US20230021059A1 - Methods of functionalizing nanoparticles - Google Patents

Methods of functionalizing nanoparticles Download PDF

Info

Publication number
US20230021059A1
US20230021059A1 US17/867,030 US202217867030A US2023021059A1 US 20230021059 A1 US20230021059 A1 US 20230021059A1 US 202217867030 A US202217867030 A US 202217867030A US 2023021059 A1 US2023021059 A1 US 2023021059A1
Authority
US
United States
Prior art keywords
nanoparticle
moiety
alkyne
compound
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
US17/867,030
Other languages
English (en)
Inventor
Kai Ma
Melik Ziya TÜRKER
Fei Wu
Feng Chen
II Thomas Courtney GARDINIER
Aranapakam M. Venkatesan
Geno J. GERMANO, Jr.
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Elucida Oncology Inc
Original Assignee
Elucida Oncology Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Elucida Oncology Inc filed Critical Elucida Oncology Inc
Priority to US17/867,030 priority Critical patent/US20230021059A1/en
Publication of US20230021059A1 publication Critical patent/US20230021059A1/en
Assigned to ELUCIDA ONCOLOGY, INC. reassignment ELUCIDA ONCOLOGY, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MA, KAI, GARDINIER, THOMAS COURTNEY, II, CHEN, FENG, GERMANO, GENO J, JR., TÜRKER, MELIK ZIYA, VENKATESAN, ARANAPAKAM M., WU, FEI
Pending legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/552—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being an antibiotic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4192—1,2,3-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/558—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a chemiluminescent acceptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1241—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins
    • A61K51/1244—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins microparticles or nanoparticles, e.g. polymeric nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • NDCs are particularly useful when targeting cancer cells with low receptor expression, that complicate delivery of a sufficient amount of drug.
  • Another advantage of NDCs is the possibility of coating the nanoparticle with an organic polymer layer, e.g., coating the nanoparticle surface with a layer of PEG groups, which can prevent adsorption of serum proteins to the nanoparticle in a physiological environment (e.g., in a subject), and may facilitate efficient urinary excretion and decrease aggregation of the nanoparticle (see, e.g., Burns et al. “Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine”, Nano Letters (2009) 9(1):442-448).
  • NDCs there are significant obstacles in the development and manufacture of NDCs. For example, it is very challenging to create NDCs that meet the stringent criteria in manufacturing controls, stability, drug release, safety, and efficacy required for clinical translation. In particular, creating a linkage between conjugated molecules (e.g., drug molecules and/or targeting ligands) and the nanoparticle carrier that meet these criteria is especially difficult.
  • conjugated molecules e.g., drug molecules and/or targeting ligands
  • the present disclosure features methods of functionalizing a nanoparticle.
  • the method can include preparation of nanoparticles suitable for conjugating compounds to the nanoparticle surface (e.g., targeting ligands, such as cancer targeting ligands, e.g., folate receptor targeting ligands, e.g., folic acid; and therapeutic agents, such as cytotoxic compounds, e.g., exatecan) to form nanoparticle drug conjugates (NDCs).
  • NDCs nanoparticle drug conjugates
  • the resulting NDCs exhibit both highly stable linkage of the conjugated molecule to the nanoparticle, and provide effective drug release in targeted biological systems, e.g., in cancer cells.
  • the method of functionalizing a nanoparticle can comprise a step of contacting a nanoparticle with a first bifunctional precursor, such as a bifunctional precursor comprising a silane moiety and another reactive group (e.g., a diene, an amine, a thiol, a hydroxyl, an azide, an alkene, or an alkyne) wherein the nanoparticle comprises a surface that is reactive with the silane (e.g., a silica surface), and wherein the contacting is under conditions suitable for reaction between the silane moiety and the nanoparticle surface (e.g., conditions described herein suitable for silane condensation), thereby forming a covalent bond between the silane moiety and a surface of the nanoparticle, and providing a nanoparticle functionalized with the reactive group (e.g., a diene, an amine, a thiol, a hydroxyl, an azide, an alkene, or an alkyne).
  • the method disclosed herein may further comprise contacting the nanoparticle functionalized with the reactive group (e.g., the nanoparticle functionalized with a diene, an amine, a thiol, a hydroxyl, an azide, an alkene, or an alkyne) with a second bifunctional precursor, wherein the second bifunctional precursor comprises a group that is reactive with the reactive group on the nanoparticle, and wherein the second bifunctional precursor comprises another functional group (e.g., an alkyne moiety (e.g., dibenzoazacyclooctyne (DBCO)), a diene, an amine, a thiol, a hydroxyl, an alkene, or an azide).
  • the reactive group e.g., the nanoparticle functionalized with a diene, an amine, a thiol, a hydroxyl, an azide
  • DBCO dibenzoazacyclooctyne
  • the second bifunctional precursor may comprise a dienophile (e.g., maleimide) that is reactive with the reactive group on the nanoparticle (e.g., a diene), and the second bifunctional precursor may also comprise an alkyne moiety (e.g., DBCO).
  • the contacting may be under conditions suitable for a reaction between the functionalized nanoparticle and the second bifunctional precursor (e.g., reaction conditions disclosed herein), thereby covalently bonding the second bifunctional precursor to the nanoparticle and providing a nanoparticle functionalized with the another functional group (e.g., an alkyne moiety, such as DBCO).
  • the method can be used to provide a nanoparticle functionalized with a plurality of the another functional group (e.g., a plurality of DBCO moieties).
  • the method may further comprise contacting the nanoparticle functionalized with the another functional group (e.g., a DBCO functionalized nanoparticle) with a compound comprising a group that is reactive with the another functional group on the nanoparticle.
  • the compound may comprise an alkyne-reactive group (e.g., an azide, diene, nitrone, or nitrile oxide), suitable for reacting with an alkyne group of an alkyne-functionalized nanoparticle.
  • the contacting may be under conditions suitable for a reaction between the alkyne moiety and the alkyne-reactive group, e.g., reaction conditions described herein, such as Click Chemistry conditions, or other cycloaddition conditions (e.g., 3+2 cycloaddition or 4+2 cycloaddition), thereby forming a nanoparticle functionalized with the compound.
  • reaction conditions described herein such as Click Chemistry conditions, or other cycloaddition conditions (e.g., 3+2 cycloaddition or 4+2 cycloaddition), thereby forming a nanoparticle functionalized with the compound.
  • the compound comprising a group that is reactive with the functional group on the nanoparticle may comprise a payload moiety (e.g., a cytotoxic drug disclosed herein, such as exatecan) or a targeting ligand (e.g., a folate receptor targeting ligand, such as folic acid).
  • a payload moiety e.
  • the method may be used to covalently attach a plurality of compounds to the nanoparticle surface.
  • the method may introduce a plurality of the compound comprising a group that is reactive with the functionalized nanoparticle, or introduce a plurality of different compounds each comprising a group that is reactive with the functionalized nanoparticle (e.g., a plurality of targeting ligands, a plurality of payload moieties, or a combination thereof), and covalently attaching said compounds to the nanoparticle.
  • each step of the method may be used to introduce a plurality of functional groups to the nanoparticle.
  • the nanoparticle can be contacted with a plurality of the first bifunctional precursor comprising a silane group and a reactive group, providing a nanoparticle functionalized with a plurality of reactive groups (e.g., a plurality of diene moieties).
  • the nanoparticle functionalized with a plurality of reactive groups can be contacted with a plurality of second bifunctional precursors, wherein the second bifunctional precursor comprises a group that is reactive with the reactive group on the nanoparticle, and wherein the second bifunctional precursor comprises another functional group (e.g., an alkyne, e.g., DBCO), thereby providing a nanoparticle functionalized with a plurality of the functional groups from the second bifunctional precursor (e.g., a plurality of DBCO moieties).
  • a plurality of reactive groups e.g., a plurality of diene moieties
  • the nanoparticle functionalized with a plurality of the functional groups from the second bifunctional precursor may then be contacted with a plurality of compounds comprising a group that is reactive with the functional group on the nanoparticle, providing a nanoparticle comprising a plurality of the compounds.
  • the nanoparticle functionalized with a plurality of the functional groups from the second bifunctional precursor may be contacted with a first plurality of compounds comprising a group that is reactive with the functional group on the nanoparticle (e.g., a targeting ligand comprising an azide), and may subsequently be contacted with a second plurality of compounds comprising a group that is reactive with the functional group on the nanoparticle (e.g., a payload-linker conjugate comprising an azide), wherein the first and second pluralities comprise structurally distinct compounds, thereby providing a nanoparticle comprising two distinct pluralities of compounds (e.g., a plurality of targeting ligands, and a plurality of payload moieties).
  • the method can comprise forming a nanoparticle functionalized with a plurality of diene moieties (e.g., cyclopentadiene moieties), e.g., by contacting a nanoparticle with a plurality of first bifunctional precursors comprising a silane moiety and a diene moiety, under conditions sufficient for a reaction between a silane moiety and a surface of the nanoparticle.
  • a plurality of diene moieties e.g., cyclopentadiene moieties
  • the method can comprise forming a nanoparticle functionalized with a plurality of alkyne moieties (e.g., DBCO moieties), e.g., by contacting a nanoparticle comprising a plurality of diene moieties (e.g., cyclopentadiene moieties) with a plurality of second bifunctional precursors comprising a dienophile (e.g., maleimide) and an alkyne moiety (e.g., DBCO), under conditions sufficient for a reaction between a diene moiety and a dienophile.
  • the method may further comprise.
  • the first compound may be a compound of Formula (D) (e.g., a compound of Formula (D-1)) disclosed herein; and the second compound may be a compound of Formula (E) (e.g., a compound of Formula (E-1)), disclosed herein.
  • the first compound may be a compound of Formula (E) (e.g., a compound of Formula (E-1)) disclosed herein, and the second compound is a compound of Formula (D) (e.g., a compound of Formula (D-1)) disclosed herein.
  • the present disclosure is also directed to a method of functionalizing a silica nanoparticle, comprising: (i) contacting a silica nanoparticle with a first bifunctional precursor, wherein the first bifunctional precursor comprises a silane moiety and a cyclopentadiene moiety, wherein the silica nanoparticle comprises a surface that is reactive with the silane moiety (e.g., a silica surface), and wherein the contacting is under conditions suitable for reaction between the silane moiety and the silica nanoparticle surface, thereby forming a covalent bond between the silane moiety and a surface of the nanoparticle, and providing a nanoparticle functionalized with a cyclopentadiene moiety; (ii) contacting the nanoparticle functionalized with a cyclopentadiene moiety with a second bifunctional precursor, wherein the second bifunctional precursor comprises an alkyne moiety (e.g., DBCO) and a dienophile (e.g., male
  • the present disclosure is also directed to a A method of functionalizing a silica nanoparticle, comprising: (i) contacting a silica nanoparticle with a first bifunctional precursor comprising a structure of Formula (A), thereby providing a nanoparticle functionalized with a cyclopentadiene moiety; (ii) contacting the nanoparticle functionalized with a cyclopentadiene moiety with a second bifunctional precursor comprising a structure of Formula (B), thereby providing a nanoparticle functionalized with an alkyne moiety; and (iii) contacting the alkyne moiety with a compound of Formula (D) or Formula (E), or both, thereby providing a nanoparticle functionalized with a targeting ligand, a payload moiety, or both.
  • the first bifunctional precursor may comprise the structure of Formula (A-1) provided herein.
  • the second bifunctional precursor may comprise a structure of Formula (B-1) provided herein.
  • the compound of Formula (D) may comprise a structure of Formula (D-1) provided herein.
  • the compound of Formula (E) may comprise a structure of Formula (E-1) provided herein.
  • the methods disclosed herein can provide a nanoparticle comprising a compound of Formula (NP-2) provided herein.
  • the average nanoparticle to (NP-2) ratio may be from about 1:1 to about 1:50 (each nanoparticle comprises an average of 1-50 units of NP-2), e.g., about 1:40, about 1:30, about 1:25, about 1:20, about 1:15, about 1:14, about 1:13, about 1:12, about 1:11, or about 1:10.
  • the method can provide a nanoparticle comprising a compound of Formula (NP-3) provided herein.
  • the average nanoparticle to (NP-3) ratio may be from about 1:1 to about 1:80 (each nanoparticle comprises an average of 1-80 units of NP-3), e.g., about 1:60, about 1:40, about 1:30, about 1:28, about 1:26, about 1:25, about 1:24, about 1:23, about 1:22, about 1:21, about 1:20, about 1:19, or about 1:18.
  • the method can provide a nanoparticle comprising a compound of Formula (NP-2) and a compound of Formula (NP-3), the structures of which are provided herein, wherein the ratio of nanoparticle:(NP-3):(NP-2) is about 1:20:10, 1:20:11, 1:20:12, 1:20:13, 1:20:14, 1:20:15, 1:21:10, 1:21:11, 1:21:12, 1:21:13, 1:21:14, 1:21:15, 1:22:10, 1:22:11, 1:22:12, 1:22:13, 1:22:14, 1:22:15, 1:23:10, 1:23:11, 1:23:12, 1:23:13, 1:23:14, 1:23:15, 1:24:10, 1:24:11, 1:24:12, 1:24:13, 1:24:14, 1:24:15, 1:25:10, 1:25:11, 1:25:12, 1:25:13, 1:25:14, or 1:25:15.
  • An advantage of the methods disclosed herein is the use of relatively stable precursors (e.g., diene-silane precursors), resulting in stable functionalized nanoparticles.
  • functionalized nanoparticles e.g., NDCs
  • other methods of functionalizing nanoparticles employ precursors that result in nanoparticles with reactive moieties on the surface of the nanoparticle, that can promote undesired reactivity that may lead to, e.g., premature release of a payload or undesired release of the targeting ligand.
  • other methods of nanoparticle functionalization use precursors that are unstable and can self-condense during reaction, causing undesired aggregation. The aggregates can be very difficult to separate from the functionalized nanoparticles.
