino and PEG-functionalized Fe-ICP-NOA particles are exceptionally colloidally stable; they can be frozen and thawed without aggregation of the particles. They are also stable to storage in 200 mM NaCI for >30 days without noticeable sedimentation or adhesion to the container sidewalls. The particle concentration and loading of oligonucleotides may be determined by UV-Vis; the ratio of A26o to A460 is a measure of the oligonucleotide loading, with a value of ~ 5.4 for bare Fe-ICP particles and 10-15 or higher for oligonucleotide-functionalized Fe-ICP-NOAs (Figure 2).

[0180] For sterilization, the Fe-ICP-NOA particles may be passed through hydrophilic PVDF filters (0.1 or 0.22 pm) with minimal loss. PES and hydrophilic PTFE also yield acceptable results.

[0181] Example 20. Special capabilities of Fe-ICP nanoparticles (FRET)

[0182] Nanotrace investigated new properties of Fe-ICP nanoparticles; for example, it was found that the core of the particle absorbs small molecules with an extremely high capacity, including fluorophores. This observation led to the consideration of ICP particles as a platform for Forster resonance energy transfer (FRET) - based on 3D molecular modeling, the pores / channels within the ICP core are able to hold different fluorophores in a “locked” conformation such that they remain within close proximity, yet held in an orientation that limits quenching seen in nonoriented fluorophores with close proximity. To test this theory, Fe-ICP particles were synthesized with a combination of two dyes that form FRET pairs together, Alexa Fluor 568 and Alexa Fluor 647, as well as Bodipy and Alexa Fluor 568. In both cases, the pair demonstrated strong FRET coupling and resulted in several fold greater fluorescence with donor excitation and receptor emission than the same fluorophores in solution. Example 21. ICP nanoparticles with metals other than iron [Gd-ICPs]

[0183] In another embodiment of the ICP core, the metal ions comprising the core are non-ferrous. The 3,4-hydroxypyridinone chelating motif has an extremely high affinity for other oxophilic metal cations such as Gd(lll) which is commonly used as a contrast agent in MRI imaging.

[0184] Briefly, 880 pL of basic D-BHP-N3 stock solution (2.28 mM) was mixed with 120 pL of gadolinium(lll)nitrate hexahydrate stock solution (10.8 mM) in a 1.5-mL polypropylene centrifuge tube. No immediate color change was observed, but the particles were shaken overnight at 37° C to encourage chelation of Gd(lll) by the ligand. The next day, the Gd-ICPs were recovered by ultrafiltration (Amicon Ultra-4 100 KDa MWCO) spinning for 3 min @ 3,000 ref x 3 spins. The Gd-ICPs were resuspended in 1 ml_ distilled water, affording a green-brown colloidal suspension of particles. Hydrodynamic diameter was measured by DLS (Figure 19). Over time, the Gd-ICPs exhibited a tendency to sediment in the container, unlike their Fe-ICP counterparts, presumably to the much larger size distribution of the as-synthesized Gd-ICPs.

[0185] Replacing some or all of the Fe(lll) atoms in the coordination polymer with Gd(lll) atoms would generate an incredibly powerful signal due to the presence of thousands of metal ions sequestered in each core. Gd(lll) MRI contrast agents in clinical use currently rely on mutidentate chelators which complex one (1) metal ion per ligand, such as in gadoteric acid (Dotarem®), creating a fundamental limitation on the amount of Gd(lll) that can be delivered per dose. ICP nanoparticles synthesized from Gd(lll) were observed to be larger than those made from Fe(lll) by DLS, suggesting that the choice of metal ion also strongly impacts the degree of polymerization and resultant size distribution of the ICP particles. Other metal ions that have been used to construct ICP nanoparticles include (but are not limited to) Cu(ll), Zn(ll), Ga(lll), Al(lll), In(lll), Cr(lll), Mn(lll), La(lll), Lu(lll), Zr(IV), and radioisotopes thereof (such as Cu64for positron emission tomography).

[0186] Example 22. Crosslinking of Fe-ICP-NOAs

[0187] The ability of the Fe-ICP-NOAs to be crosslinked by bis-cyclooctyne molecules was investigated. Due to the construction of the Fe-ICP particles via Fe(lll) complexes, they are capable of dissolution in strongly acidic environments (where H+ competes more strongly than Fe3+ for the metal binding sites) or in the presence of chelators of higher denticity, such as deferoxamine. DBCO-PEG5-DBCO (CAS No. 2363130-04-3, BroadPharm) and Sondheimer diyne (5,6,11,12-Tetradehydrodibenzo [a,e]cyclooctene) (Figure 21) were evaluated for their ability to covalently crosslink the azides present in the interior of the Fe-ICP particles to make them more resistant to chemical degradation. The ratio of each crosslinker to total azide content was varied, and the resultant particles analyzed by DLS to determine the impact of the crosslinker on the particle size distribution (and particularly, whether inter-particle crosslinks were occurring). At the highest levels tested, no inter-particle aggregation was observed.

[0188] Modest loadings of DBCO-PEG5-DBCO and Sondheimer diyne both narrowed the PDI of the ICP particles, suggesting successful crosslinking analogous to “cure shrinking” of the polymer network comprising the core. This effect was particularly pronounced when viewing the size distribution of the particles by scattering intensity (DLS), which reveals more information about the smaller fraction of larger particles in the sample, since the intensity of scattered light is proportional to the sixth power of the particle diameter.

[0189] Example 23. Dissolution of crosslinked Fe-ICP-NOAs and determination of crosslinking efficacy

[0190] To interrogate the crosslinked ICP construct, it was determined whether the covalently-crosslinked Fe-ICP-NOAs were more resistant to shedding their surface-bound oligos in response to a chemical challenge (such as deferoxamine, which chelates iron from the Fe-ICP core due to its higher denticity). 100 pL of each Fe-ICP-NOA solution (made with SEQ ID NO: 18) was treated with either Sondheimer diyne or DBCO-PEG5-DBCO (10, 20, 50 or 100 pM) (Figure 21) was mixed with 100 pL 1X PBS and an excess of deferoxamine mesylate (approximately 1 mg per sample). The samples were shaken at 37° C overnight, then passed through 50 KDa MWCO Amicon Ultra-0.5 spin filters and the filtrate absorbance at 260 nm measured for presence of free oligonucleotides. For both crosslinkers, the amount of DNA detected in the filtrate decreased at higher loadings (>20 pM) of crosslinker, indicating that the Fe-ICP core is indeed covalently crosslinked and more DNA is retained on the particle after removal of iron (Figure 22).

[0191]

[0192]

[0193] Table 3. Relative amount of free DNA shed from dissolved Fe-ICPs with increasing crosslinker concentration.

[0194] Example 24. Functionalization of Fe-ICP-N3nanoparticles to generate fluorescent Fe-ICP-NOAs

[0195] In another embodiment of the NOA, Fe-ICP-N3nanoparticles were treated with increasing amounts of BDP-FL-DBCO (CAS No. 2093197-94-3, Ambeed) alongside oligonucleotides to create fluorescent Fe-ICP-NOAs (from 5 pM to 20 pM BDP in a mixture with 100 pM total azide and 60 pM of other strained alkynes). The core is capable of absorbing large amounts of hydrophobic fluorophores and holding them in a conformation such that no quenching occurs. Unexpectedly, no BDP was observed in the filtrate upon purification by ultrafiltration, suggesting that all of it was absorbed by the Fe-ICPs, with the interior azides able to react with all of the available dye molecules.

[0196] To a 2.0-mL Eppendorf LoBind® centrifuge tube was added the following:

[0197] 1) 10 pL Fe-ICP-N3stock solution (1 mM in N3)

[0198] 2) 5 pL Triton X- 100 ( 1 % w / v)

[0199] 3) 5 pL 3’DBCO-BC22-Target DNA (SEQ ID NO: 4) (300 pM, Integrated DNA Technologies)

[0200] 4) 5 pL 3’BCN-GFP-Capture morpholino (300 pM, Gene Tools)

[0201] 5) 5, 7.5, 10, or 20 pL BDP-FL-DBCO (100 pM, Ambeed)

[0202] 6) Dilute to final volume of 0.1 mL in H2O

[0203] The mixture was shaken at 37 °C for 17h to allow the DBCO-bearing molecules to click to the surface and interior. Afterwards, the particles were purified by ultrafiltration using an Amicon Ultra-0.5 centrifugal filter with 50 kDa MWCO. The particles were resuspended in 0.2 mL H2O and the fluorescence of each was measured using a 96-well plate reader exciting at 475 nm. Notably, even at higher concentrations (50 or 100 pM), no BDP-FL-DBCO was observed in the filtrate after purification. Similar results were observed with Cyanine5.5-DBCO (Lumiprobe). All of the dye was bound by the Fe-ICPs, whereas some of the negatively-charged SulfoCyanine5.5-DBCO passed through the filter during purification by ultrafiltration.

[0204]

[0205] Table 4. Fluorescence of Fe-ICPs with increasing concentrations of BDP-FL-DBCO

[0206] Section E. “Universal” polythiol linkers for passivating noble / coinage metal nanoparticles

[0207] Nanotrace has identified several polythiol-containing compounds that provide superior stabilization of citrate-capped Au and AgNPs (and other noble metals) to harsh chemical treatments such as extremes of pH, ionic strength, and corrosive compounds such as alkali cyanides. Most of the existing noble metal nanoparticles described in the academic and patent literature rely on monothiols or disulfides to form a self-assembled monolayer (SAM) on the surface of the metal particle, displacing citrate in a predictable ligand-exchange reaction.

[0208] However, the low denticity of monothiols and disulfides results in SAMs that can be displaced by biomolecules such as glutathione (GSH) that are abundant in the human body. We sought to create a polythiol linker that (a) has a denticity of 3 or greater, (b) greatly improves the particle core’s resistance to harsh chemical environments and (c) allows for the biorthogonal copper-free click (SPAAC) conjugation of biomolecules, polymers and dyes in a “mix-and-match” fashion, such that any combination of molecules may be attached to the surface in one pot.

[0209] Nanotrace investigated the following compounds, which are mainly used as curing agents in epoxy formulations (in the case of pentaerythritol-based polythiols) or encapsulation agents (in the case of thiolated cyclodextrins). Their use as noble metal passivating agents is novel and unexpected, as the passivation only imparts colloidal stability in aqueous media when one or more of the thiols have been reacted with PEG of sufficient length: - Dipentaerythritol Hexakis(3-mercaptopropionate) (DiPETMP)

[0210] - Pentaerythritol tetra(3-mercaptopropionate) (PETMP)

[0211] - Pentaerythritol Tetrakis(mercaptoacetate) (PETMA)

[0212] - Hexakis-(6-Mercapto-6-deoxy)-a-Cyclodextrin

[0213] - Heptakis-(6-Mercapto-6-deoxy)-p-Cyclodextrin

[0214] DiPETMP was found to be the most effective at protecting Au and AgNP cores when reacted with two equivalents of bromoacetamido-PEG23-azide (CAS No. 2735663-83-7, BroadPharm). The thiolated cyclodextrins were likewise reacted with two equivalents of the bromoacetmido-PEG-azide. PETMP and PETMA were each reacted with one equivalent of PEG modifier. It was found that shorter PEGs (PEGu and PEG3, respectively) were insufficient to stabilize the particles and ligand exchange led to irreversible aggregation.

[0215] Example 25. Synthesis of “Universal Linker” Uni-2PEG23-N3(Scheme 11)

[0216] In one embodiment of the oligo(ethylene glycol) linker, a 1.5-mL polypropylene centrifuge tube was charged with 13 mg (.0164 mmol) of dipentaerythritol hexakis(3-mercaptopropionate) followed by 40 mg (.0328 mmol) of bromoacetamido-PEG23-N3. 1 mL of anhydrous DMSO was added followed by 10 pL of triethylamine as an acid scavenger (to neutralize generated HBr from the thiol SN2reaction with bromoacetamide). The mixture was shaken at 37 °C for 12 hours and then used as-is to directly functionalize Au / AgNPs. To prevent oxidation, the DMSO solution of Uni-2PEG23-Ns was stored frozen at 4°C. Solutions of Uni-2PEG23-Ns left exposed to ambient atmosphere gradually yellow over time, which is a visual indicator of disulfide (-S-S-) formation caused by oxidation. The polythiol will fully assemble on the surface of citrate-capped Au or Ag nanoparticles in 1 hour at room temperature, displacing the citrate in a ligandexchange reaction (Scheme 14) and affording azide-terminated particles for further elaboration (Figure 16). Longer reaction times are not detrimental.

[0217] Example 26. Application of “Universal Linker” Uni-2PEG23-N3 to AuNP core (Scheme 11)

[0218] A solution of citrate-capped 10 nm gold nanoparticles with a concentration of -10 nM was treated with progressively higher concentrations of Uni-2PEG23-Ns (from 25 to >300 pM), conferring greater colloidal stability with increasing polythiol concentration. Target-capture experiments suggested that higher loadings of the polythiol linker (>100 pM) generate a multilayered structure and make the conjugated oligonucleotides less accessible for hybridization.

[0219] 100 pM was chosen as the “standard” concentration for Uni-2PEG23-N3when added to a metal nanoparticle solution in the nanomolar concentration range. Conducting the SPAAC reaction of Uni-2PEG23-N3-AuNPs with BCN-morpholino oligos and DBCO-oligo(ethylene glycol) in 67% DMSO (instead of aqueous media) (Figure 15) resulted in NOAs that exhibited enhanced targetbinding capabilities. It is hypothesized that the high concentration of DMSO disrupts the coiling of oligo(ethylene glycol) chains promoted by hydrogen-bonding with water molecules, forcing the polymers into a “straightened” conformation that makes the terminal oligonucleotides more accessible for binding. Further analysis of strained alkyne-DNA by gel electrophoresis “clicked” to Uni-2PEG23-N3showed that species with 1 & 2 oligos were the major products, and small amounts of multiply-clicked oligos (3+) were also detected, indicating that Uni-2PEG23-N3has a distribution of species with multiple PEG23 modifications.

[0220] Example 27. Stabilization of copper nanoparticles by Uni-2PEG23-N3

[0221] In another embodiment of the nanomaterial core, colloidal CuNPs (Sirius Metals) were treated with Uni-2PEG23-N3analogously to the AuNPs example described above. CuNPs (including those composed of copper sulfide) are also attractive platforms for fluorescence enhancement of conjugated fluorophores, especially into the IR region. Therefore, we demonstrated that CuNPs could be functionalized with azides via for further elaboration. CuNPs treated with Uni-2PEG23-N3 exhibited a much broader size distribution compared to Au / AgNPs, presumably to their overall larger size distribution in the bare state (Figure 17).