  • the methods disclosed herein can employ relatively stable precursors, and the resulting functionalized nanoparticle (e.g., NDCs) are stable and highly pure.
  • the functionalized nanoparticles of the present disclosure can be prepared with a diene-silane precursor (such as a cyclopentadiene-silane precursor), to afford a nanoparticle functionalized with one or more diene groups.
  • the diene groups may then be reacted with a second precursor, such as a dienophile-containing precursor (e.g., a PEG-maleimide derivative, e.g., a DBCO-PEG-maleimide), causing a stable cycloadduct to form.
  • a dienophile-containing precursor e.g., a PEG-maleimide derivative, e.g., a DBCO-PEG-maleimide
  • the resulting functionalized nanoparticle comprising the cycloadduct, may optionally be reacted with one or more subsequent precursors (such as a targeting ligand precursor and/or a payload-linker conjugate precursor described herein), to further functionalize the nanoparticle.
  • a targeting ligand precursor and/or a payload-linker conjugate precursor described herein such as a targeting ligand precursor and/or a payload-linker conjugate precursor described herein
  • the diene-silane precursor, and the cycloadducts that are produced do not exhibit undesired characteristics that can be found in other functionalized nanoparticles or their precursors.
  • the functionalized nanoparticles e.g., NDCs
  • the functionalized nanoparticles prepared using the methods disclosed herein have relatively high serum stability, and can be produced in high yield and purity (e.g., free of aggregated precursor).
  • any desired ratio of payload, targeting ligand, or otherwise can be introduced to the nanoparticle. Examples
  • FIG. 9 An exemplary sequence of functionalizing a nanoparticle is depicted in FIG. 9 .
  • FIG. 1 illustrates the structure of an exemplary nanoparticle drug conjugate (NDC) that can be prepared using the methods disclosed herein.
  • NDC nanoparticle drug conjugate
  • FIG. 2 is a graph comparing efficiency between different approaches for functionalizing nanoparticles with DBCO moieties.
  • FIGS. 3 A- 3 D are RP-HPLC and SEC chromatograms.
  • FIG. 3 A provides the RP-HPLC chromatogram of DBCO-functionalized nanoparticles prepared using an amine-based bifunctional precursor, before and after incubation in PBS for 24 hours.
  • FIG. 3 B provides the RP-HPLC chromatogram of DBCO-functionalized nanoparticles prepared using a diene-based bifunctional precursor, before and after incubation in PBS for 24 hours.
  • FIG. 3 C provides the SEC chromatogram of DBCO-functionalized nanoparticles prepared using an amine-based bifunctional precursor.
  • FIG. 3 D provides the SEC chromatogram of DBCO-functionalized nanoparticles prepared using a diene-based bifunctional precursor.
  • FIG. 4 is a spectrograph from fluorescence correlation spectroscopy (FCS) of an exemplary NDC prepared using the methods disclosed herein, indicating the average hydrodynamic diameter.
  • FCS fluorescence correlation spectroscopy
  • FIG. 5 shows overlaid UV-Vis spectra of an exemplary functionalized nanoparticle at different stages of functionalization, indicating characteristic absorption peaks that can be used to verify the presence and average number of each conjugate (e.g., targeting ligand, e.g., folic acid (FA), or payload, e.g., exatecan) per particle.
  • conjugate e.g., targeting ligand, e.g., folic acid (FA), or payload, e.g., exatecan
  • FIGS. 8 A- 8 B are graphs illustrating the serum stability of exemplary NDCs prepared using methods disclosed herein.
  • FIG. 8 A compares the stability of an NDC produced using a diene-based bifunctional precursor and an NDC produced using an amine-based bifunctional precursor in 10% human serum at 37° C., over 7 days.
  • FIG. 8 B compares the stability of an NDC produced using a diene-based bifunctional precursor and an NDC produced using an amine-based bifunctional precursor in 10% mouse serum at 37° C., over 7 days.
  • FIG. 9 depicts an exemplary sequence of modifying a nanoparticle (C'Dot).
  • alkyl refers to monovalent aliphatic hydrocarbon group that may comprise 1 to 18 carbon atoms, such as 1 to about 12 carbon atoms, or 1 to about 6 carbon atoms (“C 1-18 alkyl”).
  • An alkyl group can be straight chain, branched chain, monocyclic moiety or polycyclic moiety or combinations thereof.
  • alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like.
  • Each instance of an alkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • alkynyl refers to a monovalent straight-chain or branched hydrocarbon group having from 2 to 18 carbon atoms, and one or more carbon-carbon triple bonds (“C 2-18 alkynyl”).
  • the alkynyl group may have 2 to 8 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms.
  • the one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl).
  • alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and the like.
  • Each instance of an alkynyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents, e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • heteroalkyl refers to a non-cyclic stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized.
  • the heteroatom(s) O, N, P, S, and Si may be placed at any position of the heteroalkyl group.
  • alkylene alkenylene, alkynylene, or “heteroalkylene,” alone or as part of another substituent, mean, unless otherwise stated, a divalent radical derived from an alkyl, alkenyl, alkynyl, or heteroalkyl, respectively.
  • alkenylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.
  • alkylene, alkenylene, alkynylene, or heteroalkylene group may be described as, e.g., a C 1-6 -membered alkylene, C 1-6 -membered alkenylene, C 1-6 -membered alkynylene, or C 1-6 -membered heteroalkylene, wherein the term “membered” refers to the non-hydrogen atoms within the moiety.
  • heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like).
  • alkylene and heteroalkylene linking groups no orientation of the linking group is implied by the direction in which the formula of the linking group is written.
  • the formula —C(O) 2 R′— may represent both —C(O) 2 R′— and —R′C(O) 2 —.
  • Each instance of an alkylene, alkenylene, alkynylene, or heteroalkylene group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkylene”) or substituted (a “substituted heteroalkylene”) with one or more substituents.
  • substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sul
  • heteroaryl groups include pyrrole, furan, indole, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.
  • cycloalkylene As used herein, the terms “cycloalkylene,” “heterocyclylene,” “arylene,” and “heteroarylene,” alone or as part of another substituent, mean a divalent radical derived from a cycloalkyl, heterocyclyl, aryl, and heteroaryl, respectively.
  • Each instance of a cycloalkylene, heterocyclylene, arylene, or heteroarylene may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted arylene”) or substituted (a “substituted heteroarylene”) with one or more substituents.
  • cycloalkyl is intended to include non-aromatic cyclic hydrocarbon rings, such as hydrocarbon rings having from three to eight carbon atoms in their ring structure.
  • Cycloalkyl can include cyclobutyl, cyclopropyl, cyclopentyl, cyclohexyl and the like.
  • the cycloalkyl group can be either monocyclic (“monocyclic cycloalkyl”) or contain a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic cycloalkyl”) and can be saturated or can be partially unsaturated.
  • Cycloalkyl also includes ring systems wherein the cycloalkyl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is on the cycloalkyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the cycloalkyl ring system.
  • Each instance of a cycloalkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents.
  • dipeptide refers to a peptide that is composed of two amino acid residues, which may be denoted herein as -A 1 -A 2 -.
  • dipeptides employed in the synthesis of a protease-cleavable linker-payload conjugate of the present disclosure may be selected from the group consisting of Val-Cit, Phe-Lys, Trp-Lys, Asp-Lys, Val-Lys, and Val-Ala.
  • oxo refers to an oxygen that is double bonded to carbon or another element (i.e., ⁇ O).
  • a “targeting ligand” is a molecule that can be bonded to a nanoparticle and target the nanoparticle to a tumor or cancer cell, typically by binding to the tumor or cancer cell (such as by binding to a protein expressed on the surface of the tumor or cancer cell).
  • the targeting ligand can be any suitable molecule such as a small organic molecule (e.g., folate or a folate analog), an antigen-binding portion of an antibody (e.g.
  • Fab fragment a Fab′ fragment, a F(ab′)2 fragment, a scFv fragment, a Fv fragment, a dsFv diabody, a dAb fragment, a Fd′ fragment, a Fd fragment, or an isolated complementarity determining region (CDR) region
  • an antibody mimetic e.g., an aptamer, affibody, affilin, affimer, anticalin, avimer, Darpin, and the like
  • the binding domain of a receptor e.g., an aptamer, affibody, affilin, affimer, anticalin, avimer, Darpin, and the like.
  • protecting group refers to a particular functional moiety, e.g., O, S, or N, that is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound.
  • Protecting groups may be introduced and removed at appropriate stages during the synthesis of a compound using methods that are known to one of ordinary skill in the art. The protecting groups are applied according to standard methods of organic synthesis as described in the literature (Theodora W. Greene and Peter G. M. Wuts (2007) Protecting Groups in Organic Synthesis, 4 th edition, John Wiley and Sons, incorporated by reference with respect to protecting groups).
  • oxygen protecting groups include, but are not limited to, oxygen, sulfur, nitrogen and carbon protecting groups.
  • oxygen protecting groups include, but are not limited to, methyl ethers, substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM (methylthiomethyl ether), BOM (benzyloxymethyl ether), PMBM (pimethoxybenzyloxymethyl ether), optionally substituted ethyl ethers, optionally substituted benzyl ethers, silyl ethers (e.g., TMS (trimethylsilyl ether), TES (triethylsilylether), TIPS (triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzyl silyl ether, TBDPS (t-butyldiphenyl silyl ether), esters (e.g., formate, acetate, benzoate (Bz), trifluoroacetate, dichlor
  • nitrogen protecting groups include, but are not limited to, carbamates (including methyl, ethyl and substituted ethyl carbamates (e.g., Troc), amides, cyclic imide derivatives, N-Alkyl and N-Aryl amines, imine derivatives, and enamine derivatives, etc.
  • Amino protecting groups include, but are not limited to fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), acetamide, trifluoroacetamide, etc. Certain other exemplary protecting groups are detailed herein, however, it will be appreciated that the present disclosure is not intended to be limited to these protecting groups; rather, a variety of additional equivalent protecting groups may be utilized according to methods known to one skilled in the art.
  • a nanoparticle drug conjugate may sometimes be referred to as a CDC (C'Dot-drug-conjugate), e.g., a FA-CDC, or simply as a functionalized nanoparticle.
  • silica nanoparticles can be functionalized using a method disclosed herein (including nanoparticles that are a partially comprised of silica, or completely comprised of silica).
  • the nanoparticle can have a diameter from about 0.5 nm to about 100 nm, e.g., from about 0.1 nm to about 50 nm, from about 0.5 nm to about 25 nm, from about 1 nm to about 20 nm, from about 0.8 nm to about 15 nm, from about 1 nm to about 10 nm, or from about 1 nm to about 8 nm, e.g., about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, or about 12 nm.
  • the nanoparticle may comprise a core surrounded by a shell (i.e., a core-shell nanoparticle). Alternatively, the nanoparticle may have only a core and no shell.
  • the nanoparticle may also comprise a material (e.g., on its surface) that is reactive with a silane moiety, e.g., a silica surface.
  • the shell of the nanomaterial may be a material that is reactive with a silane moiety, e.g., a silica shell.
  • the methods disclosed herein can be used to modify a silica nanoparticle that comprises a silica-based core and a silica-based shell surrounding at least a portion of the core (a core-shell silica nanoparticle).
  • the nanoparticle may be a non-mesoporous nanoparticle (e.g., a non-mesoporous core-shell nanoparticle), such as a non-mesoporous silica nanoparticle (e.g., a non-mesoporous core-shell silica nanoparticle).
  • a non-mesoporous nanoparticle e.g., a non-mesoporous core-shell nanoparticle
  • a non-mesoporous silica nanoparticle e.g., a non-mesoporous core-shell silica nanoparticle
  • the nanoparticle may comprise an organic polymer coating on its surface.
  • An organic polymer that may be attached to the nanoparticle includes, but is not limited to, poly(ethylene glycol) (PEG), polylactate, polylactic acids, sugars, lipids, polyglutamic acid (PGA), polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA), polyvinyl acetate (PVA), and combinations thereof.
  • the nanoparticle may have a layer of polyethylene glycol (PEG) molecules attached to the surface.
  • Certain organic polymer coatings can provide a nanoparticle with advantageous properties, such as properties suitable for a biological system.
  • the PEG groups may prevent adsorption of serum proteins to the nanoparticle and may facilitate efficient urinary excretion, and may decrease aggregation of the nanoparticle (see, e.g., Burns et al. “Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine”, Nano Letters (2009) 9(1):442-448, which is incorporated herein by reference in its entirety).
  • the shell of the nanoparticle may have a range of layers.
  • the shell may comprise from about 1 to about 20 layers, from about 1 to about 15 layers, from about 1 to about 10 layers, or from about 1 to about 5 layers.
  • the shell may comprise from about 1 to about 3 layers.
  • the thickness of the shell may range from about 0.5 nm to about 90 nm, e.g., from about 1 nm to about 40 nm, from about 1 nm to about 20 nm, from about 1 nm to about 10 nm, or from about 1 nm to about 5 nm.
  • the thickness of the shell may be from about 1 nm to about 2 nm.
  • the shell of the nanoparticle may cover only a portion of nanoparticle or the entire particle.
  • the shell may cover about 1 to about 100 percent, from about 10 to about 80 percent, from about 20 to about 60 percent, or from about 30 to about 50 percent of the nanoparticle.
  • the shell of the nanoparticle can comprise silica, and/or may be the reaction product of a silica forming compound, such as a tetraalkyl orthosilicate, for example tetraethyl orthosilicate (TEOS).
  • TEOS tetraethyl orthosilicate
  • the nanoparticle (e.g., the nanoparticle core and/or shell) can be substantially non-porous, meso-porous, semi-porous, or porous.
  • the nanoparticle may comprise a non-pore surface and a pore surface.
  • the pore surface may be referred to as the interior surface.
  • the nanoparticle may also have a non-pore surface (or non-porous surface).
  • the non-pore surface may be referred to herein as the exterior nanoparticle surface.
  • the pore surface (e.g., at least a portion of the pore surface) and/or the non-pore surface (e.g., at least a portion of the non-pore surface) of the nanoparticle can be functionalized.
  • the nanoparticle and/or nanoparticle shell may be non-mesoporous.
  • the nanoparticle may contain a dye, such as a fluorescent compound.
  • the dye can be contained within the nanoparticle core.
  • the nanoparticle may contain a dye covalently encapsulated in the nanoparticle.
  • the dye in a silica nanoparticle may be the reaction product of a reactive dye (e.g., fluorescent compound) and a co-reactive organo-silane compound.
  • a silica nanoparticle may contain the reaction product of a reactive dye compound and a co-reactive organo-silane compound, and silica.
  • the nanoparticles may incorporate any known fluorescent compound, such as fluorescent organic compound, dyes, pigments, or combinations thereof.
  • fluorescent compounds can be incorporated into the silica matrix of the core of a silica nanoparticle.
  • suitable chemically reactive fluorescent dyes/fluorophores are known, see for example, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, 6 th ed., R. P. Haugland, ed. (1996).
  • the fluorescent compound may be covalently encapsulated within the core of the nanoparticle.
  • the fluorescent compound can be, but is not limited to, a near infrared fluorescent (NIRF) dye that is positioned within the core of the nanoparticle, that can provide greater brightness and fluorescent quantum yield relative to the free fluorescent dye. It is well-known that the near infrared-emitting probes exhibit decreased tissue attenuation and autofluorescence (Burns et al. supra).
  • NIRF near infrared fluorescent
  • Fluorescent compounds that may be used (e.g., encapsulated by an NDC) in the present disclosure, include, but are not limited to, Cy5, Cy5.5 (also known as Cy5++), Cy2, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin, Cy7, fluorescein (FAM), Cy3, Cy3.5 (also known as Cy3++), Texas Red (sulforhodamine 101 acid chloride), LIGHTCYCLER®-Red 640, LIGHTCYCLER®-Red 705, tetramethylrhodamine (TMR), rhodamine, rhodamine derivative (ROX), hexachlorofluorescein (HEX), rhodamine 6G (R6G), the rhodamine derivative JA133, Alexa Fluorescent Dyes (such as ALEXA FLUOR® 488, ALEXA FLUOR® 5
  • Fluorescent compounds that can be used also include fluorescent proteins, such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent protein and derivatives (BFP, EBFP, EBFP2, azurite, mKalama1), cyan fluorescent protein and derivatives (CFP, ECFP, Cerulean, CyPet) and yellow fluorescent protein and derivatives (YFP, Citrine, Venus, YPet) (WO 2008/142571, WO 2009/056282, WO 1999/22026).
  • the fluorescent compound is Cy5.
  • the nanoparticle may be synthesized by the steps of: (1) covalently conjugating a fluorescent compound, such as a reactive fluorescent dye (e.g., Cy5), with a reactive moiety including, but not limited to, maleimide, iodoacetamide, thiosulfate, amine, N-hydroxysuccimide ester, 4-sulfo-2,3,5,6-tetrafluorophenyl (STP) ester, sulfosuccinimidyl ester, sulfodichlorophenol esters, sulfonyl chloride, hydroxyl, isothiocyanate, carboxyl, to an organo-silane compound, such as a co-reactive organo-silane compound, to form a fluorescent silica precursor, and reacting the fluorescent silica precursor to form a fluorescent core; or (2) reacting the fluorescent silica precursor with a silica forming compound, such as tetraalkoxysilane, to
  • the nanoparticles functionalized by methods disclosed herein may comprise a nanoparticle that comprises a silica-based core and a silica shell surrounding at least a portion of the core, and polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticle.
  • the nanoparticles may have a fluorescent compound covalently encapsulated within the core of the nanoparticle.
  • ultrasmall PEGylated silica nanoparticles referred to as C'Dots
  • C'Dots can be functionalized using the methods disclosed herein.
  • C'dots can be prepared as described previously (see, e.g., Ma, K. et al.
  • Nanoparticles for use in a method described herein may also be prepared as described in Ma, K. et al. Chem. Mater . (2013), 25:677-691; Ma, K. et al. Chem. Mater . (2016) 28:1537-1545; Ma, K. et al. Chem. Mater . (2017) 29:6840-6855; WO 2016/179260; and WO 2018/213851.
  • Each C'Dot comprises a PEGylated silica particle (approximately 6 nm in diameter) in which near-infrared fluorescent Cy5 dyes are covalently encapsulated.
  • the molecule may be a targeting ligand, such as a compound that binds to a certain receptor, e.g., a compound that binds to a folate receptor, such as folic acid or a derivative thereof.
  • a targeting ligand such as a compound that binds to a certain receptor, e.g., a compound that binds to a folate receptor, such as folic acid or a derivative thereof.
  • the method disclosed herein can be used to functionalize nanoparticles that are suitable for targeting cancer cells.
  • the compound comprising an alkyne-reactive group may be a cytotoxic compound, and can include a cleavable linker, such that the cytotoxic compound can be released in a biological system (e.g., within a cancer cell) upon cleavage of the linker.
  • the payload can be a cytotoxic drug, such as a small molecule chemotherapeutic drug, e.g., exatecan or a derivative thereof, and the linker can be a protease-cleavable linker.
  • a cytotoxic drug such as a small molecule chemotherapeutic drug, e.g., exatecan or a derivative thereof
  • the linker can be a protease-cleavable linker.
  • NDC nanoparticle drug conjugate
  • the methods disclosed herein can be used to functionalize a nanoparticle that is coated with an organic polymer.
  • the method described herein may be carried out after the nanoparticle has been coated with polyethylene glycol (post-PEGylation). This reduces the need for reaction optimization, and minimizes alterations needed in both the synthetic protocol and to the nanoparticle itself (e.g., no alterations are needed to optimize surface chemistry of the nanoparticle prior to carrying out a method disclosed herein), which is more efficient than conventional methods for nanoparticle functionalization.
  • the methods disclosed herein provide a solution to the problem of how to attach bulky molecules, such as DBCO, to the surface of a nanoparticle that is sterically hindered by an organic polymer coating (e.g., PEG layer).
  • organic polymer coating e.g., PEG layer.
  • Certain molecules, particularly those having larger size and steric hindrance, have limited access the surface of the nanoparticle, resulting in lower reactivity and hindering attachment of the molecules to the nanoparticle surface.
  • only two DBCO groups can be attached to a nanoparticle when using DBCO-PEG-silane as a bifunctional precursor, even when the DBCO-PEG-silane precursor is at a relatively high ratio in the reaction mixture (see, e.g., Example 1 and FIG. 2 ).
  • the methods disclosed can involve a two-step reaction sequence, where a small first bifunctional precursor (e.g., a precursor comprising a silane group) that has a relatively high diffusion coefficient is first bonded to the nanoparticle surface, and is then subsequently modified by another compound, e.g., an additional bifunctional precursor(s).
  • a small first bifunctional precursor e.g., a precursor comprising a silane group
  • another compound e.g., an additional bifunctional precursor(s).
  • a small bifunctional precursor comprising a silane moiety and another reactive group (e.g., a diene, an amine, a thiol, a hydroxyl, an azide, an alkene, a nitrone, a nitrile oxide, or an alkyne) can be added to a solution of a nanoparticle comprising a surface that is reactive with the silane group, under conditions suitable for reaction between the nanoparticle surface and the silane group, thereby providing a nanoparticle that is functionalized with the another reactive group.
  • another reactive group e.g., a diene, an amine, a thiol, a hydroxyl, an azide, an alkene, a nitrone, a nitrile oxide, or an alkyne
  • the conditions suitable for reaction between the silane moiety and the nanoparticle surface may comprise combining the nanoparticle and the first bifunctional precursor in an aqueous reaction medium (e.g., a reaction medium that is substantially water).
  • the reaction medium may also comprise an aprotic organic solvent, such as dimethylsulfoxide (DMSO) or acetonitrile).
  • the reaction medium may comprise no more than 20 v/v % of the aprotic solvent, e.g., less than 18 v/v %, less than 16 v/v %, less than 14 v/v %, less than 12 v/v %, less than 10 v/v %, less than 8 v/v %, less than 6 v/v %, less than 4 v/v %, less than 2 v/v %, or less than 1 v/v % of the aprotic solvent.
  • the reaction medium may also comprise a protic organic solvent, such as an alcohol (e.g., tert-butanol).
  • the reaction medium may comprise no more than 20 v/v % of the protic solvent, e.g., less than 18 v/v %, less than 16 v/v %, less than 14 v/v %, less than 12 v/v %, less than 10 v/v %, less than 8 v/v %, less than 6 v/v %, less than 4 v/v %, less than 2 v/v %, or less than 1 v/v % of the protic solvent.
  • the reaction medium may comprise a buffer (e.g., an aqueous buffer), such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the reaction medium may comprise a buffer (e.g., an aqueous buffer, such as PBS) at a final concentration between about 1 ⁇ and about 5 ⁇ , e.g., about 1 ⁇ , about 2 ⁇ , about 3 ⁇ , about 4 ⁇ , or about 5 ⁇ .
  • the reaction medium may be substantially free of an organic solvent (e.g., substantially free of organic protic solvent, substantially free of aprotic solvent, or substantially free of any organic solvent).
  • the reaction medium may be substantially free of buffer.
  • the reaction medium may be substantially water.
  • the conditions suitable for reaction between the silane moiety and the nanoparticle surface may comprise combining the nanoparticle and the first bifunctional precursor in a reaction medium for a certain period of time, e.g., between about 1 minute and about 60 minutes, between about 0.5 hours and about 24 hours, between about 0.5 hours and about 18 hours, between about 0.5 hours and about 12 hours, between about 0.5 hours and about 6 hours, between about 0.5 hours and about 4 hours, between about 0.5 hours and about 3 hours, between about 0.5 hours and about 2 hours, between about 0.5 hours and about 1 hours, or between about 1 hour and about 48 hours.
  • the conditions may comprise holding the reaction medium overnight.
  • the conditions suitable for reaction between the silane moiety and the nanoparticle surface may comprise combining the nanoparticle and the first bifunctional precursor in a reaction medium, and heating the reaction medium, e.g., to a temperature of about 20° C. or greater, e.g., about 35° C. or greater, about 40° C. or greater, about 45° C. or greater, about 50° C. or greater, about 60° C. or greater, about 70° C. or greater, about 80° C. or greater, about 90° C. or greater, about 100° C. or greater, about 110° C. or greater, or about 120° C. or greater, e.g., between about 20° C. and about 60° C., between about 40° C.
  • the conditions may comprise maintaining the nanoparticle and the first bifunctional precursor at room temperature.
  • the conditions suitable for a reaction between the silane moiety and the nanoparticle surface may comprise stirring, shaking, or applying other mixing methods to the reaction medium.
  • the first bifunctional precursor can be any suitable silane-containing compound that is reactive with the nanoparticle surface, and may comprise a functional group that will be available for further modification, e.g., after attachment of the first bifunctional precursor to the nanoparticle.
  • the functional group may be a group that is not reactive with the nanoparticle, and/or not reactive with a silane.
  • the first bifunctional precursor may comprise an alkylene group (e.g., a C 1 -C 6 alkylene group), with a silane group at one terminus, and another functional group at the other terminus (e.g., a diene, an amine, a thiol, a hydroxyl, an azide, an alkene, a nitrone, a nitrile oxide, or an alkyne).
  • an alkylene group e.g., a C 1 -C 6 alkylene group
  • silane group e.g., a silane group at one terminus
  • another functional group at the other terminus e.g., a diene, an amine, a thiol, a hydroxyl, an azide, an alkene, a nitrone, a nitrile oxide, or an alkyne.
  • the first bifunctional precursor may comprise a heteroalkylene group (e.g., a C 1 -C 6 heteroalkylene group), with a silane group at one terminus, and another functional group at the other terminus (e.g., a diene, an amine, a thiol, a hydroxyl, an azide, an alkene, a nitrone, a nitrile oxide, or an alkyne).
  • a heteroalkylene group e.g., a C 1 -C 6 heteroalkylene group
  • silane group e.g., a silane group at one terminus
  • another functional group at the other terminus e.g., a diene, an amine, a thiol, a hydroxyl, an azide, an alkene, a nitrone, a nitrile oxide, or an alkyne.
  • the first bifunctional precursor can comprise an alkylene group (e.g., a C 1 -C 6 alkylene group), with a silane group at one terminus, and a diene group at the other terminus, such as a cyclic dienyl group (e.g., a cyclopentadienyl group).
  • a cyclic dienyl group e.g., a cyclopentadienyl group
  • Any suitable diene group may be used.
  • any diene suitable to undergo a Diels-Alder (or Hetero-Diels Alder) cycloaddition may be used.
  • dienes that may be present in the first bifunctional precursor include, but are not limited to, cyclopentadiene, cyclohexadiene, furan, butadiene, and derivatives thereof.
  • moieties capable of modification e.g., by a reaction with another entity
  • the method is not limited to using Diels-Alder cycloadditions (e.g., other cycloadditions, or other bond-forming reactions, can be used, such as another bond-forming reaction described herein).
  • the first bifunctional precursor can comprise a structure of Formula (A):
  • R 1 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halo, —OR A , —NR B R C , —NO 2 , or —CN, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, may be substituted or unsubstituted; each R 2 is independently hydrogen, alkyl, halo, or —OR A ; R A , R B , and R C are each independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; n is an integer of 0 to 12; and m is an integer of 0 to 5.
  • R 1 can be alkyl, C3-alkenyl, C3-alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halo, —OR A , wherein each alkyl, C3-alkenyl, C3-alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, may be substituted or unsubstituted; each R 2 is independently hydrogen, alkyl, halo, or —OR A ; R A is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; n is an integer of 0 to 12; and m is an integer of 0 to 5.
  • R 2 may be —OR A , wherein R A is alkyl (e.g., methyl or ethyl); n may be an integer of 1 to 5 (e.g., 3); and m may be 0. In some aspects, R 2 is —OEt; n is 3; and m is 0.
  • the first bifunctional precursor can comprise the structure of Formula (A-1):
  • the first bifunctional precursor may be (3-cyclopentadienylpropyl)triethoxysilane (e.g., as demonstrated in Example 3), wherein the method provides a nanoparticle functionalized with one or more cyclopentadienyl groups.