[0222] Example 28. Other linkers based on polythiol compounds

[0223] In further embodiments of the oligo(ethylene glycol) linker, other linkers based on PETMP (Scheme 11), PETMA (Scheme 12), and cyclodextrin (Scheme 13) were synthesized in the same fashion, with the CD-linkers having 2 equivalents of PEG and the tetrathiol linkers having 1 equivalent of PEG. It was found that the CN‘ dissolution kinetics of AuNP-NOAs functionalized with Uni-2PEG23-N3(Scheme 14) exhibited the longest time to dissolve in excess sodium cyanide (multiple days) measured by the disappearance of the SPR absorbance band at 520 nm. Linkers made from tetrathiols and cyclodextrins showed a shorter dissolution time on the scale of hours, whereas traditional monothiol-protected AuNPs dissolved on the scale of minutes. Due to the stereochemistry of the cyclodextrins, all of the thiol groups are positioned on one face of the molecule, reducing the likelihood of multilayer formation.

[0224]

[0225] Scheme 12. Synthesis of universal linker PETMP-PEG23-N3

[0226]

[0227] Scheme 13. Synthesis of universal linker heptakis-SH-P-cyclodextrin-2PEG23-N3

[0228]

[0229] Scheme 14. Synthesis of polythiol-protected nanoparticles. Yellow represents a noble metal surface such as Au, Ag

[0230]

[0231] Table 5. Dissolution times of AuNP-NOAs in NaCN with different thiol surface treatments

[0232] Example 29. Functionalization of Uni-2PEG23-N3 nanoparticles to make NOAs for biodistribution studies

[0233] In another embodiment of the nanostructure, the nanomaterial consisting of Uni-2PEG23-N3-functionalized gold nanoparticles was chosen for initial biodistribution experiments. A mixture of morpholino oligonucleotides, oligo(ethylene glycol) (MW 5,000) and SulfoCy5.5-DBCO were chosen as the scaffolding components for optimal blood circulation and MS imaging.

[0234] To a 2.0-mL Lo-Bind centrifuge tube was added the following:

[0235] 1) 2119 pL AuNP-Uni-2PEG23-N3stock solution (106.2 nM)

[0236] 2) 250 pL Triton X-100 (1 % w / v)

[0237] 3) 150 pL DBCO-PEG5K (500 pM, Vector Labs) 4) 150 pL 3’BCN-GFP Capture Morpholino (SEQ ID NO: 8) (500 pM)

[0238] 5) 500 pL DBCO-SulfoCy5.5 (10 pM, Lumiprobe)

[0239] 6) Dilute to final volume of 25 mL in H₂O

[0240] The mixture was shaken at 37°C for 17 h to allow the DBCO & BCN-bearing molecules to click to the surface. Afterwards, the particles were purified by ultrafiltration using an Amicon Ultra-15 centrifugal filter with 50 kDa MWCO. The buffer was exchanged to 0.005% Tween-20 in PBS (w / v) and the particles were stored until ready for use. Notably, the fluorescence of the AuNP-NOAs produced by this method far exceeds that of the equivalent amount of free SulfoCy5.5 in solution. An average enhancement of 73-fold was found, compared to the Fe-ICP particles which demonstrated little enhancement, since they do not possess a surface plasmon resonance (SPR) band that can interact with the dyes conjugated to the surface.

[0241]

[0242] Table 6. Relative enhancement of SulfoCy5.5 fluorescence by AuNP-NOAs and Fe-ICP NOAs compared to free dye

[0243] For sterilization, the concentrated solution of functionalized AuNP-NOAs may be passed through hydrophilic PVDF syringe filters (0.10 or 0.22 pm) with minimal loss. Hydrophilic PTFE also yields acceptable results, while PES, cellulose acetate, and mixed cellulose esters (cellulose acetate / nitrocellulose) tended to result in unpredictable filter loss (20-90%).

[0244] Example 30. Enhancement of surface-bound fluorescent dyes by Uni-2PEG23-N3-NOAs

[0245] In another embodiment of the nanostructure, the nanomaterial core of was reacted with the same components of Example 29, except several Uni-2PEG23-N3NOAs with different oligonucleotide sequences were prepared, with higher loadings of SulfoCy5.5:

[0246] To a 2.0-mL Eppendorf LoBind® centrifuge tube was added the following: 1) 150 pL AuNP-Uni-2PEG23-N3 stock solution (30 nM)

[0247] 2) 5 pL Triton X-100 (1 % w / v)

[0248] 3) 5 or 10 pL DBCO-PEG5K (500 pM, Vector Labs)

[0249] 4) 5 pL 3’BCN-Morpholino (SEQ NO: 8, 9, or 10) (500 pM)

[0250] 5) 15 pL DBCO-SulfoCy5.5 (100 pM, Lumiprobe)

[0251] 6) Dilute to final volume of 0.5 mL in H2O

[0252] The mixtures were shaken at 37°C for 17 h to allow the DBCO & BCN-bearing molecules to click to the surface. Afterwards, the particles were purified by ultrafiltration using an Amicon Ultra-0.5 centrifugal filter with 50 kDa MWCO and resuspended in 0.5 mL distilled H2O, giving a total of 6 distinct particle compositions (3 sequences either with 1:1 PEG:morpholino or 2:1 PEG:morpholino). A standard curve of SulfoCy5.5-DBCO was made and the fluorescence of the purified NOAs was measured to determine the number of fluorophores per nanoparticle. The degree of enhancement was calculated by dividing the apparent concentration of SulfoCy5.5 in the purified NOA samples by its concentration in the reaction mixture (3 pM or 333 dyes / NP). The true degree of enhancement is likely much higher since it is unlikely that >300 SulfoCy5.5 molecules are physically present on each nanoparticle, a more realistic number (-100 dyes / NP) suggests an average enhancement factor of nearly 100x.

[0253]

[0254] Table 7. Relative enhancement of SulfoCy5.5 fluorescence by AuNP-Uni-2PEG23-N3-NOAs Section F. Alternative “universal” linker schemes for noble metal nanoparticles

[0255] Nanotrace has identified several other surface-modification strategies for Au / AgNPs that represent a significant improvement over the original “salt-aging” method used to functionalize nanoparticles with oligonucleotides. [v] Other polymeric linkers besides poly(ethylene glycol) have been used to successfully construct NOA particles with high loadings of oligonucleotides and characteristic in-vitro behavior (such as high cellular uptake). Primarily, poly(acrylic acid), poly(vinylpyrrolidone) and zwitterionic polymers such as poly(carboxybetaine acrylamide) have been employed to produce colloidally-stable NOA constructs, each with their own unique properties for specific applications.