  • the first bifunctional precursor may be (3-aminopropyl)triethoxysilane (e.g., as demonstrated in Example 2), wherein the method provides a nanoparticle functionalized with one or more amine groups.
  • the step of reacting the nanoparticle with the first bifunctional precursor can provide a nanoparticle functionalized with a first reactive group, which may be further modified.
  • the nanoparticle functionalized with a first reactive group may be contacted with a second bifunctional precursor that comprises a moiety reactive with the first reactive group.
  • Contacting the nanoparticle functionalized with a first reactive group with the second bifunctional precursor can be under any conditions suitable for a reaction between first reactive group and the moiety reactive with the first reactive group.
  • the conditions suitable for a reaction between first reactive group and the moiety reactive with the first reactive group can comprise an aqueous reaction medium (e.g., a reaction medium that is substantially water).
  • the reaction medium may comprise a buffer (e.g., an aqueous buffer), such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the reaction medium may comprise a buffer (e.g., an aqueous buffer, such as PBS) at a final concentration between about 1 ⁇ and about 5 ⁇ , e.g., about 1 ⁇ , about 2 ⁇ , about 3 ⁇ , about 4 ⁇ , or about 5 ⁇ .
  • the reaction medium may also comprise an aprotic organic solvent, such as dimethylsulfoxide (DMSO) or acetonitrile.
  • the reaction medium may comprise no more than 20 v/v % of the aprotic solvent, e.g., less than 18 v/v %, less than 16 v/v %, less than 14 v/v %, less than 12 v/v %, less than 10 v/v %, less than 8 v/v %, less than 6 v/v %, less than 4 v/v %, less than 2 v/v %, or less than 1 v/v % of the aprotic solvent.
  • the reaction medium may also comprise a protic organic solvent, such as an alcohol (e.g., tert-butanol).
  • the reaction medium may comprise no more than 20 v/v % of the protic solvent, e.g., less than 18 v/v %, less than 16 v/v %, less than 14 v/v %, less than 12 v/v %, less than 10 v/v %, less than 8 v/v %, less than 6 v/v %, less than 4 v/v %, less than 2 v/v %, or less than 1 v/v % of the protic solvent.
  • the reaction medium may be substantially free of an organic solvent (e.g., substantially free of organic protic solvent, substantially free of aprotic solvent, or substantially free of any organic solvent).
  • the reaction medium may be substantially water.
  • the conditions suitable for a reaction between first reactive group and the moiety reactive with the first reactive group can comprise holding the reaction mixture for a certain period of time, e.g., between about 1 minute and about 60 minutes, between about 0.5 hours and about 24 hours, between about 0.5 hours and about 18 hours, between about 0.5 hours and about 12 hours, between about 0.5 hours and about 6 hours, between about 0.5 hours and about 4 hours, between about 0.5 hours and about 3 hours, between about 0.5 hours and about 2 hours, between about 0.5 hours and about 1 hours, or between about 1 hour and about 48 hours.
  • the reaction medium may held overnight.
  • the conditions suitable for a reaction between first reactive group and the moiety reactive with the first reactive group can comprise heating the reaction medium, e.g., to a temperature of about 20° C. or greater, e.g., about 35° C. or greater, about 40° C. or greater, about 45° C. or greater, about 50° C. or greater, about 60° C. or greater, about 70° C. or greater, about 80° C. or greater, about 90° C. or greater, about 100° C. or greater, about 110° C. or greater, or about 120° C. or greater, e.g., between about 20° C. and about 60° C., between about 40° C. and about 80° C., between about 60° C.
  • the conditions may also comprise maintaining the reaction mixture at room temperature.
  • the conditions suitable for a reaction between first reactive group and the moiety reactive with the first reactive group can also comprise stirring, shaking, or applying other mixing methods to the reaction medium.
  • the first reactive group can be a diene (e.g., cyclopentadiene), and the second bifunctional precursor can comprise a dienophile (e.g., a dienophile described herein, e.g., maleimide) that is reactive with the first reactive group, wherein the contacting may be under conditions sufficient for promoting a Diels-Alder cycloaddition between a diene and dienophile.
  • a dienophile e.g., a dienophile described herein, e.g., maleimide
  • the dienophile can be any suitable dienophile, such as a moiety comprising an electron deficient alkene.
  • the dienophile may comprise a cyclic dienophile, e.g., a maleimide, a quinone, a maleic anhydride, dialkyl acetylene dicarboxylic acid, or any derivative thereof.
  • the position of the diene and dienophile can be on either the first bifunctional precursor or second bifunctional precursor, respectively.
  • first bifunctional precursor comprising a diene
  • second bifunctional precursor comprising a dienophile
  • these methods or precursors can be readily modified such that the first bifunctional precursor comprises the dienophile
  • the second bifunctional precursor comprises the diene.
  • the conjugation between the first reactive group and the second bifunctional precursor can involve any other suitable covalent bond forming reaction, which can be determined by selection of groups on the first bifunctional precursor and second bifunctional precursor.
  • the methods disclosed herein to functionalize a nanoparticle can involve etherification, amide bond formation, Click Chemistry, Diels-Alder cycloaddition, Hetero-Diels-Alder cycloaddition, 1,2-addition such as a Michael addition, Huisgen cycloaddition, nitrone-olefin cycloaddition, 3+2 cycloaddition, 4+2 cycloaddition, olefin metathesis, or any reaction described in Kolb et al. Angew. Chem. Int. Ed (2001) 40:2004-2021 (incorporated herein by reference in its entirety).
  • the method can be modified such that first reactive group (introduced by the first bifunctional precursor) can be an amine, and the second bifunctional precursor can comprise an N-hydroxysuccinimide (NHS) ester group, and the second bifunctional precursor and functionalized nanoparticle comprising amine group can be contacted under conditions sufficient for promoting an amine-ester reaction and forming a bond between the first reactive group and the second bifunctional precursor.
  • first reactive group introduced by the first bifunctional precursor
  • the second bifunctional precursor can comprise an N-hydroxysuccinimide (NHS) ester group
  • NHS N-hydroxysuccinimide
  • a click chemistry reaction could be used e.g., where the first reactive group (introduced by the first bifunctional precursor) is an azide or an alkyne, and the second bifunctional precursor is an alkyne or an azide, and the second bifunctional precursor and functionalized nanoparticle can be contacted under conditions sufficient for promoting a click chemistry reaction between the first reactive group and the second bifunctional precursor.
  • the first bifunctional precursor comprises a diene (e.g., cyclopentadiene), and the second bifunctional precursor comprises a dienophile (e.g., maleimide) that can be reacted with the diene in a Diels-Alder reaction, to attach the second bifunctional precursor to the nanoparticle (via the first bifunctional precursor), as demonstrated in Example 3, and also as depicted in FIG. 9 .
  • a diene e.g., cyclopentadiene
  • a dienophile e.g., maleimide
  • the second bifunctional precursor may comprise yet another reactive group, e.g., on the terminus of the second bifunctional precursor opposite to the moiety that is reactive with the first reactive group.
  • the second bifunctional precursor can comprise a group at one terminus that is reactive with a diene moiety (e.g., a dienophile, e.g., maleimide), and comprise another group at the other terminus suitable for further modification.
  • the another reactive group (sometimes referred to as functional group) on the second bifunctional precursor may be a moiety that is reactive with an alkyne, azide, diene, nitrone, or nitrile oxide.
  • the reactive group may be a moiety that is suitable for undergoing a click chemistry reaction.
  • the reactive group may comprise an alkyne, an azide, a diene, nitrone, or a nitrile oxide.
  • the alkyne can be a strained alkyne.
  • the reactive group may comprise a dibenzoazacyclooctyne (DBCO) group or a derivative thereof (sometimes referred to as DIBAC).
  • DBCO dibenzoazacyclooctyne
  • DIBAC dibenzocyclooctyne
  • DIBAC dibenzocyclooctyne
  • DIBO dibenzocyclooctyne group
  • the alkyne may also be a terminal alkyne.
  • an amide bond may be situated in the divalent linker between a PEG group and an alkylene group, wherein the carbon atom of the carbonyl group of the amide is covalently attached to an atom of the PEG group, and the nitrogen atom of the amide is covalently attached to an atom of the alkylene group; or the carbon atom of the carbonyl group of the amide may be attached to an atom of the alkylene group, and the nitrogen atom of the amide group may be attached to an atom of the PEG group.
  • the divalent linker e.g., heteroalkylene group
  • X is a reactive group, such as a dienophile (e.g., a moiety comprising an electron deficient alkene group; or a cyclic dienophile, e.g., a maleimide, a quinone, or maleic anhydride);
  • Y is a divalent linker (e.g., a substituted or unsubstituted alkylene or heteroalkylene group);
  • R 3 and R 4 are each independently alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halo, —OR A , —NR B R C , —NO 2 , or —CN, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl may be substituted or unsubstituted; R A , R B , and
  • the divalent linker can comprise a structure of Formula (G):
  • a 1 , A 2 , and A 3 are each independently selected from the group consisting of alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, arylene, heteroarylene, —C(O)—, —C(O)N(R)—, —N(R B )C(O)—, —N(R)—, —OC(O)—, and —C(O)O—;
  • R B is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl;
  • k, v, w, and x are each independently an integer of 0 to 10; and each independently denotes a point of attachment to another portion of the second bifunctional precursor.
  • a 1 and A 2 may each independently be —C(O)N(R B )—;
  • a 3 may be —C(O)O—;
  • each instance of R B may independently be hydrogen;
  • k, v, and w may each independently be integers of 2; and
  • x may be an integer of 4.
  • each R is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; x is an integer of 0 to 10; and each independently denotes a point of attachment to another portion of the second bifunctional precursor (e.g., an attachment to the dienophile or to the nitrogen atom of the heterocyclic ring of DBCO).
  • the divalent linker can comprise a structure of Formula (C′):
  • each R B is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; x is an integer of 0 to 10; and each independently denotes a point of attachment to another portion of the second bifunctional precursor (e.g., an attachment to the dienophile or to the nitrogen atom of the heterocyclic ring of DBCO).
  • the divalent linker can comprise a structure of Formula (C′′):
  • each R B is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; x is an integer of 0 to 10; and each independently denotes a point of attachment to another portion of the second bifunctional precursor (e.g., an attachment to the nitrogen atom of an NHS moiety, or to the nitrogen atom of the heterocyclic ring of DBCO).
  • the second bifunctional precursor may comprise a structure of Formula (B-1)
  • x is an integer of 0 to 10 (e.g., 4).
  • x may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • x is 4.
  • Reacting a diene moiety (e.g., a diene moiety covalently attached to a nanoparticle surface) with the second bifunctional precursor may provide a compound of Formula (NP-1)
  • x is an integer of 0 to 10 (e.g., 4), and wherein the silicon atom is a part of a nanoparticle.
  • x may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • x is 4.
  • the efficiency of the reaction between the second bifunctional precursor (e.g., a DBCO-containing precursor, e.g., DBCO-maleimide) and the functionalized nanoparticle (e.g., nanoparticle comprising a diene moiety) may be independent of the diffusion coefficient of the second bifunctional precursor.
  • second bifunctional precursors disclosed herein that comprise a PEG group of varying length may have similar reactivities with a functionalized nanoparticle, regardless of PEG length (see e.g., Example 4 and FIG. 2 , where employing DBCO-PEG-maleimide precursors with varying lengths of PEG did not significantly affect reactivity).
  • bifunctional precursors disclosed herein may also have a particular solubility or polarity that can avoid undesired electrostatic interactions (e.g., repulsion) with silanol groups, e.g., silanol groups on another precursor or on a nanoparticle.
  • the bifunctional precursors e.g., second bifunctional precursor
  • the method disclosed herein may be used to provide nanoparticles suitable for a particular application, such as a therapeutic application.
  • the method can be used to provide nanoparticles suitable for targeting a biological target, e.g., for use in therapy (e.g., cancer therapy), surgical navigation, imaging, diagnostics, or a combination thereof.
  • the nanoparticles may be suitable for in vivo use, or can be used ex vivo e.g., for analytical or diagnostic applications.
  • the method disclosed herein may be used to make a nanoparticle suitable for targeting a particular receptor (or group of receptors) in a biological system.
  • the method may also be used to prepare a functionalized nanoparticle (e.g., NDC) suitable for delivering a payload to a biological target (or targets).
  • a nanoparticle can be conjugated with a targeting ligand, and with a payload moiety.
  • the methods disclosed herein can be used to prepare a folate-receptor targeting NDC, e.g., for targeted cancer therapy, such as the exemplary NDC depicted in FIG. 1 .
  • Reacting a nanoparticle with the second bifunctional precursor can provide a nanoparticle comprising one or more groups suitable for conjugation with a molecule (e.g., a targeting ligand and/or payload).
  • a molecule e.g., a targeting ligand and/or payload
  • reacting the nanoparticle with a second bifunctional precursor comprising an alkyne group can provide a nanoparticle functionalized with one or more alkyne groups, wherein the alkyne groups may be further conjugated with a compound comprising an alkyne-reactive group.
  • any desired compound comprising an a reactive group may be attached to a nanoparticle disclosed herein that is functionalized with the appropriate counterpart reactive group.
  • any desired compound comprising an alkyne-reactive group may be attached to the nanoparticle functionalized with an alkyne.
  • a payload moiety e.g., comprising a cytotoxic drug, e.g., exatecan
  • linker-payload conjugate that comprises an alkyne-reactive group
  • a targeting ligand e.g., a folate receptor (FR)-targeting ligand, e.g., folic acid
  • FR folate receptor
  • any combination of compounds may be conjugated to the nanoparticle using this method.
  • a targeting ligand and a payload moiety can be conjugated to the nanoparticle.
  • Other molecules, such as labels (e.g., radiolabels or dyes), polymers, and/or macromolecules can also be conjugated to a nanoparticle using this method.
  • a nanoparticle comprising an alkyne moiety (e.g., prepared using a method disclosed herein) can be contacted with a compound comprising an alkyne-reactive group (e.g., an azide, a diene, a nitrone, or a nitrile oxide) under conditions suitable for a reaction between the alkyne moiety and the alkyne-reactive group, thereby forming a nanoparticle functionalized with the compound (e.g., a compound comprising a payload or a targeting ligand).
  • an alkyne-reactive group e.g., an azide, a diene, a nitrone, or a nitrile oxide
  • the conditions suitable for a reaction between the alkyne moiety and the alkyne-reactive group may comprise any suitable click chemistry conditions (see, e.g., Kolb et al. Angew. Chem. Int. Ed . (2001) 40:2004-2021).
  • facilitating a reaction between the alkyne moiety and the alkyne-reactive group may comprise combining the nanoparticle comprising an alkyne moiety and the compound comprising an alkyne-reactive group with a catalyst, such as a copper catalyst (e.g., CuSO 4 , CuI, CuCl, Cu(OAc) 2 , CuSO 2 , CuBr).
  • a catalyst such as a copper catalyst (e.g., CuSO 4 , CuI, CuCl, Cu(OAc) 2 , CuSO 2 , CuBr).
  • ruthenium catalyst Other metal catalysts known to effect click chemistry reactions may also be used, such as a ruthenium catalyst.
  • the reaction may be carried out without a catalyst (i.e., catalyst-free conditions, e.g., copper-free conditions).
  • catalyst-free conditions e.g., copper-free conditions.
  • the alkyne on the functionalized nanoparticle may be suitable for catalyst-free click chemistry reactions (e.g., strain-promoted cycloaddition).
  • DBCO can be used in a click chemistry reaction without a catalyst.
  • the conditions suitable for a reaction between the alkyne moiety and the alkyne-reactive group may comprise combining the nanoparticle comprising an alkyne moiety and the compound comprising an alkyne-reactive group in an aqueous reaction medium (e.g., a reaction medium that is substantially water).
  • the reaction medium may comprise a buffer (e.g., an aqueous buffer), such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the reaction medium may comprise a buffer (e.g., an aqueous buffer, such as PBS) at a final concentration between about 1 ⁇ and about 5 ⁇ , e.g., about 1 ⁇ , about 2 ⁇ , about 3 ⁇ , about 4 ⁇ , or about 5 ⁇ .
  • the reaction medium may also comprise an aprotic organic solvent, such as DMSO or acetonitrile).
  • the reaction medium may comprise no more than 20 v/v % of the aprotic solvent, e.g., less than 18 v/v %, less than 16 v/v %, less than 14 v/v %, less than 12 v/v %, less than 10 v/v %, less than 8 v/v %, less than 6 v/v %, less than 4 v/v %, less than 2 v/v %, or less than 1 v/v % of the aprotic solvent.
  • the reaction medium may also comprise a protic organic solvent, such as an alcohol, e.g., tert-butanol).
  • the reaction medium may comprise no more than 20 v/v % of the protic solvent, e.g., less than 18 v/v %, less than 16 v/v %, less than 14 v/v %, less than 12 v/v %, less than 10 v/v %, less than 8 v/v %, less than 6 v/v %, less than 4 v/v %, less than 2 v/v %, or less than 1 v/v % of the protic solvent.
  • the reaction medium may be substantially free of an organic solvent (e.g., substantially free of organic protic solvent, substantially free of aprotic solvent, or substantially free of any organic solvent).
  • the reaction medium may be substantially water.
  • the conditions suitable for a reaction between the alkyne moiety and the alkyne-reactive group may comprise combining the nanoparticle comprising an alkyne moiety and the compound comprising an alkyne-reactive group in a reaction medium for a certain period of time, e.g., between about 1 minute and about 60 minutes, between about 0.5 hours and about 24 hours, between about 0.5 hours and about 18 hours, between about 0.5 hours and about 12 hours, between about 0.5 hours and about 6 hours, between about 0.5 hours and about 4 hours, between about 0.5 hours and about 3 hours, between about 0.5 hours and about 2 hours, between about 0.5 hours and about 1 hours, or between about 1 hour and about 48 hours.
  • the reaction medium may be held overnight.
  • the conditions suitable for a reaction between the alkyne moiety and the alkyne-reactive group may comprise combining the nanoparticle comprising an alkyne moiety and the compound comprising an alkyne-reactive group in a reaction medium, and heating the reaction medium, e.g., to a temperature of about 20° C. or greater, e.g., about 35° C. or greater, about 40° C. or greater, about 45° C. or greater, about 50° C. or greater, about 60° C. or greater, about 70° C. or greater, about 80° C. or greater, about 90° C. or greater, about 100° C. or greater, about 110° C. or greater, or about 120° C.
  • reaction conditions may comprise maintaining the reaction mixture at room temperature.
  • the conditions suitable for a reaction between first reactive group and the moiety reactive with the first reactive group can also comprise stirring, shaking, or applying other mixing methods to the reaction medium.
  • the compound comprising a reactive group may comprise a targeting ligand, and may further comprise a linker (e.g., a non-cleavable linker).
  • the targeting ligand can be a folate receptor targeting ligand.
  • the targeting ligand may be a molecule that can bind to a folate receptor, e.g., a folate receptor expressed on a cancer cell or tumor.
  • the folate-receptor (FR)-targeting ligand may be folic acid, dihydrofolic acid, tetrahydrofolic acid, or a folate-receptor binding derivative thereof.
  • the folate-receptor targeting ligand can be a macromolecule, such as a protein, a peptide, an aptamer, an antibody, or an antibody fragment that can target a folate receptor.
  • the folate-receptor targeting ligand may include a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody.
  • antigen binding fragment of an antibody may be wholly or partially synthetically produced.
  • the linker can be any suitable divalent linker, such as an alkylene or heteroalkylene group.
  • the divalent linker may be a PEG group (e.g., a PEG1, PEG2, PEG3, or PEG4, group).
  • the compound comprising a reactive group may comprise a structure of Formula (D′):
  • J is a reactive group, e.g., an alkyne-reactive group; e.g., an azide, a diene, a nitrone, or a nitrile oxide);
  • T is a targeting ligand (e.g., a folate receptor-targeting ligand, e.g., folic acid or a derivative thereof);
  • y is an integer of 0 to 20 (e.g., 3).
  • y may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 2, 3, or 4.
  • the compound comprising an alkyne-reactive group may comprise a structure of Formula (D):
  • T is a targeting ligand (e.g., a folate receptor-targeting ligand, e.g., folic acid or a derivative thereof); and y is an integer of 0 to 20 (e.g., 3).
  • y may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 2, 3, or 4.
  • the compound comprising a reactive group may comprise a structure of Formula (D-1′):
  • J is a reactive group (e.g., an alkyne-reactive group; e.g., an azide, a diene, a nitrone, or a nitrile oxide);
  • y is an integer of 0 to 20 (e.g., 3).
  • y may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 2, 3, or 4.
  • the compound comprising an alkyne reactive group may comprise a structure of Formula (D-1):
  • y is an integer of 0 to 20 (e.g., 3).
  • y may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 2, 3, or 4.
  • the compound comprising a reactive group may comprise a payload moiety, and may further comprise a linker (e.g., a cleavable linker).
  • the payload moiety can be a molecule with a therapeutic use, such as for use in cancer therapy.
  • the payload moiety can be a cytotoxic compound, e.g., a small molecule with cytotoxic properties.
  • Exemplary payloads include enzyme inhibitors (see e.g., “A Review of Evaluation of Enzyme Inhibitors in Drug Discovery,” Robert A. Copeland, John Wiley and Sons, Hoboken, N.J.), such as topoisomerase inhibitors (e.g., exatecan, SN-38, topotecan, irinotecan, camptothecin, belotecan, indenoisoquinoline, phenanthridines, indolocarbazoles, and analogs thereof), dihydrofolate reductase inhibitors, thymidylate synthase inhibitors, DNA intercalators, DNA minor groove binders, tubulin disruptors, DNA cleavers, anthracyclines, vinca drugs, mitomycins (e.g., mitomycin-C, mitomycin-A), bleomycins, cytotoxic nucleosides, pteridines, diynenes, enediynes, podophyllo
  • the payload may be exatecan, SN-38, topotecan, irinotecan, belotecan, 9-amino camptothecin, etoposide, camptothecin, taxol, esperamicin, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4-9-diene-2,6-diyne-13-one, podophyllotoxin, anguidine, vincristine, vinblastine, duocarmycin, a pyrrolobenzodiazepine, morpholine-doxorubicin, N-(5,5-diacetoxy-pentyl) doxorubicin, a compound described in U.S. Pat. No.
  • a functional group e.g., amine, hydroxyl, or sulfhydryl
  • amine, hydroxyl, or sulfhydryl may be appended to the drug at a position which has minimal or an acceptable effect on the activity or other properties of the drug.
  • the linker may be any suitable divalent linker, and can comprise a portion that is cleavable under certain conditions.
  • the linker can be a self-immolative linker capable of releasing the payload in vitro or in vivo under certain conditions.
  • the linker may be a protease-cleavable linker (e.g., cleavable by a protease such as cathepsin or trypsin), a pH-sensitive linker, or a redox-sensitive linker.
  • the protease-cleavable linker can be a cathepsin B (Cat-B)-cleavable linker.
  • the linker may comprise a peptide moiety (e.g., a dipeptide), a PEG spacer, and/or a self-immolative moiety (e.g., p-aminobenzyloxycarbonyl (PABC)), e.g., for effective cleavage in a cell, such as a cancer cell.
  • a peptide moiety e.g., a dipeptide
  • PEG spacer e.g., a PEG spacer
  • a self-immolative moiety e.g., p-aminobenzyloxycarbonyl (PABC)
  • PABC p-aminobenzyloxycarbonyl
  • the cleavable linker may be selectively cleaved at a desired location or after a selected time (e.g., upon entry into a targeted cancer cell).
  • the compound comprising a reactive group may comprise a structure of Formula (E′):
  • the compound comprising an alkyne reactive group may comprise a structure of Formula (E):
  • L is a cleavable linker moiety (e.g., a protease-cleavable linker moiety); P is a payload moiety (e.g., a cytotoxic drug, e.g., exatecan); and y is an integer of 0 to 20 (e.g., 9).
  • L may be a protease-cleavable linker moiety
  • P may be exatecan
  • y may be an integer of 0 to 20 (e.g., 7, 8, 9, 10, or 11).
  • the cleavable linker moiety can comprises a structure of Formula (F).
  • each instance of [AA] is a natural or non-natural amino acid residue; z is an integer of 1 to 5; w is an integer of 1 to 4 (e.g., 2 or 3); and each denotes a point of attachment to another portion of the compound comprising a reactive group (e.g., an alkyne-reactive group; e.g., an attachment to a PEG group, an attachment to an alkyne-reactive group, or an attachment to a payload moiety).
  • a reactive group e.g., an alkyne-reactive group; e.g., an attachment to a PEG group, an attachment to an alkyne-reactive group, or an attachment to a payload moiety.
  • -[AA] w - may comprise Val-Lys, Val-Cit, Phe-Lys, Trp-Lys, Asp-Lys, Val-Arg, or Val-Ala, and z may be 2, wherein one denotes a point of attachment to the oxygen atom of a PEG group, and the other denotes a point of attachment to the nitrogen atom of exatecan.
  • the cleavable linker moiety can comprises a structure of Formula (F-1):
  • one denotes a point of attachment to another portion of the compound comprising a reactive group (e.g., an alkyne-reactive group; e.g., an attachment to a PEG group, an attachment to an alkyne-reactive group, or an attachment to a payload moiety).
  • a reactive group e.g., an alkyne-reactive group; e.g., an attachment to a PEG group, an attachment to an alkyne-reactive group, or an attachment to a payload moiety.
  • a reactive group e.g., an alkyne-reactive group
  • an attachment to a PEG group e.g., an attachment to a PEG group
  • an attachment to an alkyne-reactive group e.g., an attachment to a PEG group
  • an attachment to an alkyne-reactive group e.g., an attachment to a payload moiety
  • the compound comprising a reactive group may comprise a structure of Formula (E-1′):
  • J is a reactive group (e.g., an alkyne-reactive group; e.g., an azide, a diene, a nitrone, or a nitrile oxide); and y is an integer of 0 to 20 (e.g., 9).
  • a reactive group e.g., an alkyne-reactive group; e.g., an azide, a diene, a nitrone, or a nitrile oxide
  • y is an integer of 0 to 20 (e.g., 9).
  • the compound comprising an alkyne reactive group may comprise a structure of Formula (E-1):
  • y is an integer of 0 to 20 (e.g., 9).
  • Reacting an alkyne moiety e.g., an alkyne covalently attached to a nanoparticle
  • a compound comprising an alkyne-reactive group e.g., a compound of Formula D, e.g., D-1
  • NP-2 a compound of Formula (NP-2):
  • x is an integer of 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, e.g., 4), and y is an integer of 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 3), and the silicon atom is a part of the nanoparticle (e.g., bonded with the silica shell of a core-shell silica nanoparticle).
  • x is an integer of 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 4), and y is an integer of 0 to 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 9), and the silicon atom is a part of the nanoparticle (e.g., bonded with the silica shell of a core-shell silica nanoparticle).
  • NDC An exemplary NDC comprising a payload (exatecan) linked via a protease-cleavable linker, and the mechanism of cleavage of the linker and release of the payload, is depicted in Scheme 1.
  • an NDC produced using a method disclosed herein may have an average nanoparticle to payload (e.g., exatecan or a salt or analog thereof) ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:32, 1:34, 1:36, 1:38, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, or 1:80.
  • an average nanoparticle to payload e.g., exatecan or a salt or analog thereof
  • the average number of exatecan molecules on each nanoparticle may be between about 5 and about 10, between about 10 and about 15, between about 15 and about 20, between about 20 and about 25, or between about 25 and about 30, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 exatecan molecules per nanoparticle.
  • An NDC produced using a method disclosed herein may have an average nanoparticle to targeting ligand (e.g., folic acid) ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, or 1:30.
  • ligand e.g., folic acid
  • the nanoparticle can be functionalized with a first bifunctional precursor, followed by conjugation to a second bifunctional precursor comprising an alkyne-reactive group, thereby providing a nanoparticle functionalized with an alkyne-reactive group (e.g., azide, a diene, a nitrone, or a nitrile oxide). Then, compounds (e.g., targeting ligands or payloads) comprising an alkyne may be reacted with the nanoparticle to conjugate the compounds to the nanoparticle.