[0256] Example 31. Synthesis of polyfacrylic acid) / poly(ethylene qlycol)-azide capped silver “intermediate” nanoparticles

[0257] In one embodiment, silver nanoparticles are employed as an attractive core for fluorescent imaging due to dramatic surface plasmon resonance-enhanced emission of nearby fluorophores. Polycarboxylic acids such as citric acid are commonly used to stabilize silver nanoparticles in aqueous solution. Poly(acrylic acid) [PAA] was selected as a potential stabilizer for AgNPs. Bifunctional PAA terminated with a-mercapto and co-bromo groups was selected for this purpose (Mw = 2,600 g / mol, Polymer Source Inc). Unexpectedly, it was found that treating citrate-capped AgNPs with this polymer resulted in unstable aggregates, presumably due to competition between the thiol groups and the carboxylic acid groups of PAA leading to unpredictable surface organization. Addition of a small amount (1:9 molar equivalents) of PEG modifier (lipoamido-PEG-azide, MW 2,000, Nanosoft Polymers) led to dramatically improved stability, likely due to interstitial hydrogen-bonding with the carboxylic acid groups of PAA enforcing a ‘standing up’ conformation of the scaffolding. The terminal co-bromo groups were substituted in-situ with NaN3(10 mM) to yield colloidally stable, azide-terminated AgNPs with an extremely high charge density and tolerance to high ionic strength, extremes of pH, and freezing / thawing (Scheme 15).

[0258]

[0259] Scheme 15. Synthesis of AgNP-PAA-N3intermediate particle showing termini of surface-bound polymers (right)

[0260] To a 15-mL polypropylene centrifuge tube was added 10 mL of 20 nm citrate-capped silver nanoparticle solution (.76 nM, CytoDiagnostics) followed by 900 pL of thiolated, bromoterminated poly(acrylic acid) (1mM in H2O), 100 pL of lipoamido-PEG2000-azide (Nanosoft Polymers), and 100 pL of 500mM sodium azide in H2O. The mixture was shaken overnight at room temperature, and the particles were purified by ultrafiltration (Amicon Ultra-1550 KDa MWCO) followed by repeated washing with H2O. The particles were resuspended in a final volume of 1 mL H2O and the concentration of the particle stock was measured by UV-Vis taking the absorbance at 400 nm.

[0261] Example 32. Synthesis of polyfacrylic acid) capped silver nanoparticles with FITC in one pot

[0262] In another embodiment, the synthesis of fluorescent PAA / PEG-AgNP NOAs can be simplified by using a PEG modifier already functionalized with a fluorescent dye. For this purpose, LA-PEG-FITC (MW 2000, Nanosoft Polymers) was employed. To a 15-mL polypropylene centrifuge tube was added 2 mL of 20 nm citrate-capped silver nanoparticle solution (.76 nM, CytoDiagnostics) followed by 90 pL of thiolated, bromo-terminated poly(acrylic acid) (1mM in H2O), 10 pL of lipoamido-PEG2000-FITC (Nanosoft Polymers), 20 pL of 500mM sodium azide in H2O, and 10 pL of 1X PBS. The mixture was shaken overnight at room temperature, and the particles were purified by ultrafiltration (Amicon Ultra-1550 KDa MWCO) with repeated washing with H2O. The particles were resuspended in a final volume of 1 mL H2O and the concentration was measured by UV-Vis taking the absorbance at 400 nm. These intermediate particles already demonstrate enhanced fluorescence without the need for a cyclooctyne dye to be conjugated in a separate reaction. The PAA-N3-PEG-FITC intermediate AgNPs were then functionalized with 3’DBCO-BC22-Target DNA (SEQ ID NO: 4) in the presence of 0.3M NaCI and again purified by ultrafiltration before in vitro experiments were performed.

[0263] Triple-negative breast cancer cells (MDA-231) were treated with 100 pM of the PAA-PEG-FITC-DNA-AgNP-NOAs and imaged on a confocal microscope over several hours, showing cellular uptake via endocytosis as expected for a NOA particle with a dense DNA scaffolding (Figure 14), affording extremely high-resolution imaging of the endosomal compartments over long time-scales due to the low laser power required. Other dyes found to exhibit significant fluorescence enhancement by AgNP-NOAs include Alexa Fluor 647® and Cyanine 5 / Cyanine 5.5.

[0264] Example 33. Synthesis of zwitterionic polyfcarboxybetaine acrylamide)-thiol-modified AuNPs

[0265] In one embodiment of the NOA, another class of oligomer chosen are zwitterionic oligomers: their global net neutral charge makes them an attractive choice for in vivo therapeutic use while the high local density of ion pairs confers colloidal stability and repulsion of serum proteins. An oligomer consisting of poly(acrylamide) with pendant carboxy betaine groups (CBZ) was selected as a candidate for NOA construction (Polymer Source, Inc). The presence of a-amino and cu-trithiocarbonate groups allowed for further elaboration and reaction with AuNPs. Initially, reaction of the oligomer “as-received” with citrate-capped AuNPs led to irreversible aggregation. It was concluded that the trithiocarbonate group did not sufficiently interact with the AuNP surface to create a well-ordered monolayer of CBZ oligomers.

[0266] The trithiocarbonate ester was cleaved using NaNs, adapting a procedure from the literature (Scheme 16). [iv] Briefly, a 1.81 mM solution of the CBZ-trithiocarbonate oligomer (Mw= 5,500 g / mol, Dp = 18) in H2O was prepared. Then, 0.55 mL of CBZ-TTC was mixed with 0.1 mL of 500 mM sodium azide and 0.35 mL 1X PBS. The mixture was shaken overnight at 37° C, then used directly in the next step. The thiol-terminated CBZ-SH solution (20 pL, 1mM) was mixed with 1 mL of 10 nm AuNPs (9.1 nM, CytoDiagnostics). Unexpectedly, the color of the nanoparticles immediately red-shifted, indicative of aggregation. Before the nanoparticles were discarded, the color blue-shifted back to its original state after approximately 30 minutes of shaking at room temperature. It was presumed that the zwitterionic groups are the first to interact with the AuNP surface and induce aggregation, but the Au-thiol bond is more thermodynamically stable and the particles revert back to their non-aggregated state when the oligomer chains form a monolayer via the co-terminal thiol (scheme 17). The AuNPs were shaken overnight at 37° C in the presence of CBZ-SH, then purified using an Amicon Ultra-4 centrifugal filter device with 100 KDa MWCO. The recovered CBZ-SH-AuNPs showed no sign of red-shifting or aggregation and were extremely colloidally stable, even on extended storage under ambient conditions (> 6 months).

[0267]

[0268] Scheme 16. Synthesis of CBZ-SH polymer from trithiocarbonate precursor Example 34. Azide-functionalization of zwitterionic polyfcarboxybetaine acrylamide)-thiol-modified AuNPs

[0269] In further elaboration of the nanomaterial core, the a-amino groups of the zwitterionic oligomer were modified by incubating the particles in a basic buffer (0.5M HEPES, pH 9.0) and introducing an excess of (100-1000 equivalents) of azide-NHS ester modifier (Azido-PEG4-NHS, TCI America) or similar azide-modifying reagent (Azide-PEG3-Tos, BroadPharm), followed by recovery by ultrafiltration.