  • a first bifunctional precursor followed by conjugation to a second bifunctional precursor comprising an alkyne-reactive group, thereby providing a nanoparticle functionalized with an alkyne-reactive group (e.g., azide, a diene, a nitrone, or a nitrile oxide).
  • compounds e.g., targeting ligands or payloads
  • Functionalized nanoparticles produced using methods disclosed herein can exhibit uniform morphology and narrow size distribution, e.g., as measured by transmission electron microscopy (TEM).
  • the nanoparticles may be characterized by TEM, or UV spectroscopy, e.g., to identify absorption peaks characteristic of a dye or ligand in or on the nanoparticle.
  • the purity of the nanoparticles can be assessed using chromatography, e.g., reversed-phase high performance liquid chromatography (RP-HPLC) and size-exclusion chromatography.
  • RP-HPLC reversed-phase high performance liquid chromatography
  • the methods disclosed herein are also capable of providing highly consistent nanoparticle characteristics across various reaction scales, and batch-to-batch.
  • protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions.
  • suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in Greene et al. Protecting Groups in Organic Synthesis , Second Edition, Wiley, New York, 1991, and references cited therein.
  • the (3-mercaptopropyl)trimethoxysilane, 2-[methoxy(polyethyleneoxy)6-9propyl]trimethoxysilane, and (3-cyclopentadienylpropyl)triethoxysilane precursors were purchased from Gelest. 10 ⁇ PBS was purchased from Thermo Fisher Scientific. Human cathepsin-B was purchased from Millipore Sigma. Human and mouse serum were purchased from BIOIVT (New York). All other chemicals were purchased from Millipore Sigma. Cy5-C'Dot was prepared following the protocol described in Ma, K. et al. Chemistry of Materials (2015) 27(11):4119-4133, incorporated herein by reference in its entirety.
  • a 633 nm solid-state laser was used to excite the Cy5 dye encapsulated in the silica core of C'Dots.
  • RP-HPLC coupled to a photodiode array (PDA) detector was used to assess the purity of nanoparticle conjugates (including FA-CDC), using a commercially available Waters Xbridge Peptide BEH C18 column (50 ⁇ 4.6 mm).
  • the mobile phase for the separation is a gradient elution starting at a composition of 85% A (0.1 vol % trifluoroacetic acid in deionized water)/15% B (acetonitrile) and the composition is linearly changed to 5% A/95% B over a course of 15 minutes.
  • RP-HPLC separates molecules with different polarities and is suitable as an analytical method for FA-CDC because of its ultrasmall sub-10 nm particle size.
  • the nanoparticles are well separated from other chemical moieties such as ligands that are non-covalently associated with the nanoparticles and degraded or force released components.
  • Different chemical moieties are identified based on elution time and unique UV/Visible spectra collected using the PDA detector.
  • the impurity content in a sample can be quantified using calibration curves developed with reference standard materials for key impurities.
  • SEC Size Exclusion Chromatography
  • Cy5-C'Dot was diluted with deionized water to a desired concentration, typically between 15 to 30 ⁇ M, in a round-bottom flask with a stir bar.
  • Silanol groups on the surface of the nanoparticle under the organic polymer coating are not easily accessible to bulky molecules, such as DBCO-PEG-silane.
  • the result is that when using DBCO-PEG-silane as a bifunctional precursor (e.g., as in Example 1), only one or two DBCO groups can be attached to each nanoparticle (e.g., C'Dot), even at a high reaction ratio of DBCO-PEG-silane to nanoparticle (see FIG. 2 ). Therefore, an alternative two-step reaction was designed, employing relatively small silane molecules with a high diffusion coefficient, depicted in Scheme 3.
  • Cy5-C'Dot was diluted with deionized water to a desired concentration, typically between 15 to 30 ⁇ M, in a round-bottom flask with a stir bar.
  • APTMS (3-Aminopropyl)triethoxysilane
  • DBCO-PEG4-NHS ester in DMSO was added to reach a desired particle to DBCO molar ratio, and to further conjugate to the amine groups on C'Dots via amine-ester reaction. The reaction was left at room temperature overnight, and then concentrated and purified using GPC.
  • this two-step reaction makes use of the fast diffusion of small silane molecules to enhance their reactivity to the silanol groups under the PEG layer, permitting more than 20 DBCO groups to be easily attached onto each C'Dot (see FIG. 2 ).
  • amine-based DBCO-C'Dots produced using this approach exhibited limited stability. For example, a considerable amount of DBCO detached from C'Dots after incubation in PBS at 37° C. overnight (see FIG. 3 A ), which may be due to the hydrolysis of the amide bonds between the DBCO groups and C'Dot, accelerated by residual primary amine groups on the C'Dot surface.
  • amine-silane molecules can self-condense during reaction due to the electrostatic attraction between their positively charged amine portion and negatively charged silanol portion. Amine-silane aggregates are difficult to remove from C'Dot solution by size-exclusion chromatography (SEC) (see FIG. 3 C ), and can lead to undesired downstream effects. For example, if administered, these aggregates may lead to kidney retention due to the positive charge of amine.
  • SEC size-exclusion chromatography
  • DBCO-Cy3 molecules were further conjugated to azide-functionalized C'Dots, attaching Cy3 to particles via click chemistry reaction between DBCO and azide; b The presence of alkyne groups on C'dot was verified by the specific absorbance peak of alkyne at 290 nm; c Obtained after optimization of synthesis protocol; d DBCO-Cy3 molecules were further conjugated to azide-functionalized C'Dots, attaching Cy3 to particles via click chemistry reaction between DBCO and azide.
  • Example 3 The use of a Diels-Alder reaction was identified in Example 3 as a suitable replacement for the amine-ester reaction.
  • the amine-silane used in the method described in Example 2 can be replaced with (3-cyclopentadienylpropyl)triethoxysilane (“diene-silane”) to first functionalize C'Dots with cyclopentadiene groups
  • DBCO-PEG-NHS ester used in the method of Example 2 can be replaced with a DBCO-PEG-maleimide.
  • Cy5-C'Dot was diluted with deionized water to a desired concentration, typically between 15 to 30 ⁇ M, in a round-bottom flask with a stir bar.
  • 3-Cyclopentadienylpropyl)triethoxysilane (cyclopentadiene) was first diluted 100 ⁇ in DMSO and then added into the reaction under stirring, to reach a desired particle to cyclopentadiene molar ratio. After overnight reaction, 10 ⁇ PBS was added into the reaction to achieve a final concentration of 1 ⁇ PBS.
  • DBCO-maleimide precursor (DBCO-mal, DBCO-PEG4-mal, DBCO-PEG12-mal, or DBCO-sulfo-mal) was dissolved in DMSO and added into the reaction to reach a desired particle to DBCO molar ratio. After mixing for about 30 min to 1 hour, the reaction mixture was heated to 80° C. while stirring overnight. The reaction solution was then concentrated and purified using gel permeation chromatography (GPC) to obtain diene-based DBCO-C'Dot.
  • GPC gel permeation chromatography
  • the neutral charge of the cyclopentadiene groups averts hydrolysis of the amide bonds in the linkage, that can be accelerated by the primary amine on C'Dots when using an amine-silane as a precursor.
  • the C'Dots produced using this method are highly stable (see FIG. 3 B ).
  • using cyclopentadiene groups greatly diminishes the self-condensation of silane during the reaction (see FIG. 3 D ), and improves the stability, size homogeneity, reaction yield, and purity of the functionalized nanoparticles (e.g., DBCO-C'Dots).
  • the reaction efficiency of the DBCO-containing precursor is independent of its diffusion coefficient. For example, even when DBCO-maleimide precursors with varying PEG spacer lengths and molecular weights were employed in functionalizing C'Dots, the number of DBCO groups attached to each of the resulting C'Dots remained consistent (see FIG. 2 ). This is a departure from typical methods involving other bifunctional precursors, where the reaction efficiency can be greatly influenced by the molar mass of silanes.
  • NDCs comprising an alkyne moiety e.g., DBCO
  • DBCO alkyne moiety
  • a compound comprising a reactive group e.g., an alkyne-reactive group (e.g., an azide).
  • These compounds can include payload-linker conjugate precursors, e.g., as described in Synthetic Example 1, and/or folate targeting ligand precursors, e.g., as described in Synthetic Example 2.
  • the DBCO-C'Dots produced by the methods disclosed herein were conjugated with azide-functionalized folic acid-linker conjugates (suitable as a targeting ligand for targeting cancers expressing folate receptor (FR)), and azide-functionalized exatecan-linker conjugates (exatecan is a topoisomerase I inhibitor drug, suitable for treating cancer).
  • the folic acid moiety was attached through a non-cleavable linker, while the exatecan moiety was attached through a cathepsin-B (Cat-B) cleavable linker (Cat-B is an enzyme present in cell lysosomes and overexpressed in a variety of malignancies).
  • DBCO-C'Dot was diluted with deionized water to a desired concentration, typically between 15 to 30 ⁇ M, in a round-bottom flask with a stir bar.
  • a desired concentration typically between 15 to 30 ⁇ M
  • folate-PEG-azide was dissolved in DMSO and added into the reaction under stirring, to achieve a desired number of ligands per particle.
  • the reaction was left at room temperature overnight, and the conversion rate was checked by in-process HPLC purity test (typically above 95%). If the purity was below 95%, the reaction solution would be concentrated and purified using GPC.
  • the folic acid functionalized C'Dot was isolated as an intermediate to determine the number of folic acid ligands per particle using UV-Vis.
  • Cleavable linker-exatecan conjugate was dissolved in DMSO and added into the particle solution under stirring, to achieve a desired number of payloads per particle. The reaction was left at room temperature overnight, and then reaction solution was concentrated and purified using GPC to obtain FA-CDC.
  • the resulting targeted-NDCs exhibited uniform morphology and narrow size distribution under transmission electron microscopy (TEM) with an average core size of 3.9 nm.
  • the average hydrodynamic diameter of the NDCs was determined by fluorescence correlation spectroscopy (FCS) to be 6.4 nm ( FIG. 4 ), consistent with TEM as the 2.5 nm difference is attributed to the organic PEG layer that cannot be observed in TEM.
  • the final purified NDCs had a purity above 99.0% as characterized by both reversed-phase high performance liquid chromatography (RP-HPLC) and SEC ( FIGS. 6 A- 6 ). These quality attributes of NDCs were demonstrated to be highly consistent from batch to batch across various reaction scales (Table 2).
  • the UV-Vis spectrum of folic acid functionalized C'Dot before attaching exatecan payloads was used to determine the concentration of the particles and the concentration of FR-targeting ligands by application of the Beer-Lambert Law at 647 nm (corresponding to covalently encapsulated Cy5) and 360 nm (corresponding to folic acid targeting ligands), respectively.
  • the number of FR-targeting ligands per particle was determined by dividing the molar concentration of FR-targeting ligands by the particle concentration. The number of FR-targeting ligands have been confirmed to remain unchanged in subsequent processing to produce FA-CDC.
  • the UV-Vis spectrum of FA-CDC can be used to determine the concentration of FA-CDC particles, as well as the concentration of cleavable exatecan by application of the Beer-Lambert Law at 647 nm and 360 nm (corresponding to both folic targeting ligands and exatecan) after subtraction of the absorbance of FR-targeting ligands at 360 nm. The concentration of exatecan was then divided by the concentration of FA-CDC particles to determine the number of exatecan per particle.
  • NDCs produced herein were assessed after incubation in 0.9% saline, PBS, human plasma (10%), and mouse plasma (10%) at 37° C. in a shaking dry bath for different time periods.
  • plasma proteins in the samples were removed by precipitation, through addition of an equivalent volume of cold acetonitrile, followed by centrifugation at 10000 rpm in an Eppendorf 5425 microcentrifuge. Following centrifugation, the clear supernatant was transferred from the centrifuge tube into a clear total recovery HPLC vial. The supernatant free of any visible aggregation was diluted with an equivalent volume of deionized water to adjust the sample matrix to match the starting conditions of the HPLC separation and avoid loss of sensitivity. The purity and impurity of each sample is then quantified by RP-HPLC as described above. Stability in human and mouse serum of NDCs produced by a method of Example 2 and Example 4 are provided in FIGS. 8 A- 8 B .
  • Nanoparticle conjugate samples (e.g., FA-CDC) were prepared for forced release by first diluting an aliquot of the FA-CDC sample to 2 ⁇ M concentration and then incubating with activated recombinant human Cathepsin-B in a shaking dry bath at 37° C.
  • Recombinant human Cathepsin-B was prepared as follows: 4 ⁇ L of 0.33 ⁇ g/ ⁇ L Cathepsin-B was added to an activation buffer consisting of dithiothreitol and (MES) and adjusted to pH 5.0. Appropriate pH was confirmed with a pH test strip after preparation. After 24 hours of incubation, the force released free exatecan is determined by the percentage of the integrated area that is present at the free exatecan elution time in RP-HPLC. The results of this study are provided in FIG. 7 .
  • exatecan drugs More than 95% of exatecan drugs remain on the NDCs for up to 7 days in mouse and human plasma, obtained by the UV-Vis spectra of the NDC peaks in RP-HPLC chromatograms. Meanwhile, an independent RP-HPLC assay monitoring free exatecan suggested that the released exatecan was below detection limit of RP-HPLC, i.e., 0.02%, and the absence of non-desired free drug further demonstrates their high plasma stability.
  • the targeted-NDCs also exhibited high storage stability at 4° C. in 0.9% saline. Their purity, size distribution, and hydrodynamic diameter were characterized by RP-HPLC, SEC and FCS respectively, and remained unchanged over 6 months under storage condition. Such high storage stability is another key parameter important for both clinical translation and commercial manufacture.
  • Linker-payload conjugate precursors suitable for use in the methods disclosed herein can be synthesized according to the following exemplary protocols.
  • reaction mixture was quenched with water (15 mL) and extracted with 10% methanol in chloroform (2 ⁇ 20 mL). The combined organic layers were dried over anhydrous sodium sulfate (Na 2 SO 4 ) and concentrated under reduced pressure.
  • Targeting ligand precursors suitable for use in the methods disclosed herein can be prepared according to the following exemplary synthetic protocols.
US17/867,030 2020-10-27 2022-07-18 Methods of functionalizing nanoparticles Pending US20230021059A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/867,030 US20230021059A1 (en) 2020-10-27 2022-07-18 Methods of functionalizing nanoparticles

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
US202063105995P 2020-10-27 2020-10-27
US202063116393P 2020-11-20 2020-11-20
US202063117110P 2020-11-23 2020-11-23
US202163155043P 2021-03-01 2021-03-01
US202163222181P 2021-07-15 2021-07-15
US202163242201P 2021-09-09 2021-09-09
US202163254837P 2021-10-12 2021-10-12
PCT/US2021/056611 WO2022093794A1 (en) 2020-10-27 2021-10-26 Methods of functionalizing nanoparticles
US17/867,030 US20230021059A1 (en) 2020-10-27 2022-07-18 Methods of functionalizing nanoparticles

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/056611 Continuation WO2022093794A1 (en) 2020-10-27 2021-10-26 Methods of functionalizing nanoparticles

Publications (1)

Publication Number Publication Date
US20230021059A1 true US20230021059A1 (en) 2023-01-19

Family

ID=78725635

Family Applications (5)

Application Number Title Priority Date Filing Date
US17/867,025 Active US11744897B2 (en) 2020-10-27 2022-07-18 Folate receptor targeted nanoparticle drug conjugates and uses thereof
US17/867,030 Pending US20230021059A1 (en) 2020-10-27 2022-07-18 Methods of functionalizing nanoparticles
US18/183,305 Pending US20230263898A1 (en) 2020-10-27 2023-03-14 Folate receptor targeted nanoparticle drug conjugates and uses thereof
US18/183,328 Active US11957760B2 (en) 2020-10-27 2023-03-14 Folate receptor targeted nanoparticle drug conjugates and uses thereof
US18/297,200 Pending US20230241243A1 (en) 2020-10-27 2023-04-07 Carrier particle-drug conjugates, self-immolative linkers, and uses thereof

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US17/867,025 Active US11744897B2 (en) 2020-10-27 2022-07-18 Folate receptor targeted nanoparticle drug conjugates and uses thereof

Family Applications After (3)

Application Number Title Priority Date Filing Date
US18/183,305 Pending US20230263898A1 (en) 2020-10-27 2023-03-14 Folate receptor targeted nanoparticle drug conjugates and uses thereof
US18/183,328 Active US11957760B2 (en) 2020-10-27 2023-03-14 Folate receptor targeted nanoparticle drug conjugates and uses thereof
US18/297,200 Pending US20230241243A1 (en) 2020-10-27 2023-04-07 Carrier particle-drug conjugates, self-immolative linkers, and uses thereof

Country Status (10)

Country Link
US (5) US11744897B2 (ja)
EP (5) EP4106820B1 (ja)
JP (4) JP2023516111A (ja)
KR (2) KR20230097087A (ja)
AU (2) AU2021369485A1 (ja)
CA (1) CA3195153A1 (ja)
DK (1) DK4103236T3 (ja)
ES (2) ES2963150T3 (ja)
FI (1) FI4103236T3 (ja)
WO (3) WO2022093800A2 (ja)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11744897B2 (en) 2020-10-27 2023-09-05 Elucida Oncology, Inc. Folate receptor targeted nanoparticle drug conjugates and uses thereof

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024003002A1 (en) 2022-06-27 2024-01-04 Diaccurate N-substituted indole derivatives and conjugates for the treatment of cancer
CN115028551B (zh) * 2022-07-15 2024-01-05 成都普康生物科技有限公司 一种叠氮-九甘醇-丙酸的制备方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100179075A1 (en) * 2008-07-30 2010-07-15 Life Technologies Corporation Particles for use in supported nucleic acid ligation and detection sequencing