[0270] Example 35. Oligonucleotide functionalization of zwitterionic polvfcarboxybetaine acrylamide)-thiol-modified AuNPs & loading characterization

[0271] In another embodiment of the nanostructure, the CBZ-N3particles were conjugated with DNA and morpholino oligonucleotides to afford CBZ-NOAs (Scheme 17). Briefly, to a 1.5-mL polypropylene centrifuge tube was added 120 pL CBZ-N3nanoparticle stock (~30 nM), 5 pL 3’BCN-GFP-Capture morpholino oligo stock (SEQ ID NO: 8) (300 pM), 2.8 pL 3’DBCO-GFP-Capture DNA stock (SEQ ID NO: 3) (532 pM), 5 pL of Triton X-100 (1% w / v), and 400 pL of 1X PBS. A duplicate mixture was made, but with added NaCI to a total of 350 mM. The mixture was shaken overnight at 37° C, then recovered by ultrafiltration (Amicon Ultra-4, 100 KDa MWCO). Recovery of the particles was nearly 100% with none sticking to the sidewalls of the polypropylene container(s) or the cellulose acetate filter material. In order to quantify the number of oligos per CBZ-NOA, the particles were dissolved with excess sodium cyanide and the solution passed through a NAP-5 column (Cytiva) equilibrated in 1X PBS and the DNA concentration was measured by UV-Vis. The CBZ-NOAs made in PBS (-150 mM total salt) had an average of 33 oligos per particle, whereas the CBZ-NOAs made in 350 mM NaCI had an average of 70 oligos per particle. Their dense packing of zwitterionic species affords low background and makes them attractive scaffolds for in-situ hybridization, immunohistochemistry, and other fluorescence-based techniques.

[0272]

[0273] Scheme 17. Synthesis of azide-terminated CBZ-AuNPs and functionalization with cyclooctyne-DNA. (n=18)

[0274] Example 36. Unexpected in vitro cytotoxicty of CBZ-NOAs

[0275] In an application of the nanostructure made from zwitterionic oligomers, cells treated with CBZ-NOAs experienced dramatic cell death in under 24 hours. This result prompted further investigation of zwitterionic cargo as a potential cytotoxic agent for therapeutic NOAs.

[0276] Example 37. Synthesis of sulfobetaine zwitterion-terminated DNA strands (Scheme 20

[0277] In another embodiment of the nanomaterial core, NOAs were synthesized bearing oligonucleotides “capped” with either a terminal amine or sulfobetaine moiety, presenting a positively-charged or zwitterionic surface to the surrounding chemical environment, respectively. It was found that NOAs functionalized with these 5’ terminal modifications exhibited superior target-capture efficiency as well as cellular internalization (Figure 4). DNA was synthesized with a 3’ lipoamide and 5’ C6-amino modifier (5'-DMS(O)MT-Amino-Modifier C6, Glen Research) which was deprotected on the solid supports using 3% trichloroacetic acid in dichloromethane, followed by incubation with 1,4-butanesultone (10% in MeCN) for 17 hours (SEQ ID NO: 14). The optimal functionalization percentage is between 10 and 100% of amino groups converted to zwitterionic groups. Related compounds such as 1,3-propanesultone may also be used to install zwitterionic groups on amino-modified oligonucleotides.

[0278]

[0279] Scheme 18. Synthesis of 5’ zwitterion (sulfobutylbetaine) terminated oligonucleotide strands.

[0280] Example 38. Uni-2PEG23-N3-morpholino NOAs for gene knockdown

[0281] In another embodiment of the nanostructure, a nanomaterial core consisting of Uni-2PEG23-N3-functionalized gold nanoparticles was chosen for / ri vivo knockdown experiments. Morpholino oligonucleotides and Alexa Fluor 647® were chosen as the scaffolding components for optimal knockdown efficiency and fluorescence imaging.

[0282] To a 2.0-mL Lo-Bind centrifuge tube was added the following:

[0283] 1) 0.5 mL AuNP-Uni-2PEG23-N3stock solution (~10 nM)

[0284] 2) 5 pL 3’BCN-Survivin Capture Morpholino (SEQ ID NO: 15) (300 pM)

[0285] 3) 5 pL DBCO-Alexa Fluor® 647 (100 pM, Lumiprobe)

[0286] The mixture was shaken at 37°C for 17 h to allow the strained alkyne-bearing molecules to click to the surface. Afterwards, the particles were purified by ultrafiltration using an Amicon Ultra-15 centrifugal filter with 50 kDa MWCO. The particles were stored at 4° C protected from light until ready for use.

[0287] The resultant nanoparticles were added to a solution of MDA “triple negative” breast cancer cells at a concentration of 5 nM, 500 pM, and 50 pM NOAs targeting survivin mRNA and incubated in DM EM containing 10% fetal bovine serum and 1% penicillin / streptomycin for 3 days. In parallel, cells were also treated with a standard transfection protocol from Gene Tools, Endo-Porter. After 3 days of incubation, cells incubated with Endo-Porter and 10,000 nM morpholino clearly exhibited a rounded apoptotic phenotype. Likewise, cells treated with 5 nM NOAs showed a similarly dramatic apoptotic phenotype, while untreated cells remained healthy and proliferative. Visualization of the SulfoCy5.5-labeled probes was performed using an Evos M7000 epifluorescent microscope, which revealed perinuclear localization of the nanoparticles, a well-documented and characteristic pattern after endocytosis of nucleotide-functionalized nanoparticles.

[0288] Section G. NOAs for biomolecule detection

[0289] Nanotrace has identified an unmet need for fluorophores with higher fluorescence and signal-to-noise ratio for staining experiments. For instance, most commercially-available antibodies used for this purpose typically have 1-2 fluorophores conjugated to the protein, leading to weak signal when detected by microscopy or flow cytometry. Nanotrace has exploited the nanoparticle core and oligomeric scaffold to create a nanoconjugate capable of addressing the sources of decreased signal to noise in molecular biological assays.

[0290] In this embodiment, the architecture is such that the fluorophores are placed internally, sterically and / or electrostatically protected by the much larger oligomers of the scaffold (Scheme 19). Likewise, in embodiments of this architecture which utilize a core, the core material and fluorophore distance from that core can be controlled via the oligonucleotide scaffolding to place the fluorophore at a distance which results in fluorescence boosting plasmonic coupling, known as metal enhanced fluorescence (MEF). This architecture was deployed in the live-cell experiment shown in Figure 1, with the fluorophores buried beneath the oligonucleotide strands affording intense fluorescence with low background. The successful endocytosis and subsequent intracellular imaging of these nanoparticles required specific placement of the fluorophore such that 1) the external portion of the nanoconstruct presented a densely arranged scaffold of oligonucleotides to the external environment, in order to maintain the ability of the nanoparticles to be endocytosed into live cells, and 2) holding the fluorophore a specific distance from the silver core, which was essential to maintain with nm-scale precision in order to achieve MEF.

[0291]

[0292] Scheme 19. Mechanism of fluorescent dye shielding by intermediate azide-scaffold NOA functionalization strategy.

[0293] Example 39: Metal Enhanced Fluorescence

[0294] The enhancement of NOA-scaffolded fluorophores is readily observed by quantifying the fluorescence of a nanoparticle solution throughout the fluorophore scaffolding process (Figure 6, right). An aqueous solution with the DNA sequence 3’Thiol T10-BC22 Capture (SEQ ID NO: 16) in water provided a dynamic fluorescence response upon the addition of 20nm gold nanoparticles. In this embodiment, the fluorescence level of the solution drops immediately after nanoparticle addition, as the DNA / fluorophore strands nonspecifically adsorb to the nanoparticle surface. However, over the course of two hours the strands orient with the thiol attaching to the gold surface and the negative backbones of DNA repelling one another, causing each conjugated strand to form a radial orientation, lifting the attached fluorophore away from the nanoparticle surface. The result is a dramatic and measurable increase in fluorescence after two hours, which is boosted to levels beyond that of a solution with the same number of fluorophores present but no nanoparticles. Using this scaffolding approach, fluorophores can be paired with core materials and sizes that optimally couple with the chosen fluorophore for MEF, and then placed with precision at a distance which significantly enhances the fluorescence response while maintaining low background due to the internal nature of the scaffolded fluorophore. We observed over 63,000x higher fluorescence compared to the same fluorophores in solution with optimally paired fluorophore-core combinations and scaffolds (Figure 6, left). This precision can also be used for non-fluorescent detection molecules, such as gadolinium (MRI), radioisotopes (PET, metal ions (ICP-MS), etc. The MEF of NOA constructs can also be used to enhance advanced imaging modalities such as stochastic optical reconstruction microscopy (STORM) microscopy, stimulated emission depleted (STED) microscopy, and fluorescence lifetime imaging microscopy (FLIM). As an example, NOAs were imaged using STORM in a biological sample: Fixed U2OS cells were treated with 1nM NOA constructs consisting of a silver core and AF647-DBCO conjugated to the surface using a thiol-polyacrylic acid-azide linker, co-adsorbed to the nanoparticle surface with stabilizing thiol-polyethylene glycol. NOAs bound onto biological surfaces were then imaged on a Zeiss Elyra 7 using 639nm excitation (Figure 11). Notably, typical imaging of AF647 dyes with this technique require exposure times in the range of 20-50ms in order to capture enough photons from blinking fluorophores. However, the greatly enhanced fluorescence of the NOAs were observable with an incredibly short 1ms exposure time, and with the acquisition of 10,000 frames was able to reconstruct a single molecule localization image with resolution of approximately 20nm. This represents a several-fold improvement in acquisition time of a powerful imaging technique due to the enhancement of NOA-bound fluorophores, with minimal sacrifice of resolution due to the increased size of the NOA construct.

[0295] Example 40: Metal Enhanced FRET and FLIM, and Multicolor Effects

[0296] The precise placement of fluorophores in proximity of a metal NOA core also enables other fluorescence techniques which are ordinarily difficult or impossible to achieve with non-scaffolded fluorophores. In one embodiment, the polyvalency of the oligomer scaffold is exploited to place multiple colors of fluorophores on a single NOA probe (Figure 7). As an example, a 20nm gold nanoparticle was functionalized with the DNA sequence 3’Thiol T10-BC22 Capture (SEQ ID NO: 16) by incubating the nanoparticles with 500 strands per particle, combined with 500 strands of LA-PEG2K-FITC in.01% (v / v) Tween-20 and.01% sodium dodecyl sulfate (w / v). The solution was mixed at 50°C for two hours, after which sodium chloride was added to the solution gradually overnight at room temperature to a maximum of 300 mM NaCI. Particles were then purified by centrifugation at 21,000 x ref for 20 min at 4° C and removing the supernatant. This purification step was performed 3x, after which 10 flares of the sequence 5’Cy3-BC22-Flare (SEQ ID NO: 17) were added per particle. The solution was heated to 70°C for 10 minutes and cooled overnight to room temperature. Centrifugation purification was again performed to remove unbound flares.

[0297] Solutions of the resulting particles were analyzed using a Zeiss LSM 980 confocal microscope with a spectral detector. Using a 514nm excitation wavelength, the emission spectrum of the particles was observed and compared to LA-PEG2K-FITC in solution and reference spectra of all three fluorophores. While the LA-PEG2K-FITC matched well with documented emission of fluorescein isothiocyanate, the metal core of the NOA, which has a plasmon resonance maximum of 520nm, generated a redshift of the FITC spectrum of approximately 20nm. Likewise, Cy3 demonstrated a measurable, though lesser redshift. Importantly, the excitation of this nanoconjugate with one laser resulted in three distinct fluorescence peaks. This is partly a result of the broad excitation wavelengths of FITC and Cy3, and partially due to FRET between Cy3 and Cy5, which through hybridization of the Cy3 carrying flare to the Cy5-carrying capture strand, are placed between 7-9nm away from one another. Importantly, if FRET efficiency is a desirable quality, then this method of scaffolding permits localization of one or both fluorophores to be closer to one another and / or further from the core. As such, the NOA construct was made into both a spectral-shifted as well as multicolor FRET fluorescent probe. This concept can be extended to other colors available for fluorescence detection. With just standard blue, green, yellow, red, and far-red channels, the theoretical number of combinations extends to 31 spectrally-distinguishable NOA constructs (Figure 8), a number which increases with advanced fluorophores that extend into the NIR / IR spectrum or further into the UV.

[0298] Example 41: Detectable NOAs in molecular biology assays

[0299] Further examples of the utility of these constructs can be readily observed in fixed cell assays. Typical molecular biological assays such as immunocytochemistry (ICC), immunohistochemistry (IHC), and fluorescence in-situ hybridization (FISH) rely on the specific recognition by a molecule such as antibody or DNA oligonucleotide conjugated to a fluorophore. However, this exposes said fluorophores to the external chemical environment, providing opportunities for the exposed fluorophore to nonspecifically bind to non-targeted structures or molecules. However, the NOA scaffold allows the fluorophore to be buried in a shielded position and reduce interactions with external molecules. This capability is illustrated in figure 27: when fixed MDA-MB 231 cells are treated with 10 nM of the fluorophore SulfoCy5.5 for 30 minutes, the fluorophore rapidly attaches to the mitochondrial membrane, despite having no targeting moiety. This is likewise true for the oligomer LA-PEG5K-Sulfocy5.5, which does not have a well-defined internal and external portion and therefore retains the mitochondrial labeling. Mitochondrial labeling is also observable for gold nanoparticles functionalized with the same LA-PEG5K-Sulfocy5.5, as the 3’ end of the oligomer is chemisorbed to the gold surface, leaving numerous fluorophores exposed to the environment for binding. Critically, when a SulfoCy5.5-conjugated flare is hybridized to a NOA consisting of neutral morpholino capture strands which sterically shield the bound flares’ fluorophores, mitochondrial localization is no longer observed. This feature can be exploited in functional biological assays, as illustrated in figure 10: Typical immunocytochemistry relies on the treatment of fixed cells with a primary antibody to bind to a targeted molecule (in this example, p-actin), and a subsequent secondary antibody to bind to the primary (Left). After such processes, the proximity of the secondary antibody can be directly imaged with fluorescence microscopy, and the location of the primary antibody (and targeted molecule) inferred to be colocalized. However, fluorophore-labeled antibodies suffer due to their necessarily exposed fluorophores, and are also limited in utility to specific animal species. As such, an alternative detection method utilizes NOAs (figure 10, right). In such an assay, the primary or secondary antibody can be functionalized with a short oligonucleotide strand, which may be a single sequence or a component of several sequences comprising a “barcode.” Then, after treatment with the oligonucleotide-functionalized antibody, a NOA with complementary sequence can be introduced to bind onto the oligonucleotide-functionalized antibody. As such, specific staining can be observed while mitigating non-specific binding, and the NOA has the further advantage that it is not limited in scope to particular animal species or tissues.

[0300] In the case of fluorescent flare-bearing NOAs, fluorophores are appended on the internal portion of the attached oligomer scaffold. The radial arrangement of the oligomer strands ensures that the fluorophores are protected from the external environment by the steric bulk of the densely packed oligomer strands and (in the case of charged oligonucleotides / oligomers) the accompanying cloud of salt ions. This prevents non-specific interactions of the fluorophores with external biomolecules, improving the specificity of target detection (scheme 20).

[0301]

[0302] hybridization

[0303] Scheme 20. Hybridization of complementary flares: the cargo is held “internally,” shielded by the scaffolding

[0304] Section H. Miscellaneous Example 34. Small-molecule backfill for improved stability

[0305] Nanotrace has identified numerous small molecules with high affinity for noble / coinage metal surfaces that are capable of acting as a “backfill” to cover any areas of the NP surface left 'bare’ or otherwise unprotected by the surface passivation treatments previously claimed. This renders the core less susceptible to degradation. These include:

[0306] Bismuthiol

[0307] Trithiocyanuric acid

[0308] Bis-DMTD (Vanlube 829, CAS No. 72676-55-2)

[0309] Thiosalicylic acid

[0310] Fluorescent Labeling and Fluorophore Shielding

[0311] In the “intermediate particle” examples demonstrated in this patent, wherein a bare nanoparticle core is functionalized with terminal azido groups for further elaboration, the architecture is such that co-functionalization of the particle with a combination of oligonucleotides, polymers, and fluorophores creates a “tiered” architecture where the fluorophores are sterically protected by the much larger oligonucleotides and polymers (Scheme 19). This architecture was deployed in the live-cell experiment shown in Figure 1, with the fluorophores buried beneath the oligonucleotide strands affording intense fluorescence with low background.

[0312] In the case of fluorescent flare-bearing NOAs, fluorophores are appended on the internal portion of the attached oligonucleotide / oligomer strands. The radial arrangement of the oligomer strands ensures that the fluorophores are protected from the external environment by the steric bulk of the densely packed oligonucleotide strands and (in the case of charged oligonucleotides / polymers) the accompanying ionic cloud of salt. This prevents non-specific interactions of the fluorophores with external biomolecules, improving the specificity of target detection (scheme 20).

[0313] Salt Ion Shielding Mechanism

[0314] The close packing of the oligomer strands is facilitated by the presence of salt ions, which create a charge-shielding effect. These ions neutralize the negative charges present on the backbone phosphate groups (in the case of DNA and other charged oligonucleotides), reducing electrostatic repulsion between neighboring strands (Scheme 21). This tight packing is essential for maintaining the integrity of the steric hindrance that protects the fluorophores, enables cooperative binding from the multiple strands, and also forces the oligomers into a conformation with fewer degrees of freedom for movement relative to oligomers that are not templated on such a surface. This forced conformation reduces the entropic barrier of the templated strands to bind to complementary target sequences, thereby creating stronger bonds between capture and target strands.

[0315]

[0316] Scheme 21. Increasing surface packing of charged oligos with increasing ionic strength.

[0317] In the case of neutral oligonucleotide-based NOAs (such as those with morpholino oligonucleotides), aprotic organic solvents (such as DMSO) disrupt coiling of co-adsorbed PEG molecules as well as non-specific hydrogen-bonding of the morpholino nucleobases, creating a “shielding” effect analogous to salt ions in water and leading to improved target-capture ability.

[0318] A therapeutic construct described herein comprises a plurality of oligomer strands having an oligomeric structure, wherein the oligomer strands arrange to create a three-dimensional nanostructure with a shielded internal portion and an exposed external portion, and wherein the plurality of oligomer strands contains at least one nucleic acid analogue species with enhanced nuclease resistance; and one or more therapeutic agents nonrandomly positioned within the shielded internal portion of the structure; wherein the combined arrangement of the oligomer strands sterically and / or electrostatically maintains the therapeutic agent in a pharmacologically inactive or reduced-activity state by inhibiting interaction with a non-target microenvironment, and wherein the construct exhibits enhanced stability against nuclease degradation compared to a construct utilizing only DNA or RNA oligomer strands.

[0319] A target-activated therapeutic construct described herein comprises a plurality of oligomer strands having an oligomeric structure, wherein the oligomer strands arrange to create a three-dimensional structure with a shielded internal portion and an exposed external portion; one or more therapeutic agents nonrandomly positioned within the shielded internal portion of the structure; and a recognition moiety incorporated into at least one of the oligomer strands, wherein said recognition moiety specifically binds to a targeted biomolecule, wherein the arrangement of the oligomer strands sterically and / or electrostatically maintains the therapeutic agent in a pharmacologically inactive or reduced-activity state by inhibiting interaction with a non-target microenvironment; and wherein said binding of the recognition moiety to the targeted biomolecule induces a conformational change in the oligomer structure that exposes or releases the one or more therapeutic agents from the internal portion, thereby restoring or increasing their pharmacological activity. Optionally, the recognition moiety is a therapeutic oligonucleotide (e.g., antisense, siRNA, or guide RNA, including nucleotide analogues) that specifically binds to a targeted RNA or DNA strand, thereby initiating a first therapeutic effect (e g., gene regulation or target degradation); and one or more therapeutic agents nonrandomly positioned within the shielded internal portion is a small molecule drug (e.g., a chemotherapeutic agent), the release of which initiates a second therapeutic effect, thereby providing a synergistic combination therapy. The therapeutic agent is an oligonucleotide, which upon target recognition is displaced or otherwise activated in accordance with its structure, sequence, and / or composition. The plurality of oligomer strands may further comprise a non-nucleic acid component selected from the group consisting of oligomeric and polymeric ethylene glycol, oligomeric and polymeric saccharides, poly(vinylpyrrolidone), poly(acrylic acid), polyanions, polycations, polyzwitterions, or peptides. The non-nucleic acid component may be used to generate an “intermediate” particle which is then functionalized with nucleic acids or may be added alongside the nucleic acid. The internal portion of the oligomeric structure is oriented around a nanoparticle core, the nanoparticle core being comprised of one or more metals, silica, polymeric material, lipid, protein, polypeptide, dendrimer, or combination thereof. Alternatively, the internal portion of the oligomeric structure is oriented by a chemical coordination of one end of each oligomer. The core portion is capable of further binding or association with added molecules which are not part of the oligomeric shell, and wherein the added molecules are bound with non-random coordination or orientation. This coordination / orientation may impart chemical, biological, photonic, magnetic, or other effects which may not be observed without said coordination. The oligomeric shell may be comprised of one or more oligomeric entities that contribute to the biological, chemical, photonic, magnetic, or other interaction with the surrounding environment. The external region of the oligomeric structure may contain moieties which influence the biodistribution within a body and / or localization within a cell and / or organelle. The internal region of the oligomeric structure contains moieties which, upon recognition of a specific target molecule or condition, are released and / or exposed and subsequently influence the biodistribution within a body and / or localization within a cell and / or organelle.

[0320] An intermediate nanostructure as described herein includes may be colloidally stable in aqueous buffers, does not degrade after repeated freezing / thawing cycles, and is capable of further elaboration with cyclooctyne-bearing nucleic acid oligomers, therapeutic agents, fluorescent dyes, and other molecules with biological activity via copper-free click reaction, involving simple mixing of components without any other ancillary reagents. The nanostructure is comprised of a metal nanoparticle core functionalized with a mixture of azide-terminated poly(acrylic acid) and polyethylene glycol) oligomers each bearing thiol or disulfide moieties for anchoring to the metal surface. The intermediate nanostructure may be comprised of a metal nanoparticle core functionalized with a polythiol-terminated poly(ethylene glycol)-azide linker, where the polythiol portion possesses (3) or more free thiol groups for anchoring to the metal surface. Further, the intermediate nanostructure is comprised of a metal nanoparticle core functionalized with a zwitterionic polymer bearing a thiol or disulfide group at one terminus and an azido group at the opposite terminus.

[0321] The anticipated methods of organelle-targeted gene modification within the cell of an individual includes, administering a therapeutic construct of the present invention to the individual; and concurrently or sequentially delivering an effector protein or mRNA encoding an effector (e.g., a Cas effector construct or base editor construct) that is capable of acting synergistically with the oligonucleotide from the therapeutic construct within a target organelle (e.g., the nucleus or mitochondria) to affect sequence-specific biological function, such as genome modification. The method further comprises a monitoring guide release by detecting change of guide oligonucleotide fluorescence or by biochemical isolation of mitochondria and quantification of guide oligonucleotide by RT-qPCR. The methods of manufacturing a three-dimensional therapeutic construct described herein include providing a plurality of oligomer strands; providing at least one therapeutic agent; and assembling the oligomer strands and at least one therapeutic agent to form a three-dimensional structure, wherein said assembling positions the at least one therapeutic agent internally and nonrandomly within said structure, such that said therapeutic agent is sterically and / or electrostatically shielded by an external portion formed by the oligomer strands, wherein the binding of the therapeutic to a target induces a conformational change and results in the release or exposure of at least one therapeutic agent.

[0322] The methods of synthesizing an activated ester linker described herein include reaction of a halomethylated benzoic acid derivative with a metal azide to form an azidomethyl benzoic acid intermediate, and coupling the intermediate carboxylic acid with / V-hydroxysuccinimide in a reaction sequence that does not require inert atmosphere or chromatographic purification (Scheme 1). Other / V-hydroxyimides or similar activated esters may be substituted for / V-hydroxysuccinimide, including but not limited to A / -hydroxy-5-norbornene-2,3-dicarboximide (HONB), / V-hydroxynaphthalimide, A / -hydroxyphthalimide, / \ / -Hydroxy-A / -phenylbenzamide, / V-hydroxyglutarimide, para-nitrophenol, pentafluorophenol, and tetrafluorophenol.

[0323] The methods of preparing a therapeutic conjugate described herein include conjugating a therapeutic agent to an oligonucleotide via a copper-free click-chemistry reaction between a cyclooctyne-modified oligonucleotide and the therapeutic agent modified with the activated ester linker described herein. Other therapeutic agents can likewise be converted into an azidoderivative using the linker described herein, including but not limited to the following (including prodrugs of said agents): Monomethyl auristatin E (MMAE), 7-ethyl-10-hydroxycamptothecin (SN-38), temozolomide, bortezomib, ixazomib, pomalidomide, lenalidomide and any other chemical compound with ATC code L01, L02, L03 or L04 (antineoplastic and immunomodulating agents), which are therapeutic subgroups of the WHO’s Anatomical Therapeutic Chemical Classification System. Other unclassified cytotoxic agents may be considered, including but not limited to: 2,4-dinitrophenol, staurosporine, cantharidin, norcantharidin, anethole tritihone, desmethylanethole trithione, strychnine, and brucine.

[0324] Described herein is a kit for detecting a target molecule in a sample, the kit comprising: the three-dimensional probe and at least one buffer solution for contacting the probe with the sample. As used in this specification and in the appended claims, the singular forms include the plural forms. For example, the terms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. Additionally, the term "at least" preceding a series of elements is to be understood as referring to every element in the series. The inventions illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the future shown and described or any portion thereof, and it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions herein disclosed can be resorted by those skilled in the art, and that such modifications and variations are considered to be within the scope of the inventions disclosed herein. The inventions have been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the scope of the generic disclosure also form part of these inventions. This includes the generic description of each invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised materials specifically resided therein. In addition, where features or aspects of an invention are described in terms of the Markush group, those schooled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. It is also to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of in the art upon reviewing the above description. The scope of the invention should therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. Those skilled in the art will recognize or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A Nanoparticle-Oligomer Array (NOA) composition comprising:a core;a plurality of oligomer strands wherein the oligomer strands are nucleic acids which are densely arrayed on the core and extending from the core to create a three-dimensional scaffold; andat least one functional moiety,wherein said scaffold is configured to precisely position said functional moiety at a desired location relative to the core and / or an external surface of the composition.

2. The composition of claim 1, wherein the core is a nanoparticle core.

3. The composition of claim 1, the functional moiety is a therapeutic agent, prodrugs of a therapeutic, diagnostic or a detectable moiety.

4. The composition of claim 4, wherein the detectable moiety is a fluorophore or a fluorophore mixture.

5. A colloidal nanostructure comprising:a. a nanomaterial core; andb. a scaffolding of synthetic oligomers, wherein the oligomers are connected to the core and arranged to form a three-dimensional shell around the core,wherein the nanostructure is less than 200 nm in diameter.

6. The colloidal nanostructure of claim 5, further comprising a flare oligonucleotide partially or fully hybridized to the scaffolding oligomers, wherein the flare oligonucleotide is covalently attached to a cargo molecule at its 5’ terminus, 3’ terminus or internally, positioned within the scaffolding or exposed on the external surface.

7. The colloidal nanostructure of claim 5, wherein the synthetic oligomers are selected from the group consisting of single-stranded oligonucleotides, oligo(ethylene glycol), oligo(propylene glycol), oligo(acrylic acid), oligo(methacrylic acid), oligo(1 -vinylpyrrolidone), and oligo(acrylamido(carboxybetaine)).

8. The colloidal nanostructure of claim 7, wherein the single-stranded oligonucleotides are nuclease-resistant and selected from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), phosphorodiamidate morpholino oligonucleotides (PMOs), tricyclo-DNA (tcDNA), bicyclo-DNA (bcDNA), constrained ethyl LNA (cEt-LNA), transbridged nucleic acids (trans-BNAs), and spirocyclopropylene bridged nucleic acids (scpBNA).

9. The colloidal nanostructure of claim 8, wherein the core is a metallic element coated with an azide-functionalized coating selected from the group consisting of: a binary mixture of o-thiol and w-azide terminated oligo(acrylic acid) with thiol-terminated oligo(ethylene glycol), oligo(ethylene glycol) possessing multiple thiol groups and one or more azido groups, a thiol or disulfide-terminated zwitterionic oligomer and an oligonucleotide with one or more thiol or disulfide groups, and also containing an azide group.

10. The colloidal nanostructure of claim 6, wherein the cargo molecule is an azide-containin therapeutic or other chemically active agent derivatized with N-succinimidyl-(4-azidomethyl)benzoate (AMB-NHS) or an N-hydroxyimide analogthereof.

11. The colloidal nanostructure of claim 10, wherein the therapeutic agent is an antineoplastic or immunomodulating agent (ATC codes L01 -L04) or a cytotoxic agent, including but not limited to lenalidomide, bortezomib, doxorubicin, paclitaxel, staurosporine, and cantharidin.

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