Family Cites Families (77)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US1002942A (en) 1910-08-29 1911-09-12 Edward Hiram Taylor Hair-ironing comb.
US3870791A (en) 1972-04-24 1975-03-11 Heskel M Haddad Solid state ophthalmic medication delivery method
US3867519A (en) 1972-04-27 1975-02-18 Alza Corp Bioerodible drug delivery device
US4051842A (en) 1975-09-15 1977-10-04 International Medical Corporation Electrode and interfacing pad for electrical physiological systems
DE2626348C3 (de) 1976-06-11 1980-01-31 Siemens Ag, 1000 Berlin Und 8000 Muenchen Implantierbare Dosiereinrichtung
US4136177A (en) 1977-01-31 1979-01-23 American Home Products Corp. Xanthan gum therapeutic compositions
US4255415A (en) 1978-11-22 1981-03-10 Schering Corporation Polyvinyl alcohol ophthalmic gel
US4383529A (en) 1980-11-03 1983-05-17 Wescor, Inc. Iontophoretic electrode device, method and gel insert
US4931279A (en) 1985-08-16 1990-06-05 Bausch & Lomb Incorporated Sustained release formulation containing an ion-exchange resin
US4668506A (en) 1985-08-16 1987-05-26 Bausch & Lomb Incorporated Sustained-release formulation containing and amino acid polymer
US4788603A (en) 1985-10-19 1988-11-29 Fuji Photo Film Co., Ltd. Camera for sequentially photographing a subject using a reference optical system and a telescopic optical system
US4713224A (en) 1986-03-31 1987-12-15 The Boc Group, Inc. One-step process for purifying an inert gas
US5198560A (en) 1990-04-27 1993-03-30 Bristol-Myers Squibb Company Cytotoxic bicyclo[7.3.1]tridec-4-ene-2,6-diyne compounds and process for the preparation thereof
US6214345B1 (en) 1993-05-14 2001-04-10 Bristol-Myers Squibb Co. Lysosomal enzyme-cleavable antitumor drug conjugates
GB2330907A (en) 1997-10-28 1999-05-05 Applied Imaging Int Ltd A karyotyper and methods for producing karyotypes
EP2343047B2 (en) * 2000-02-08 2021-03-17 Rice University Optically-active nanoparticles for use in therapeutic and diagnostic methods
US6548264B1 (en) 2000-05-17 2003-04-15 University Of Florida Coated nanoparticles
EP1243276A1 (en) 2001-03-23 2002-09-25 Franciscus Marinus Hendrikus De Groot Elongated and multiple spacers containing activatible prodrugs
US20040101822A1 (en) 2002-11-26 2004-05-27 Ulrich Wiesner Fluorescent silica-based nanoparticles
ES2395082T3 (es) 2003-01-27 2013-02-08 Endocyte, Inc. Conjugado de folato-vinblastina como medicamento
KR101520209B1 (ko) 2003-11-06 2015-05-13 시애틀 지네틱스, 인크. 리간드에 접합될 수 있는 모노메틸발린 화합물
WO2005082023A2 (en) 2004-02-23 2005-09-09 Genentech, Inc. Heterocyclic self-immolative linkers and conjugates
US8084001B2 (en) 2005-05-02 2011-12-27 Cornell Research Foundation, Inc. Photoluminescent silica-based sensors and methods of use
US9913917B2 (en) 2005-12-22 2018-03-13 Visen Medical, Inc. Biocompatible fluorescent metal oxide nanoparticles
EP1995327A1 (en) 2007-05-21 2008-11-26 Humboldt Universität zu Berlin Probe for detecting a particular nucleic acid sequence
US20110028662A1 (en) 2007-08-31 2011-02-03 Hybrid Silica Technologies, Inc. Peg-coated core-shell silica nanoparticles and methods of manufacture and use
DE102007052517A1 (de) 2007-10-29 2009-04-30 Autoimmun Diagnostika Gmbh ELISPOT-Verfahren mit zwei Filtersystemen
PT2281006T (pt) 2008-04-30 2017-12-06 Immunogen Inc Agentes de reticulação e as suas utilizações
EP2349274A4 (en) 2008-09-17 2014-12-17 Endocyte Inc CONJUGATES OF ANTIFOLATES BINDING THE FOLATE RECEPTOR
CN101721372B (zh) * 2008-10-10 2012-02-01 陈东 金壳包覆的中空介孔二氧化硅球及其制备方法和在肿瘤治疗方面的用途
CN102459062B (zh) 2009-04-15 2017-03-22 康奈尔大学 通过二氧化硅致密化改进的荧光二氧化硅纳米颗粒
ES2639310T3 (es) 2009-07-02 2017-10-26 Sloan-Kettering Institute For Cancer Research Nanopartículas fluorescentes basadas en sílice
WO2011042564A1 (en) 2009-10-09 2011-04-14 Universite De Strasbourg Labelled silica-based nanomaterial with enhanced properties and uses thereof
RU2582235C2 (ru) 2010-12-29 2016-04-20 Ф.Хоффманн-Ля Рош Аг Низкомолекулярные конъюгаты для внутриклеточной доставки нуклеиновых кислот
US9011735B2 (en) 2010-12-30 2015-04-21 Ut-Battelle, Llc Volume-labeled nanoparticles and methods of preparation
EP2634179A1 (en) 2012-02-28 2013-09-04 Sanofi Functional PLA-PEG copolymers, the nanoparticles thereof, their preparation and use for targeted drug delivery and imaging
WO2013192609A1 (en) 2012-06-22 2013-12-27 Cornell University Mesoporous oxide nanoparticles and methods of making and using same
CN103566377A (zh) 2012-07-18 2014-02-12 上海博笛生物科技有限公司 癌症的靶向免疫治疗
US20160018404A1 (en) 2013-02-20 2016-01-21 Cornell University Multilayer fluorescent nanoparticles and methods of making and using same
WO2014145606A1 (en) 2013-03-15 2014-09-18 Sloan-Kettering Institute For Cancer Research Multimodal silica-based nanoparticles
HUE047329T2 (hu) * 2013-08-13 2020-04-28 Univ Northwestern Peptiddel konjugált részecskék
SI3038650T1 (sl) 2013-08-30 2021-11-30 Immunogen, Inc. Protitelesa in preiskave za odkrivanje folatnega receptorja 1
US10986997B2 (en) 2013-12-31 2021-04-27 Memorial Sloan Kettering Cancer Center Systems, methods, and apparatus for multichannel imaging of fluorescent sources in real time
DK3148591T3 (da) 2014-05-29 2020-04-14 Memorial Sloan Kettering Cancer Center Nanopartikel-lægemiddelkonjugater
WO2016100376A1 (en) * 2014-12-15 2016-06-23 The Regents Of The University Of California Nanotherapeutic for treating infections caused by intracellular and extracellular pathogens
BR112017012621A2 (pt) 2014-12-15 2018-01-02 Univ Cornell peptídeos cíclicos com seletividade de ligação nervosa melhorada, nanopartículas ligadas com os referidos peptídeos cíclicos, e o uso dos mesmos para a produção de imagens de tecido nervoso in vivo em tempo real
KR20180002645A (ko) 2015-04-07 2018-01-08 메모리얼 슬로안-케터링 캔서 센터 나노입자 면역컨쥬게이트
US11291737B2 (en) 2015-05-04 2022-04-05 Cornell University Ultrasmall nanoparticles and methods of making and using same
AU2016262107B2 (en) 2015-05-12 2021-01-21 Blinkbio, Inc. Silicon based drug conjugates and methods of using same
EP3302568B1 (en) 2015-05-29 2023-12-06 Memorial Sloan Kettering Cancer Center Methods of treatment using ultrasmall nanoparticles to induce cell death of nutrient-deprived cancer cells via ferroptosis
CZ307488B6 (cs) 2015-11-20 2018-10-10 Univerzita Karlova V Praze Sloučeniny na bázi cyklamu, jejich konjugáty, cílicí konjugáty, koordinační sloučeniny, farmaceutický přípravek je obsahující, způsob jejich přípravy a jejich použití
WO2017106525A1 (en) 2015-12-15 2017-06-22 Memorial Sloan Kettering Cancer Center Imaging systems and methods for tissue differentiation, e.g., for intraoperative visualization
WO2017156191A1 (en) 2016-03-08 2017-09-14 Los Gatos Pharmaceuticals, Inc. Composite nanoparticles and uses thereof
JP7455510B2 (ja) 2016-04-29 2024-03-26 メモリアル スローン－ケタリング キャンサー センター 悪性脳腫瘍における標的化粒子の浸透、分布および応答のための組成物及び方法
CN109475567A (zh) 2016-06-09 2019-03-15 布兰克生物股份有限公司 基于硅烷醇的治疗有效载荷
WO2018003739A1 (ja) 2016-06-30 2018-01-04 レナセラピューティクス株式会社 機能的リガンドを含む核酸複合体
CN109475279A (zh) 2016-07-07 2019-03-15 纪念斯隆凯特琳癌症中心 用于颗粒驱动的、基于知识的且预测性癌症放射基因组学的成像系统和方法
IL254053B (en) * 2016-08-19 2021-03-25 Univ Nat Taiwan Nanoparticles of hollow silica with thermalized biologically active components, a process for their preparation and their applications
ES2921236T3 (es) 2016-11-25 2022-08-22 Mabwell Shanghai Bioscience Co Ltd Enlazador de amida maleica disustituida para la conjugación de anticuerpo y fármaco y método de preparación y uso del mismo
TW201825121A (zh) 2016-11-30 2018-07-16 美國紀念斯隆-凱特琳癌症中心 抑制劑—功能化超微小奈米粒子及其方法
US20200155710A1 (en) 2017-04-10 2020-05-21 Cornell University Sulfur- or heavy atom-containing nanoparticles, methods of making same, and uses thereof
EP3624777A4 (en) 2017-05-19 2021-03-10 Cornell University FUNCTIONALIZED NANOPARTICLE AND METHOD FOR MANUFACTURING AND USING THEREOF
JP2020520953A (ja) 2017-05-25 2020-07-16 メモリアル スローン ケタリング キャンサー センター ジルコニウム−８９で標識した超小型ナノ粒子およびその方法
US20210145985A1 (en) 2017-06-23 2021-05-20 Memorial Sloan Kettering Cancer Center Method of imaging in vivo tissues using nanoparticles comprising a reference dye and a sensor dye
WO2019034176A1 (zh) 2017-08-18 2019-02-21 四川百利药业有限责任公司 一种喜树碱-抗体偶联物
EP3700577A1 (en) 2017-10-23 2020-09-02 Mablink Bioscience Ligand-drug-conjugate comprising a single molecular weight polysarcosine
JP2018070626A (ja) * 2017-11-24 2018-05-10 カリフォルニア インスティテュート オブ テクノロジー 標的化ナノ粒子
US20200383943A1 (en) 2017-12-04 2020-12-10 Memorial Sloan Kettering Cancer Center Methods of cancer treatment via regulated ferroptosis
CA3094313A1 (en) 2018-04-06 2019-10-10 Seattle Genetics, Inc. Camptothecin peptide conjugates
WO2019195858A1 (en) 2018-04-06 2019-10-10 Cornell University Inorganic nanocages, and methods of making and using same
WO2019213456A1 (en) 2018-05-02 2019-11-07 Cornell University Ultrasmall nanoparticles and methods of making, using and analyzing same
US11666411B2 (en) 2018-05-10 2023-06-06 Memorial Sloan Kettering Cancer Center Systems for augmented reality surgical and clinical visualization
CN111001012A (zh) 2018-10-19 2020-04-14 四川百利药业有限责任公司 一种亲水碳酸酯型抗体偶联药物
EP3956394A4 (en) 2019-04-15 2023-01-04 Cornell University FUNCTIONALIZED SILICA NANORINGS, PROCESS FOR THEIR MANUFACTURE AND USE
CA3138058A1 (en) 2019-05-20 2020-11-26 Matthew T. Burger Mcl-1 inhibitor antibody-drug conjugates and methods of use
WO2021092065A1 (en) 2019-11-04 2021-05-14 Cornell University Ultrasmall nanoparticles and methods of making, using and analyzing same
FI4103236T3 (fi) 2020-10-27 2023-11-09 Elucida Oncology Inc Folaattireseptoriin kohdennettuja nanopartikkelilääkekonjugaatteja sekä niiden käyttöjä

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100179075A1 (en) * 2008-07-30 2010-07-15 Life Technologies Corporation Particles for use in supported nucleic acid ligation and detection sequencing

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Chen et al. (Nat. Commun. 2018, 9, 1414, p1-11 and Supporting Information) *
Destito et al. (Chemistry & Biology 2007, 14, 1152-1162) *
Kettenback et al. (Med. Chem. Commun. 2016, 7, 654-657) *
Liu et al. (Biomacromolecules 2016, 17, 173-182) *
Ma et al. (Chem. Mater. 2017, 29, 6840-6855) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11744897B2 (en) 2020-10-27 2023-09-05 Elucida Oncology, Inc. Folate receptor targeted nanoparticle drug conjugates and uses thereof
US11957760B2 (en) 2020-10-27 2024-04-16 Elucida Oncology, Inc. Folate receptor targeted nanoparticle drug conjugates and uses thereof

Also Published As

Publication number Publication date
ES2963003T3 (es) 2024-03-22
DK4103236T3 (en) 2023-11-13
CA3195153A1 (en) 2022-05-05
US11957760B2 (en) 2024-04-16
US20220378924A1 (en) 2022-12-01
EP4106820B1 (en) 2023-08-16
EP4257156A3 (en) 2024-01-17
EP4275758A2 (en) 2023-11-15
KR20230097088A (ko) 2023-06-30
US20230263898A1 (en) 2023-08-24
AU2021369485A1 (en) 2023-06-08
EP4237008A2 (en) 2023-09-06
US20230263899A1 (en) 2023-08-24
EP4106820A1 (en) 2022-12-28
WO2022093793A1 (en) 2022-05-05
EP4257156A2 (en) 2023-10-11
JP2023153424A (ja) 2023-10-17
KR20230097087A (ko) 2023-06-30
AU2021370647A1 (en) 2023-06-08
EP4275758A3 (en) 2024-02-21
EP4103236A1 (en) 2022-12-21
WO2022093800A3 (en) 2022-06-02
US20230241243A1 (en) 2023-08-03
WO2022093794A9 (en) 2022-06-09
JP2023133440A (ja) 2023-09-22
ES2963150T3 (es) 2024-03-25
EP4103236B1 (en) 2023-08-16
WO2022093794A1 (en) 2022-05-05
FI4103236T3 (fi) 2023-11-09
JP2023515759A (ja) 2023-04-14
US11744897B2 (en) 2023-09-05
WO2022093800A2 (en) 2022-05-05
JP2023516111A (ja) 2023-04-18
JP7349578B2 (ja) 2023-09-22

Similar Documents

Publication Publication Date Title
US20230021059A1 (en) Methods of functionalizing nanoparticles
Fan et al. Targeted anticancer prodrug with mesoporous silica nanoparticles as vehicles
US10172951B2 (en) Dendrimer based nanodevices for therapeutic and imaging purposes
TWI412367B (zh) 化學鏈接劑與可裂解基質以及其之綴合物
Shukla et al. HER2 specific delivery of methotrexate by dendrimer conjugated anti-HER2 mAb
KR102051038B1 (ko) 나노입자 약물 컨쥬게이트
Dharmatti et al. Surface orchestration of gold nanoparticles using cysteamine as linke r and folate as navigating molecule for synaphic delivery of doxorubicin
US9486535B2 (en) Methods of making and using nanostructures
De Matos et al. Inhibitor-conjugated harmonic nanoparticles targeting fibroblast activation protein
CN114929281A (zh) 超小型纳米颗粒及其制备、使用和分析方法
CN116669773A (zh) 功能化纳米颗粒的方法
JP2022515884A (ja) 切断可能なリンカーを含む化合物及びその使用
US20230158154A1 (en) Conjugates comprising a phosphorus (v) and a camptothecin moiety
US20220241429A1 (en) Antifolate-Carrying Nanoparticles and Their Use in Medicine
Sánchez García et al. Glycyrrhetinic acid-functionalized mesoporous silica nanoparticles for the co-delivery of DOX/CPT-PEG for targeting HepG2 cells

Legal Events

Date Code Title Description
STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

AS Assignment

Owner name: ELUCIDA ONCOLOGY, INC., NEW JERSEY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:MA, KAI;TUERKER, MELIK ZIYA;WU, FEI;AND OTHERS;SIGNING DATES FROM 20230629 TO 20230811;REEL/FRAME:064577/0256

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED