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WO2023004364A2 - Nanomatériau à auto-assemblage pour la détection, l'imagerie ou le traitement du cancer - Google Patents

Nanomatériau à auto-assemblage pour la détection, l'imagerie ou le traitement du cancer Download PDF

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Publication number
WO2023004364A2
WO2023004364A2 PCT/US2022/073964 US2022073964W WO2023004364A2 WO 2023004364 A2 WO2023004364 A2 WO 2023004364A2 US 2022073964 W US2022073964 W US 2022073964W WO 2023004364 A2 WO2023004364 A2 WO 2023004364A2
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self
seq
assembling components
probe
motif
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WO2023004364A8 (fr
WO2023004364A3 (fr
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Adem YILDIRIM
Jared FISCHER
Bruce Branchaud
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Oregon Health and Science University
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Oregon Health and Science University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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    • 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
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/62—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 a protein, peptide or polyamino acid
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    • A61K47/6905—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 colloid or an emulsion
    • A61K47/6907—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 colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
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    • A61K49/001—Preparation for luminescence or biological staining
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    • 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
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    • A61K49/0017—Fluorescence in vivo
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    • 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
    • A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
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    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
    • A61K49/0076—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 dispersion, suspension, e.g. particles in a liquid, colloid, emulsion
    • A61K49/0082—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 dispersion, suspension, e.g. particles in a liquid, colloid, emulsion micelle, e.g. phospholipidic micelle and polymeric micelle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
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    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1806—Suspensions, emulsions, colloids, dispersions
    • A61K49/1809—Micelles, e.g. phospholipidic or polymeric micelles
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2610/00—Assays involving self-assembled monolayers [SAMs]

Definitions

  • the current disclosure provides self-assembling nanomaterials and uses thereof in the detection, imaging, and treatment of cancer.
  • the self-assembled nanomaterials include a plurality of self-assembling components.
  • Each self-assembling component is amphiphilic, including a hydrophobic self-assembly motif operatively connected to a hydrophilic motif, whereby, upon dissolution in an aqueous solution, the self-assembling components form a micellar structure and generally orient so that the hydrophilic motifs remain in contact with the aqueous solution, thereby forming the self-assembled nanomaterial.
  • Molecular imaging methods have many potential benefits in cancer detection and management, from early detection of solid malignancies to monitoring patient response to therapy and from patient stratification to image-guided surgery.
  • a key challenge in the field is the efficient delivery of functional molecules to tumors.
  • An ideal imaging agent would demonstrate cancer-specific accumulation with extended retention in the tumor tissue.
  • Such imaging agents would provide a high signal-to-noise ratio for extended time periods enabling accurate detection of small ( ⁇ 1 cm) tumors or metastatic sites and assessing biomarker levels.
  • current clinical imaging modalities are limited to >5 mm tumors and cannot accurately identify tumor margins.
  • small molecular probes can easily extravasate from circulation and penetrate deep into solid malignancies, they can rapidly be cleared from circulation and tissues.
  • rationally designed nanomaterials can remain in circulation for up to several days and an ideal nanomaterial would avoid removal from circulation by the liver or kidneys.
  • tumor accumulation is usually low due to their larger sizes, limiting deep tissue penetration.
  • EISA enzyme instructed self-assembly
  • hydrolysis of the substrate on a small molecule or nanoparticle by tumor-specific enzyme triggers their self-assembly into larger aggregates enabling kinetic entrapment of the functional molecules within the tumor tissue.
  • various EISA methods have been applied to develop contrast agents for several imaging modalities, including fluorescence, photoacoustic, magnetic resonance imaging (MRI), and positron emission tomography (PET).
  • existing EISA methods require a self-assembling component that is cleavable by a protease, adding complexity to the EISA method.
  • the current disclosure provides self-assembling nanomaterials for the detection, imaging, and treatment of cancer.
  • the self-assembled nanomaterials include a plurality of selfassembling components.
  • each self-assembling component is amphiphilic, including a hydrophobic self-assembly motif operatively connected to a hydrophilic motif, whereby, upon dissolution into an aqueous solution, the self-assembly motifs collocate and generally orient the hydrophilic motifs of each self-assembling component to remain in contact with the aqueous solution such that the self-assembling components form a micellar structure creating the self-assembled nanomaterial.
  • the self-assembling component further includes a functional molecule operatively connected to the self-assembling component.
  • the functional molecule includes a dye for detection and/or imaging.
  • the functional molecule is a drug or a prodrug.
  • the functional molecule is attached to the hydrophilic motif.
  • the self-assembly motif is optionally operatively connected to the hydrophilic motif by a spacer.
  • the spacer includes a linker and/or a substrate having a cleavage site.
  • the linker includes a glycine, a glycine-histidine, or a proline-rich linker.
  • the optional substrate having a cleavage site is cleaved by a protease.
  • a protease can be used to detect protease activity or elicit an effect where protease activity is high (e.g., at a tumor site).
  • a functional molecule e.g., a dye or drug
  • FIGs. 1A, 1B Schematic showing an embodiment of a self-assembling component.
  • This self-assembling component is composed of four modules: i) a self-assembly motif, ii) a spacer, iii) a hydrophilic motif, and iv) a functional molecule.
  • the functional molecule is conjugated to the hydrophilic motif, but it can be conjugated to the other parts of the self-assembling components as well.
  • FIG. 1B) A schematic representing self- assembly into a probe and the release of the functional molecule in the presence of a target protease.
  • the functional molecule is a quenched dye and the spacer is a substrate that functions as a protease cleavage site, the protease can cleave the substrate causing fluorescence and aggregation of the partially disassembled probe.
  • the functional molecule is a drug and the spacer is a substrate that functions as a protease cleavage site, the drug can be released in the protease expressing locations such as in solid tumors.
  • a “probe” refers to a self-assembled nanomaterial.
  • FIG. 2 Molecular structure of an example self-assembling component (or probe).
  • This probe is composed of the following motifs: i) palmitoyl-GGGH (SEQ ID NO: 1), self-assembly motif, ii) AANG (SEQ ID NO: 3) substrate, which can be specifically cleaved by legumain, iii) ECEE (SEQ ID NO: 10), hydrophilic motif, and iv) Indocyanine green (ICG), near-infrared (NIR) dye as the functional molecule. ICG was conjugated to the cysteine residue on the hydrophilic motif through a thiol-maleimide conjugation reaction.
  • short peptides such as GGGH (SEQ ID NO: 1) are referred to as part of the self-assembly motif. They may also be considered a linker, linking the self-assembly motif to an adjacent portion of the self-assembling component.
  • FIGs. 3A-3F Liquid chromatography-mass spectrometry (LC-MS) data of example peptides, showing successful peptide functional molecule conjugation.
  • FIG. 3A Asp-Probe
  • FIG. 3B Zwitter-Probe
  • FIG. 3C MMP
  • FIG. 3D Matriptase
  • FIG. 3E NoSA
  • FIG. 3F NoSubs (No Substrate)
  • FIGs. 4A-4C Transmission electron microscopy (TEM) images of different peptide probes (See FIG. 5 for peptide sequences and conjugated molecules).
  • FIG. 4A ICG conjugated peptides with different spacers.
  • FIG. 4B ICG conjugated peptides with different hydrophilic motifs.
  • FIG. 4C Glu-Probe conjugated with Cy7 dye or doxorubicin (DOX).
  • FIG. 5 Structures of the self-assembling components used in the experimental examples. Lower case letters are symbols of d amino acids ami 1 indicates that the residue is amidated.
  • FIG. 6 TEM images of the Glu-Probe (also referred to as ECEE probe) (50 mM) after 2 h of incubation in the presence or absence of 2.5 pg/mL legumain. In the absence of legumain, the probe forms micelle structures with sizes around 5-10 nm. The probe formed micron-sized aggregated structures after incubating with legumain.
  • ECEE probe also referred to as ECEE probe
  • FIGs. 7A-7C show that FIG. 7A) LC and FIG. 7B) MS data of Glu-Probe after 2 h of incubation in the presence or absence of 2.5 pg/mL legumain. For the sample incubated with legumain an additional peak appeared which corresponds to the cleavage product of the Glu-Probe.
  • FIG. 7C LC data of d-amino probe showing that this probe was not cleaved by legumain as expected for peptides of d-amino acids.
  • FIGs. 8A-8D Representative fluorescence spectra showing fluorescence recovery of the Glu-Probe (50 mM) after 2 h of incubation with 2.5 pg/mL legumain. Hydrolysis of the probe by legumain resulted in a fluorescence intensity enhancement of more than 100- fold.
  • FIG. 8B Kinetics of probe (50 mM) cleavage at different probe concentrations. Probe hydrolysis was mostly completed after 1 h of incubation.
  • FIG. 8C Fluorescence intensity enhancement of the probe at different legumain concentrations showing a fairly linear increase with increasing legumain concentration.
  • FIGs. 9A-9D Mice were intravenously (IV) injected with probes into Balb/c mice containing 4T1 breast cancer cells located in the mammary fat pad.
  • FIG. 9A Glu-Probe specifically accumulates in the tumor. Free ICG is rapidly cleared from the mouse and does not accumulate in the tumor.
  • FIG. 9B Tumor accumulation peaks at 12 h after injection for Glu-Probe and gradually dissipates.
  • FIG. 9C Tumor signal to background ratio over time, which reaches a maximum around day 3.
  • FIG. 9D Total detected ICG intensity for all probes throughout this experimental example.
  • FIG. 10 Mice were intravenously injected with Glu-Probe at different concentrations in 100 pL of phosphate buffered saline (PBS) into Balb/c mice containing 4T 1 breast cancer cells located in the mammary fat pad. Tumor fluorescence signal increased linearly with the increasing probe concentration.
  • PBS phosphate buffered saline
  • FIG. 12 Blood circulation time of the Glu-Probe and free ICG in mice. While free ICG was quickly cleared from the circulation, the probe was still detectable in the circulation even after 7 days.
  • FIGs. 13A, 13B Blood toxicity did not change for white blood cells (WBC), red blood cells (RBC), platelets (PLT), Hemoglobin (HGB) and hematocrit (HCT).
  • WBC white blood cells
  • RBC red blood cells
  • PHT platelets
  • HGB Hemoglobin
  • HCT hematocrit
  • FIG. 13B In addition, there was no change in the liver toxicity measured by alanine serum transferase or creatinine levels.
  • FIG. 14A, 14B Tumor accumulation (FIG. 14A) and tumor to background ratio (FIG. 14B) of different probes.
  • FIG. 15 The Glu-Probe can target a wide range of cell types: HCT-116 (human colon), BxPC-3 (human pancreatic), LS174 T (human colon), A375 (human melanoma), B16F10 (mouse melanoma), MCF7 (human breast).
  • A375 cells have luciferase and luciferase signal (top) matches where probe signal is (bottom).
  • A375 cells also move to the local lymph node and can be visualized by both luciferase and probe.
  • B16F10, LS174T, MCF7 tumors were smaller than 5 mm.
  • cancer cells were injected either subcutaneously or intradermally.
  • A375 melanoma cells also contained luciferase and metastasize to the local lymph node. Tumors were allowed to form.
  • the Glu-Probe 50 nmole was then injected IV and imaged with an IVIS® (Xenogen Corporation, Hopkinton, MA) machine for fluorescence (red-yellow) or luminescence (blue-green-red).
  • FIG. 16 Mice were injected with HCT116 cells and tumors were allowed to grow to different sizes. Glu-Probe with ICG signal was measured with IVIS® (Xenogen Corporation, Hopkinton, MA) for fluorescence (red-yellow) and positively correlates with tumor size.
  • IVIS® Xenogen Corporation, Hopkinton, MA
  • FIG. 17 Patient derived xenografts were implanted subcutaneously in mice and allowed to form 0.5-1 cm mass.
  • the Glu-Probe 50 nmole was injected and specifically went to the PDX in both pancreatic and colon cancers. Probe was imaged using the IVIS® (Xenogen Corporation, Hopkinton, MA) machine for fluorescence (red-yellow).
  • FIG. 18 Targeting transgenic mouse models of cancer.
  • a transgenic model of breast cancer mouse mammary tumor virus (MMTV) mouse model
  • MMTV mammary tumor virus
  • Probe was imaged using the IVIS® (Xenogen Corporation, Hopkinton, MA) machine for fluorescence (red-yellow).
  • IVIS® Xenogen Corporation, Hopkinton, MA
  • the probe was able to predict the location of tumor before the tumor was visible or palpable (arrows).
  • the signal was specific for the tumor compared to other normal tissues.
  • FIG. 19 Tumor signal of the Glu-Probe (50 nmole) 2 days after IV injection for different mouse models detected using an IVIS® (Xenogen Corporation, Hopkinton, MA) system.
  • FIG. 20 Ape min mice were injected with probe at 4 months of age and the intestines were analyzed 2 days later. Small intestinal adenomas and colon polyps had higher fluorescent signal than the surrounding normal intestine. In addition, by first marking all adenomas and polyps using the photograph image and then overlaying the identified tumors phenotypically and fluorescently revealed 27/27 small intestinal adenomas and 3/3 colon polyps were positive both phenotypically and fluorescently.
  • FIG. 21 Glu-Probe accumulation in occult 4T1 tumors with submillimeter sizes.
  • FIGs. 22A-22D Metastasis detection using the Glu-Probe (50 nmole).
  • FIG. 22A Detection of HCT-116 kidney metastasis.
  • FIG. 22B HCT-116 lung metastasis. Probe was imaged using the IVIS® (Xenogen Corporation, Hopkinton, MA) machine for fluorescence (red-yellow). HCT116 cancer cells containing luciferase were injected IV. Probe containing ICG was injected IV after metastases were established. Probe was imaged using the IVIS® (Xenogen Corporation, Hopkinton, MA) machine for fluorescence (red-yellow) and luminescence (blue-green-red).
  • IVIS® Xenogen Corporation, Hopkinton, MA
  • FIG. 22C Probe targeted and visualized lung metastases containing 7,500 cells based upon luminescence of HCT cells in 12 well plate and imaged with IVIS® (Xenogen Corporation, Hopkinton, MA).
  • FIG. 22D 4T1 lung metastasis.
  • FIGs. 23A, 23B FIG. 23A
  • the 4T1 tumors were imaged after 2 days of Glu-Probe or free ICG injection using a fluorescence imaging setup. The fluorescence signal was only observed in the tumor area at a clinically relevant exposure time (0.5 ms) where there was no signal in the free ICG injected tumor.
  • FIG. 23B Imaging metastasis. 4T1 cells were injected intravenously to establish experimental metastases in the lung. Mice were euthanized one day after probe injection. Lungs were imaged using both an IVIS® (Xenogen Corporation, Hopkinton, MA) and an image-guided surgery setup. A metastatic lesion smaller than 1 mm was clearly visible in both imaging modalities.
  • FIG. 24 RG2 cells were injected orthotopically into the rat brain. Gliomas were established and the Glu-Probe (500 nmole) was injected IV. Two days after probe injection, the first gadolinium contrast agent was injected IV and MRI imaging was performed 5 min after gadolinium injection. Then rats were sacrificed and organs were harvested. Brain sections were imaged using IVIS® (Xenogen Corporation, Hopkinton, MA) and a fluorescence guided surgery setup. Brain tissue sections were also imaged under a fluorescence microscope to visualize the probe (green, ICG) and tumor and normal cells (blue).
  • IVIS® Xenogen Corporation, Hopkinton, MA
  • FIG. 25 Photoacoustic imaging was performed on wild type mice containing 4T1 breast tumors. Tumors were imaged before probe injection and after probe injection. Mock injected showed no change in photoacoustic properties, but Glu-Probe-Cy7 and PA-ICG probes showed a 100-200% increase in photoacoustic signal.
  • FIG. 26 RG2 cells were injected orthotopically into the rat brain. Gliomas were allowed to form. Rats were then IV injected with MRI/NIR Probe containing both Gadolinium and ICG.
  • FIG. 27 4T1 cells were injected into the mammary fat pad of wild type mice. Once tumors were established, some mouse fat pads were injected with lipopolysaccharide (LPS) to induce inflammation and others were not. Then Glu-Probe (50 nmole) was injected IV and imaging was performed 2 days after injection. 4T1 cancers always had the strongest signal. Fat pads with just inflammation showed an increased signal compared to untreated fat pads.
  • FIG. 28 Comparison of performance of the Glu-Probe with commercially available products; MMPsense, 800CW-2DG, and cRGD-ICG using 4T1 mouse model.
  • FIGs. 29A-29C Improved chemotherapy using the DOX conjugated probe in 4T1 mouse model.
  • Three weekly injections of free DOX or Glu-Probe-DOX (5 mg/kg of DOX) were applied, and relative tumor size and mouse weight were measured at different time points. Both the free DOX and Glu-Probe-DOX slowed the tumor growth compared with control (FIG. 29A). There was no statistically significant difference between free DOX or the Glu-Probe- DOX. Importantly, it was found that conjugation of DOX to the probe largely reduces its side effects (FIG. 29B and FIG. 29C).
  • FIG. 30 Mice were induced to have senescent cells via injection of LPS in the mammary fat pad region. Senescence is shown by p16-luciferase expression (red). The Glu- Probe was injected IV and probe signal (blue) overlapped with senescent signal as indicated by arrows.
  • FIG. 31 Mice were injected intradermally with either dividing or senescent A375 cells containing luciferase. Mice were imaged for luciferase just before treatment in the IVIS® (Xenogen Corporation, Hopkinton, MA) (blue-green-red). Mice were treated IP with a single dose of probe containing Glu-Probe-SN38. Mice were then imaged 3 weeks after and treated mice showed loss of senescent cells (arrow with triangle), while untreated mice did not show loss of senescent cells (arrow with star).
  • FIGs. 32A, 32B Absorbance (FIG. 32A) or fluorescence (FIG. 32B) spectra of different probes (10 mM) in PBS, bovine serum albumin (BSA) solution (10 mg/mL), or 20% mouse plasma.
  • BSA bovine serum albumin
  • FIGs. 33A, 33B Time dependent absorbance (FIG. 33A) or fluorescence (3 FIG. 3B) spectra of different probes (10 pM) in 20% mouse plasma.
  • FIG. 34 Fluorescence quenching of BSA fluorescence in the presence different amounts of probes.
  • FIG. 35 Fluorescence spectra of Glu-Probe after incubating with different proteins.
  • FIG. 36 Proposed mechanism of improved tumor accumulation for Glu-Probe and similar probes.
  • FIG. 37 Reducing angiogenesis decreases the tumor accumulation of the Glu-Probe in 4T1 tumors.
  • FIG. 38 Tumor accumulation of the Glu-Probe in Matrigel® plugs.
  • I VIS® Xenogen Corporation, Hopkinton, MA
  • FIG. 38 Tumor accumulation of the Glu-Probe in Matrigel® plugs.
  • I VIS® Xenogen Corporation, Hopkinton, MA
  • White light and IVIS® Xenogen Corporation, Hopkinton, MA
  • FIG. 39 Accumulation of Glu-Probe at the wound site in wild type mice at different time points. Probe (50 nmole) was injected 2 days before imaging for each time point.
  • FIG. 40 Cellular uptake of the probe was studied using 4T1 cells expressing red fluorescent protein (red channel). Fluorescein (green channel) conjugated Glu-Probe- Fluorescein and probe without the self-assembly motif (NoSA-fluorescein) were prepared as described elsewhere herein. Confocal imaging showed that the self-assembly motif is needed for the cellular uptake.
  • FIG. 41 4T1 Cells were preincubated with cytochalasin D, Filipin, Heparan, wortmannin or chlorpromazine.
  • Filipin inhibits lipid raft or caveolae mediated endocytosis.
  • Chlorpromazine inhibits clathrin mediated endocytosis.
  • Cytochalasin D inhibits phagocytosis.
  • Wortmannin inhibits PI3K mediated endocytosis.
  • Heparin inhibits heparin sulfate proteoglycan binding for cell entry.
  • FIG. 42 Table of probes with random spacers.
  • FIG. 43 Tumor signal of the probes with different spacers in 4T1 mouse model 2 days after injection. All probes demonstrated similar accumulation in the tumor.
  • FIG. 44 Table of probes with only hydrophilic domain.
  • FIG. 45 Tumor signal of the probes with only hydrophilic domain and the NoSA probe in 4T1 mouse model 2 days after injection. Except 6K and 2D all probes demonstrated similar tumor accumulation with NoSA probe. These results showed that at least 3 glutamic or aspartic acids are needed for high tumor accumulation.
  • FIG. 46 Table of probes with different hydrophilic domains.
  • FIG. 47 Tumor signal of the probes with different hydrophilic motifs in 4T1 mouse model 2 days after injection. Except for Zwitter-Probe-3 all probes demonstrated high tumor accumulation which was comparable with the Asp-Probe (also referred to as DCDD probe).
  • FIG. 48 Table of probes with different hydrophobic domains indicates that it is different from all other probes. For these two probes, the self-assembly domain is in the c- terminal. Their sequences are EEAANVFFC (SEQ ID NO: 55) and RRAANVFFC (SEQ ID NO: 56), respectively.
  • FIG. 49 FIG. 49.
  • FIG. 50 Sequences of exemplary linkers, substrates, hydrophilic motifs, and peptide combinations.
  • FIG. 51 Table of self-assembling components and their associated self-assembly motif, linker, substrate, and hydrophilic motif.
  • FIG. 52 Table of functional molecules and exemplary self-assembling components.
  • the self-assembled nanomaterials include a plurality of self-assembling components.
  • each self-assembling component includes a hydrophobic self-assembly motif operatively connected to a hydrophilic motif. That is, the self-assembling components are “amphiphilic” or “amphipathic”, having both a hydrophobic and a hydrophilic portion.
  • the hydrophobic self-assembly motifs from each self-assembling component collocate and generally orient the hydrophilic motifs to remain in contact with the aqueous solution, forming a micellar structure.
  • Self-assembled as used herein means a multi-subunit nanomaterial formed from subunit monomers that, under suitable conditions, form the multi-subunit nanomaterial.
  • the self-assembly motif includes any hydrophobic moiety such that the hydrophobic moieties of a plurality of self-assembling components form the core of micellar structure upon dissolution in an aqueous solution.
  • the self-assembly motif includes a saturated hydrocarbon, an unsaturated hydrocarbon, an aromatic hydrocarbon, a fluorocarbon, a hydrophobic amino acid, or combinations thereof.
  • the saturated hydrocarbon is a long chain saturated hydrocarbon.
  • the long chain saturated hydrocarbon includes at least 8 carbons in length (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
  • the saturated hydrocarbon includes lauroyl, tridecanoyl, myristoyl, pentadecenoyl, palmitoyl, heptadecanoyl, stearoyl, nonadecanoyl, heneicosanoyl, or behenoyl.
  • hydrophobic amino acids include F, Y, A, V, I, L, or W, or combinations thereof.
  • the self-assembly motif includes a peptide sequence.
  • the peptide sequence includes GGGH (SEQ ID NO: 1), GGGh (SEQ ID NO: 2), PPPP (SEQ ID NO: 21), VFFC (SEQ ID NO: 39), or FFY.
  • the self-assembly motif includes palmitoyl, palmitoyl-GGGH (SEQ ID NO: 1), palmitoyl-GGGh (SEQ ID NO: 2), palmitoyl- PPPP (SEQ ID NO: 21), or fluorenylmethoxycarbonyl (Fmoc)-FFY.
  • the self-assembly motif includes any hydrophobic moiety that can collocate upon dissolution in an aqueous solution including waxes, lipids, cholesterol, or steroid hormones.
  • the hydrophilic motif includes a hydrophilic moiety.
  • the hydrophilic motif can include a peptide and/or a polyethylene glycol (PEG).
  • the hydrophilic motif includes a peptide having the sequence ECEE (SEQ ID NO: 10), ecee (SEQ ID NO: 14), DCDD (SEQ ID NO: 11), KCKK (SEQ ID NO: 12), KCEK (SEQ ID NO: 13), RCRR (SEQ ID NO: 27), DDCDD (SEQ ID NO: 24), DDGDDCDD (SEQ ID NO: 25), KEKEKECK (SEQ ID NO: 23), ECEE wherein the C terminal is amidated (SEQ ID NO: 22), KKKGCGKKK (SEQ ID NO: 28), DGCGD (SEQ ID NO: 29), DDGCGDD (SEQ ID NO: 30), DDDGCGDDD (SEQ ID NO: 31), EEGCGEE (SEQ ID NO: 32), EKEKEG
  • the hydrophilic motif includes polyethylene glycol (PEG).
  • the hydrophilic motif includes 3-10 PEG molecules (e.g., 3, 4, 5, 6, 7, 8, 9 or 10).
  • the hydrophilic motif includes PEG 6 or glycine (G)-PEG, for example, G-PEG3, G-PEG4, G-PEG5, G-PEG6, G-PEG7, G-PEGs, G-PEGg, or G-PEG10 ⁇
  • the hydrophilic motif includes G-PEG &
  • the self-assembling component further includes a functional molecule operatively connected to the self-assembling component.
  • the functional molecule includes a dye for detection and/or imaging.
  • the dye is a near-infrared (NIR) dye.
  • NIR fluorescence is a light wavelength of 650 nm to 1500 nm. Skilled persons will understand that NIR fluorescence facilitates in vivo fluorescent imaging of tissue because of its ability to penetrate targeted tissues while having relatively lower autofluorescence from adjacent, or non-targeted, tissues.
  • the NIR dye includes indocyanine green (ICG), sulfo-Cy7-maleimide (Cy7) dye, 800CW, IR-783, IR-820, IR-786, 3,3-diethylthiatricarbocyanine (DTTC) iodide, and HIDC (2- [5-(1 ,3-dihydro-1 ,3,3-trimethyl-2H-indol-2-ylidene)-1 ,3-pentadienyl]-1 ,3,3-trimethyl-3H- indolium) iodide.
  • the NIR dye includes indocyanine green (ICG), sulfo-Cy7-maleimide (Cy7) dye, or IR-783.
  • the dye includes two functional molecules.
  • the dye includes an NIR fluorescent dye attached to an MRI contrast agent to create and MRI/NRI Probe.
  • an MRI/NRI Probe includes the functional molecules ICG and Gd-DOTA.
  • dyes can be used to prepare a self-assembling component for PET/SPECT imaging.
  • a dye can include any detectable label that can be used for detection and/or imaging in vivo and that can be operatively connected to a self-assembling component.
  • a detectable label can include fluorophores, affinity tags, radiolabels, or contrast agents.
  • an MRI contrast agent is Gd-DOTA.
  • the functional molecule is a drug or prodrug.
  • the drug is an anti-cancer drug.
  • an anti-cancer drug includes alkylating agents, nitrosoureas, antimetabolites, anthracyclines, topoisomerase I inhibitors, topoisomerase II inhibitors, mitotic inhibitors, and corticosteroids.
  • alkylating agents include altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, and trabectedin.
  • nitrosoureas include carmustine, lomustine, and streptozocin.
  • antimetabolites include azacitidine, 5-fluorouracil (5-FU), 6- mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine (Ara-C), decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate, thioguanine, and trifluridine/tipiracil combination.
  • anthracyclines include daunorubicin, doxorubicin, doxorubicin liposomal, epirubicin, idarubicin, and valrubicin.
  • topoisomerase I inhibitors also called camptothecins
  • topoisomerase II inhibitors include etoposide (VP-16), mitoxantrone, and teniposide.
  • mitotic inhibitors include cabazitaxel, docetaxel, Nab-paclitaxel, paclitaxel, vinblastine, vincristine, vincristine liposomal, and vinorelbine.
  • corticosteroids include prednisone, methylprednisolone, and dexamethasone.
  • other anticancer drugs include all-trans-retinoic acid, arsenic trioxide, asparaginase, bleomycin, dactinomycin, mitomycin-C, eribulin, hydroxyurea, ixabepilone, mitotane, omacetaxine, pegaspargase, procarbazine, romidepsin, taxol, and vorinostat.
  • the drug is functionalized, for example, with an iodine.
  • a functionalized doxorubicin includes N-(lodoacetamido)-Doxorubicin (Dox).
  • the drug includes CL2-SN-38 (contains the topoisomerase I inhibitor, SN-38).
  • the self-assembly motif is operatively connected to the hydrophilic motif by a spacer.
  • the spacer includes a linker and/or a substrate.
  • a “linker” as referred to herein connects two distinct molecules.
  • the distinct molecules can possess hydrophobic properties, hydrophilic properties, can be proteolytically cleavable, and/or can be naturally expressed and assembled as separate molecules.
  • a number of strategies may be used to covalently link molecules together. These include polypeptide linkages between N- and C-termini of proteins or protein domains, linkage via disulfide bonds, and linkage via chemical cross-linking reagents.
  • the linker is a linker peptide, generated by recombinant techniques or peptide synthesis.
  • the linker peptide should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity.
  • the linker is from 1 to 50 amino acids in length or 1 to 30 amino acids in length.
  • linkers of 1 to 20 amino acids in length may be used.
  • Exemplary peptide linkers include G, GGSGGSGG (SEQ ID NO: 17), AGQGLR (SEQ ID NO: 18), FGSGKD (SEQ ID NO: 19), and TGGYPVE (SEQ ID NO: 20).
  • a proline-rich linker is a peptide sequence having more proline residues than would be expected based on chance alone.
  • a proline-rich linker is one having at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51% proline residues.
  • proline-rich linkers include fragments of proline-rich salivary proteins (PRPs).
  • a proline-rich linker includes PPPP (SEQ ID NO: 21).
  • the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (Gly x Ser y ) n , wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10).
  • n is an integer including, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more.
  • the linker is (Gly Ser) 4 (SEQ ID NO: 61), (Gly Ser) 3 (SEQ ID NO: 62), (Gly 4 Ser) 2 (SEQ ID NO: 63), (Gly 4 Ser) !
  • the spacer region is (EAAAK) n (SEQ ID NO: 71) wherein n is an integer including 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.
  • peptide linkers include AAEPKSS (SEQ ID NO: 72), AAEPKSSDKTHTCPPCP (SEQ ID NO: 73), or GGGGDKTHTCPPCP (SEQ ID NO: 74).
  • AAEPKSS SEQ ID NO: 72
  • AAEPKSSDKTHTCPPCP SEQ ID NO: 73
  • GGGGDKTHTCPPCP SEQ ID NO: 74
  • non-proteinaceous polymers including polyethylene glycol (PEG) m , (polypropylene glycol) m , (polyoxyalkylenes)m, or copolymers of polyethylene glycol and polypropylene glycol, may be used as linkers where m is an integer of at least one but less than 150.
  • the spacer includes a substrate.
  • the substrate refers to a peptide sequence having a cleavage site.
  • the cleavage site results in cleavage of the self-assembling component by a protease.
  • the substrate includes the sequence AANG (SEQ ID NO: 3), aanG (SEQ ID NO: 4), AARG (SEQ ID NO: 6), AGFSL (SEQ ID NO: 8), PLGVR (SEQ ID NO: 7), AANGGC (SEQ ID NO: 26), or AAN.
  • a protease includes legumain, CathepsinG, Matriptase, MMP-2, MMP-9, MMP-13, ADAM10, ADAM17, trypsin, chymotrypsin, pepsin, thermolysin, elastase, furin, Cathepsin B, Caspase 3, Caspase 7, Caspase 10, pyroglutamate aminopeptidase, acylamino-acid-releasing enzyme, Cathepsin C, carboxypeptidase, clostripain, subtilisin, proteinase K, and pancreatin.
  • Embodiments utilizing a substrate can be used to detect protease activity or elicit an effect where protease activity is high. These methods include administering self-assembled nanomaterials having self-assembling components, wherein the self-assembling components include a self-assembly motif operatively connected to a hydrophilic motif by a substrate, and wherein the hydrophilic motif is operatively connected to a functional molecule. In their micellar structure, the effect of the functional molecule is quenched or not released.
  • “released” means that the functional molecule is no longer trapped by the self-assembled nanomaterial but is able to have activity within its environment.
  • when the functional molecule is released it can be attached to other components such as a linker, a substrate, or any other molecule that does not impede its activity (e.g., fluorescence or tumor killing).
  • the functional molecule is a dye (e.g., NIR dye).
  • NIR dye e.g., NIR dye
  • the dye is able to generate and convey a fluorescent signal.
  • aggregation of the cleaved self-assembling components generates and conveys a stronger fluorescent signal at tumor sites (FIG. 1 B).
  • the functional molecule is a drug.
  • the hydrophilic motif is disconnected from the self-assembling component and the drug is released. Because proteases are highly expressed in tumor environments, the drug is released and is active in these tumor environments and can thereby treat the tumor.
  • the self-assembling component includes a self-assembly motif, a hydrophilic motif, and a functional molecule.
  • the functional molecule is a fluorescent dye such as ICG or Cy7 or a drug such as doxorubicin or a drug such as taxol.
  • the self-assembling component includes a spacer.
  • the spacer includes a linker and/or a substrate that can be cleaved by a protease.
  • protease includes legumain, matriptase, or matrix metalloproteinase (MMP). The cleavage of the substrate can free a functional molecule and may induce aggregation of the self-assembled nanomaterial and improve its tumor accumulation.
  • any of the self-assembled nanomaterials or self-assembling components described herein, in any exemplary format, can be formulated alone or in combination into compositions for administration to subjects.
  • “probes” refer to self-assembled nanomaterials in a form suitable for an intended use.
  • “probes” refer to self-assembled nanomaterials in a form suitable for an intended use and in a form suitable for administration to a subject.
  • a pharmaceutical composition can include the self-assembled nanomaterials or selfassembling components of any of the embodiments disclosed herein and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is one that does not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration.
  • Exemplary pharmaceutically acceptable carriers are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990.
  • compositions can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by U.S. FDA Office of Biological Standards and/or other relevant foreign regulatory agencies.
  • a pharmaceutically acceptable carrier includes absorption delaying agents, antioxidants, binders, buffering agents, bulking agents or fillers, chelating agents, coatings, disintegration agents, dispersion media, gels, isotonic agents, lubricants, preservatives, salts, solvents or co-solvents, stabilizers, surfactants, and/or delivery vehicles.
  • a pharmaceutical composition can further include pharmaceutically acceptable salts.
  • compositions include self-assembled nanomaterials or selfassembling components of at least 0.1% w/v or w/w of the composition; at least 1% w/v or w/w of composition; at least 10% w/v or w/w of composition; at least 20% w/v or w/w of composition; at least 30% w/v or w/w of composition; at least 40% w/v or w/w of composition; at least 50% w/v or w/w of composition; at least 60% w/v or w/w of composition; at least 70% w/v or w/w of composition; at least 80% w/v or w/w of composition; at least 90% w/v or w/w of composition; at least 95% w/v or w/w of composition; or at least 99% w/v or w/w of composition.
  • compositions disclosed herein can be formulated for administration by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion.
  • the compositions disclosed herein can further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, sublingual, and/or subcutaneous administration.
  • kits can include components to practice, for example, the methods described herein.
  • kits can include self-assembling components (or components thereof (e.g., PEG) wherein the self-assembling components are not linked to a functional molecule.
  • kits can include self-assembling components (or components thereof (e.g., PEG) wherein the self-assembling components are not linked to a functional molecule, but the functional molecule is part of the kit.
  • kits can include self-assembling components that include a functional molecule linked to the self-assembling components.
  • the kit includes the compositions disclosed herein.
  • the kit may include material(s), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or other material useful in administration, detection, imaging, treatment, or conducting any other step of the methods described herein.
  • material(s) which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or other material useful in administration, detection, imaging, treatment, or conducting any other step of the methods described herein.
  • the kit can be tailored to include materials necessary for detection, imaging, or treatment.
  • the kit for imaging includes materials for high-resolution fluorescence imaging, NIR fluorescence imaging, photoacoustic imaging, and/or image-guided surgery imaging.
  • Photoacoustic imaging is a process of delivering light energy to cells or a tissue to cause a thermoelastic expansion in the cells or tissue that generates ultrasound waves that are then detected by a transducer to produce images of optical absorption contrast within the cells or tissues.
  • Subjects include, e.g., humans, veterinary animals (dogs, cats, reptiles, birds), livestock (e.g., horses, cattle, goats, pigs, chickens), and research animals (e.g., monkeys, rats, mice, fish).
  • livestock e.g., horses, cattle, goats, pigs, chickens
  • research animals e.g., monkeys, rats, mice, fish.
  • compositions When used as a treatment to deliver a drug or a prodrug as a functional molecule, the compositions provide a therapeutically effective amount.
  • Therapeutically effective amounts include effective amounts and/or provide prophylactic and/or therapeutic treatments.
  • an “effective amount” is the amount of a composition necessary to result in a desired physiological change in the subject. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically-significant effect in an animal model or in vitro assay relevant to the assessment of a condition’s development, progression, and/or resolution.
  • a condition includes cancer expressing high protease activity.
  • a “prophylactic treatment” includes a treatment administered to a subject who does not display signs or symptoms of a condition or displays only early signs or symptoms of a condition such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the condition further. Thus, a prophylactic treatment functions as a preventative treatment against a condition. In particular embodiments, prophylactic treatments reduce, delay, or prevent the worsening of a condition.
  • a “therapeutic treatment” includes a treatment administered to a subject who displays symptoms or signs of a condition and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the condition.
  • the therapeutic treatment can reduce, control, or eliminate the presence or activity of the condition and/or reduce control or eliminate side effects of the condition.
  • administering Function as an effective amount, prophylactic treatment, or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.
  • therapeutically effective amounts provide anti-cancer effects.
  • Anti-cancer effects include a decrease in the number of cancer cells, decrease in the number of metastases, prevented or reduced metastases, a decrease in tumor volume, inhibited tumor growth, an increase in life expectancy, prolonged subject life, induced chemo- or radiosensitivity in cancer cells, inhibited cancer cell proliferation, reduced cancer- associated pain, and/or reduced relapse or re-occurrence of cancer following treatment.
  • a “tumor” is a swelling or lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells).
  • Tumor cell is an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be benign, pre-malignant or malignant.
  • the self-assembled nanomaterials can similarly be used to deliver drugs to a tumor site.
  • self-assembled nanomaterials can deliver drugs to a tumor site for one day, two days, three days, four days, five days, six days, seven days, or for more than a week.
  • the accumulated self-assembled nanomaterials can release loaded drugs slowly and specifically to the tumors for extended periods.
  • methods for treating a cancer in subjects includes administering to the subject a composition including the self-assembling or self-assembled components including an anti-cancer drug.
  • Compositions for treatment of cancer including the self-assembled nanomaterial or self-assembling components can deliver drugs with better efficiency and reduced side effects.
  • Self-assembled nanomaterial disclosed herein can be used for in vivo, ex vivo, or in vitro detection or imaging of cancer cells and/or tumors.
  • the tumors have high protease activity (e.g., tumor environments).
  • detection is for research, diagnostic, and/or prognostic uses.
  • methods of detection include administering an effective amount of a composition disclosed herein having a dye as the functional molecule.
  • a composition of the presently disclosed subject matter includes a label that can be detected in vivo.
  • In vivo imaging or detection methods generally use non-invasive methods such as fluorescence, scintigraphic methods, magnetic resonance imaging, autoradiographic detection, or radioimmunoguided systems.
  • non-invasive methods includes methods employing administration of a contrast agent to facilitate in vivo imaging. In vivo imaging can be useful in the staging and treatment of malignancies.
  • methods for detecting a high protease activity environment (e.g., tumor site) in subjects includes (a) administering to the subject a composition including the self-assembled nanomaterials including a substrate and a dye; and (b) detecting the dye to thereby detect the high protease activity environment (e.g., tumor site).
  • methods for imaging a high protease activity environment in subjects includes (a) administering to the subject a composition including the self-assembled nanomaterials including a substrate and a dye; and (b) detecting the dye to thereby image the high protease activity environment (e.g., tumor site).
  • Exemplary imaging modalities include high-resolution fluorescence imaging, NIR fluorescence imaging, photoacoustic imaging, scintigraphic imaging (e.g., Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET)), magnetic resonance imaging (MRI), autoradiographic detection, or radioimmunoguided surgery and imaging.
  • scintigraphic imaging e.g., Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET)
  • MRI magnetic resonance imaging
  • autoradiographic detection e.g., radioimmunoguided surgery and imaging.
  • time sufficient for protease cleavage refers to a temporal duration that permits a protease to come into contact with and cleave the substrate, thus releasing the functional molecule from its quenched state or inhibited state.
  • the cleaved self-assembling components upon cleavage by a protease, form aggregates, enabling kinetic entrapment of functional molecules within the tumor tissue.
  • “Kinetic entrapment” as used herein means the physical entrapment of a molecule, especially a biomolecule, at a locus due to non-covalent cross-linking bonding (or interactions) such as tt-p (pi-pi) effects with other molecules at the locus.
  • self-assembled nanomaterials accumulate in tumors, facilitating the detection, imaging, and/or treatment of cancer.
  • self-assembled nanomaterials can generate a strong NIR fluorescent signal with high signal to noise ratios enabling the detection of small tumors and metastatic sites with sizes down to 1.0 millimeter (mm).
  • self-assembled nanomaterials can detect small tumors and metastatic sites with sizes of 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, or more than 5mm.
  • self-assembled nanomaterials can detect small tumors and metastatic sites in vivo, in xenografts, in syngeneic orthografts, in experimental metastatic disease, and in transgenic cancer models.
  • self-assembled nanomaterials can detect the tumor signal for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days post administration. In particular embodiments, self-assembled nanomaterials can detect the tumor signal up to 7 days post administration. In particular embodiments, self-assembled nanomaterials are cleared from non-tumor tissues in one day, in two days, or in three days post administration. In particular embodiments, self-assembled nanomaterials are cleared from non-tumor tissues in 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 15 hours, 20 hours, or 1 day post administration. In particular embodiments, self-assembled nanomaterials are cleared from non-tumor tissues in a day post administration.
  • therapeutically effective amounts can be initially estimated based on results from in vitro assays and/or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest.
  • the actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of condition, stage of condition, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.
  • Useful doses can range from 0.1 nmoles to 20 pmoles.
  • a dose can range from 2.5 nmoles to 50 nmoles.
  • a dose can include 2.5 nmoles, 3 nmoles, 5 nmoles, 7 nmoles, 10 nmoles, 15 nmoles, 20 nmoles, 25 nmoles, 30 nmoles, 35 nmoles, 40 nmoles, 45 nmoles, or 50 nmoles.
  • a dose can include 200 nmoles.
  • a dose can include 10 pmoles.
  • Useful doses can range from 0.1 to 5 pg/kg or from 0.5 to 1 pg /kg.
  • a dose can include 1 pg /kg, 15 pg /kg, 30 pg /kg, 50 pg/kg, 55 pg/kg, 70 pg/kg, 90 pg/kg, 150 pg/kg, 350 pg/kg, 500 pg/kg, 750 pg/kg, 1000 pg/kg, 0.1 to 5 mg/kg or from 0.5 to 1 mg/kg.
  • a dose can include 1 mg/kg, 10 mg/kg, 30 mg/kg, 50 mg/kg, 70 mg/kg, 100 mg/kg, 300 mg/kg, 500 mg/kg, 700 mg/kg, 1000 mg/kg or more.
  • Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly).
  • a treatment regimen e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly.
  • the treatment protocol may be dictated by a clinical trial protocol or an FDA-approved treatment protocol.
  • compositions described herein can be administered by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion.
  • Routes of administration can include intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, oral, subcutaneous, and/or sublingual administration.
  • self-assembling components can be operably to solid supports (or “solid phase”) in order to form the micellar structure.
  • solid supports include microbeads, nanoparticles, dendrimers, surfaces, and membranes.
  • percent homology when used to describe an amino acid sequence or a nucleic acid sequence, relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Percent homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application.
  • BLAST basic local alignment search tool
  • Embodiments disclosed herein can have 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to sequences disclosed herein.
  • amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids.
  • a conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
  • Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1 : Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Val) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr; Group
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982).
  • amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and/or mutations that do not affect the function of an encoded product to a statistically-significant degree.
  • D-amino acids or “dd-amino acids” as used herein are amino acids where the stereogenic carbon alpha to the amino group has the D-configuration. Skilled persons will understand that generally only L-amino acids are utilized by mammals and thus, are generally non-reactive to mammalian enzymatic activity, including protease activity.
  • a fusion protein is a recombinant protein containing amino acid sequence from at least two unrelated proteins that have been joined together, via a peptide bond, to make a single protein.
  • the unrelated amino acid sequences can be joined directly to each other or they can be joined using a linker sequence.
  • proteins are unrelated, if their amino acid sequences are not normally found joined together via a peptide bond in their natural environment(s) (e.g., inside a cell).
  • the amino acid sequences of bacterial enzymes such as B.
  • stearothermophilus dihydrolipoyl acyltransferase E2p
  • the amino acid sequences of HIV- 1 gp120 or gp41 glycoproteins are not normally found joined together via a peptide bond.
  • the selfassembling components can be referred to as fusion molecules.
  • polypeptide is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and includes any chain or chains of two or more amino acids.
  • terms including, “peptide,” “dipeptide,” “tripeptide,” “protein,” “enzyme,” “amino acid chain,” and “contiguous amino acid sequence” are all encompassed within the definition of a “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with, any of these terms.
  • polypeptides that have undergone one or more post-translational modification(s), including for example, glycosylation, acetylation, phosphorylation, amidation, palmitoylation, derivatization, proteolytic cleavage, post-translation processing, or modification by inclusion of one or more non-naturally occurring amino acids.
  • post-translational modification(s) including for example, glycosylation, acetylation, phosphorylation, amidation, palmitoylation, derivatization, proteolytic cleavage, post-translation processing, or modification by inclusion of one or more non-naturally occurring amino acids.
  • Conventional nomenclature exists in the art for polynucleotide and polypeptide structures.
  • one-letter and three-letter abbreviations are widely employed to describe amino acids: Alanine (A; Ala), Arginine (R; Arg), Asparagine (N; Asn), Aspartic Acid (D; Asp), Cysteine (C; Cys), Glutamine (Q; Gin), Glutamic Acid (E; Glu), Glycine (G; Gly), Histidine (H; His), Isoleucine (I; lie), Leucine (L; Leu), Methionine (M; Met), Phenylalanine (F; Phe), Proline (P; Pro), Serine (S; Ser), Threonine (T; Thr), Tryptophan (W; Trp), Tyrosine (Y; Tyr), Valine (V; Val), and Lysine (K; Lys).
  • an “ECEE peptide” (SEQ ID NO: 10) is a peptide having the sequence of Glu-Cys-Glu-Glu
  • a “DCDD peptide” (SEQ ID NO: 11) is a peptide having the sequence of Asp-Cys-Asp-Asp
  • a “KCKK peptide” (SEQ ID NO: 12) is a peptide sequence of Lys-Cys-Lys-Lys
  • a “KCEK peptide” (SEQ ID NO: 13) is a peptide sequence of Lys-Cys-Glu- Lys
  • a “G-PEG6 peptide” is a Glycine bound to six repeating polyethylene glycol linkers.
  • operatively connected refers to two distinct molecules connected by a spacer (e.g., a linker) or that are chemically bound to each other covalently.
  • a plurality of self-assembling components to form a self-assembled nanomaterial each of the plurality of self-assembling components including: a hydrophobic self-assembly motif operatively connected to a hydrophilic motif.
  • hydrophobic self-assembly motif of each of the plurality or a subset thereof includes a saturated hydrocarbon, an unsaturated hydrocarbon, an aromatic hydrocarbon, a fluorocarbon, a hydrophobic amino acid, or combinations thereof.
  • hydrophobic self-assembly motif includes palmitoyl-GGGH (SEQ ID NO: 1), palmitoyl-GGGh (SEQ ID NO: 2), palmitoyl-PPPP (SEQ ID NO: 21), or Fmoc-FFY.
  • hydrophilic motif of each of the plurality or a subset thereof includes a hydrophilic peptide and/or a polyethylene glycol (PEG).
  • hydrophilic peptide includes the sequence ECEE (SEQ ID NO: 10), ecee (SEQ ID NO: 14), DCDD (SEQ ID NO: 11 ), KCKK (SEQ ID NO: 12), KCEK (SEQ ID NO: 13), RCRR (SEQ ID NO: 27), DDCDD (SEQ ID NO: 24), DDGDDCDD (SEQ ID NO: 25), KEKEKECK (SEQ ID NO: 23), ECEE wherein the C terminal is amidated (SEQ ID NO: 22), KKKGCGKKK (SEQ ID NO: 28), DGCGD (SEQ ID NO: 29), DDGCGDD (SEQ ID NO: 30), DDDGCGDDD (SEQ ID NO: 31), EEGCGEE (SEQ ID NO: 32), EKEKEGCGKEKEK (SEQ ID NO: 33), EE, RR, ECE, EGEE (SEQ ID NO: 15), or CGEKEE (SEQ ID NO: 10), ecee (SEQ ID NO: 14
  • linker includes a polyethylene glycol (PEG) linker.
  • AANG SEQ ID NO: 3
  • aanG SEQ ID NO: 4
  • AARG SEQ ID NO: 6
  • AGFSL SEQ ID NO: 8
  • PLGVR SEQ ID NO: 7
  • AANGGC SEQ ID NO: 26
  • AAN AAN
  • protease includes legumain, CathepsinG, Matriptase, MMP-2, MMP-9, MMP-13, ADAM10, ADAM17, trypsin, chymotrypsin, pepsin, thermolysin, elastase, furin, Cathepsin B, Caspase 3, Caspase 7, Caspase 10, pyroglutamate aminopeptidase, acylamino-acid-releasing enzyme, Cathepsin C, carboxypeptidase, clostripain, subtilisin, proteinase K, or pancreatin.
  • the protease includes legumain, CathepsinG, Matriptase, MMP-2, MMP-9, MMP-13, ADAM10, ADAM17, trypsin, chymotrypsin, pepsin, thermolysin, elastase, furin, Cathepsin B, Caspase 3, Caspase
  • linker is a peptide having the sequence G, GGSGGSGG (SEQ ID NO: 17), AGQGLR (SEQ ID NO: 18), FGSGKD (SEQ ID NO: 19), or TGGYPVE (SEQ ID NO: 20) and the substrate is a peptide having the sequence AANG (SEQ ID NO: 3), aanG (SEQ ID NO: 4), AARG (SEQ ID NO: 6), AGFSL (SEQ ID NO: 8), PLGVR (SEQ ID NO: 7), AANGGC (SEQ ID NO: 26), or AAN.
  • the spacer includes the sequence GGGHAANG (SEQ ID NO: 75), GGGHAARG (SEQ ID NO: 76), GGGHAGFSL (SEQ ID NO: 77), GGGHPLGVR (SEQ ID NO: 78), GGGhaanG (SEQ ID NO: 79), or GGGHAANGGC (SEQ ID NO: 80).
  • GGGHAANG SEQ ID NO: 75
  • GGGHAARG SEQ ID NO: 76
  • GGGHAGFSL SEQ ID NO: 77
  • GGGHPLGVR SEQ ID NO: 78
  • the NIR dye includes a Indocyanine green (ICG) dye, a sulfo-Cy7-maleimide (Cy7), an 800CW dye, an IR- 783 dye, an IR-820, an IR-786, a 3,3-diethylthiatricarbocyanine (DTTC) iodide, or an HIDC (2- [5-(1 ,3-dihydro-1 ,3,3-trimethyl-2H-indol-2-ylidene)-1 ,3-pentadienyl]-1 ,3,3-trimethyl-3H- indolium) iodide.
  • ICG Indocyanine green
  • Cy7 sulfo-Cy7-maleimide
  • 800CW dye an IR- 783 dye
  • an IR-820 an IR-786
  • DTTC 3,3-diethylthiatricarbocyanine
  • HIDC 2,5-(1 ,3-dihydro-1
  • anti-cancer drug includes an alkylating agent, a nitrosourea, an antimetabolite, an anthracycline, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a mitotic inhibitor, or a corticosteroid.
  • alkylating agent includes altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, or trabectedin.
  • antimetabolite includes azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine (Ara-C), decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate, thioguanine, or trifluridine/tipiracil combination.
  • the antimetabolite includes azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine (Ara-C), decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate, thi
  • anthracycline includes daunorubicin, doxorubicin, doxorubicin liposomal, epirubicin, idarubicin, orvalrubicin.
  • topoisomerase I inhibitor includes irinotecan, irinotecan liposomal, topotecan, or CL2-SN-38.
  • topoisomerase II inhibitor includes etoposide (VP-16), mitoxantrone, and teniposide.
  • mitotic inhibitors include cabazitaxel, docetaxel, Nab-paclitaxel, paclitaxel, vinblastine, vincristine, vincristine liposomal, or vinorelbine.
  • anti-cancer drug includes all-trans-retinoic acid, arsenic trioxide, asparaginase, bleomycin, dactinomycin, mitomycin-C, eribulin, hydroxyurea, ixabepilone, mitotane, omacetaxine, pegaspargase, procarbazine, romidepsin, taxol, or vorinostat.
  • hydrophobic self-assembly motif of each of the plurality or a subset thereof includes a palmitoyl molecule and the hydrophilic motif includes the peptide sequence ECEE (SEQ ID NO: 10), ecee (SEQ ID NO: 14), DCDD (SEQ ID NO: 11), KCKK (SEQ ID NO: 12), KCEK (SEQ ID NO: 13), RCRR (SEQ ID NO: 27), DDCDD (SEQ ID NO: 24), DDGDDCDD (SEQ ID NO: 25), KEKEKECK (SEQ ID NO: 23), ECEE wherein the C terminal is amidated (SEQ ID NO: 22), KKKGCGKKK (SEQ ID NO: 28), DGCGD (SEQ ID NO: 29), DDGCGDD (SEQ ID NO: 30), DDDGCGDDD (SEQ ID NO: 31), EEGCGEE (SEQ ID NO: 32), EKEKEGCG
  • a self-assembled nanomaterial formed from the plurality of self-assembling components of any of embodiments 33-36.
  • a pharmaceutical composition including the self-assembled nanomaterial of embodiments 62 or 63 and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition including the self-assembled nanomaterial of embodiments 65 or 66 and a pharmaceutically acceptable carrier.
  • a method for detecting a tumor in a subject including administering a therapeutically effective amount of the pharmaceutical composition of embodiment 64 to the subject, and detecting the dye, thereby detecting the tumor in the subject.
  • the tumor is a colon cancer tumor, a pancreatic cancer tumor, a melanoma, a breast cancer tumor, a kidney cancer tumor, a lung cancer tumor, or a glioma.
  • the detecting includes high-resolution fluorescence imaging, NIR fluorescence imaging, photoacoustic imaging, scintigraphic imaging (e.g., Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET)), magnetic resonance imaging (MRI), autoradiographic detection, or radioimmunoguided surgery imaging.
  • high-resolution fluorescence imaging NIR fluorescence imaging, photoacoustic imaging, scintigraphic imaging (e.g., Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET)), magnetic resonance imaging (MRI), autoradiographic detection, or radioimmunoguided surgery imaging.
  • SPECT Single Photon Emission Computed Tomography
  • PET Positron Emission Tomography
  • MRI magnetic resonance imaging
  • autoradiographic detection or radioimmunoguided surgery imaging.
  • a method of treating cancer in a subject in need thereof including: administering a therapeutically effective amount of the pharmaceutical composition of embodiment 67 to the subject thereby treating cancer in the subject in need thereof.
  • EISA Enzyme-instructed self-assembly
  • peptide-based materials where peptides are rationally designed to self- assemble into large aggregates upon cleavage of a hydrophilic part of the peptide by the target enzyme such as proteases or alkaline phosphatases. This enables in situ formation of large aggregates in the tumor site, improving the tumor accumulation and retention of the peptide.
  • EISA approaches are also limited by the expression level of the targeted enzyme. While a few probes that target more universal hallmarks of cancer, such as low pH, hypoxia, or aerobic glycolysis (i.e., Warburg effect) have been developed, achieving cancer-specific accumulation with broad tumor applicability has remained as a challenge.
  • a self-assembled nanoprobe that can specifically accumulate in a broad range of solid tumors and enable their high-resolution fluorescent, photoacoustic, or magnetic resonance imaging (MRI) was developed.
  • the self-assembled nanoprobe is composed of a near infrared (NIR) fluorescent dye and an amphiphilic peptide, forming 5-10 nm micelles when dispersed in aqueous solutions.
  • NIR near infrared
  • amphiphilic peptide forming 5-10 nm micelles when dispersed in aqueous solutions.
  • the tumor signal was detectable for up to 14 days, but was mostly cleared from other organs in a day.
  • the strong and durable signal generated by the self-assembled nanoprobe enabled the detection of small tumors ( ⁇ 1 mm) and metastatic or early lesions invisible to the eye.
  • the self-assembled nanomaterial outperformed several commercially available tumor imaging products such as MMPsenseTM and 800CW 2- DG (deoxyglucose).
  • This tumor-specific nanoprobe will be clinically beneficial in image-guided surgery and photoacoustic imaging of cancer by allowing cleaner tumor margins and earlier detection of occult lesions, respectively.
  • the probe developed here can be easily repurposed for other imaging modalities such as positron emission tomography (PET) or single-photon emission computerized tomography (SPECT) by conjugating different functional molecules such as radioactive isotopes.
  • PET positron emission tomography
  • SPECT single-photon emission computerized tomography
  • the probe can be used to deliver chemotherapeutic drugs with better efficiency and reduced side effects.
  • the probe could be conjugated to a chemotherapy drug, doxorubicin, to significantly reduce its side effects without reducing its efficacy.
  • the self-assembling component is composed of 3 major components; a self- assembly motif, a hydrophilic motif, and a functional molecule (dye, drug gadolinium complex, etc.) ⁇
  • a spacer motif randomly selected or rationally designed amino acid sequences
  • One function of the spacer is that it can be specifically cleaved by a protease, such as legumain, matriptase, or matrix metalloproteinases (MMPs).
  • MMPs matrix metalloproteinases
  • Peptide functional molecule conjugation and characterization Peptides were obtained commercially from companies such as GenScript. Then functional molecules with maleimide or N-hydroxysuccinimide (NHS) ester are conjugated to a cysteine or lysine, or residue of the peptide, respectively.
  • peptides and maleimide functionalized molecules such as ICG-maleimide
  • DMSO dimethyl sulfoxide
  • buffer solution with a pH of 6.5-7.
  • peptides and NHS functionalized molecules are mixed in a buffer with a pH of 7.4-8.5.
  • This buffer solution can contain up to 50% DMSO to solubilize the peptide and the functional molecule.
  • the same protocol can be used to conjugate molecules with halogen modifications (i.e., Cl, I, Br) to peptides with lysine or cysteine residues.
  • halogen modifications i.e., Cl, I, Br
  • N-(lodoacetamido)-Doxorubicin and IR-783 are two examples of such molecules.
  • the mixtures are shaken at 400 rpm at RT for 2 to 18 h and purified through dialysis (1 or 2 kDa cutoff) against phosphate buffered saline (PBS) (10 mM, pH 7.4) or water.
  • PBS phosphate buffered saline
  • LC-MS liquid chromatography-mass spectrometry
  • Morphology of the peptides is investigated using transmission electron microscopy (TEM).
  • TEM transmission electron microscopy
  • the peptide conjugates dissolved in PBS, DMSO, or water (0.5 to 10 mM) are stored at -20 °C.
  • Other bioconjugation reactions such as azide - alkyl or trans-cyclooctenes (TCO) - tetrazine coupling can be used to attach functional molecules to peptides.
  • FIG. 2 The molecular structure of an example self-assembling component with an ICG dye modification is shown in FIG. 2.
  • This probe Glu-Probe or also referred to as ECEE was used in most of the experiments presented below unless otherwise specified.
  • FIGs. 3A-3F shows the LC-MS data for some example peptide conjugates.
  • TEM images of several peptide conjugates were also provided in FIGs. 4A-4C.
  • TEM analysis showed that changing the peptide spacer did not affect the morphology of the peptide ICG conjugates with ECEE (SEQ ID NO: 10) hydrophilic motif. All the peptides formed micelles with sizes around 5-10 nm (FIG. 4A).
  • hydrophilic motif has a profound effect on morphology. While negatively charged motifs (Glu-Probe or Asp-Probe) resulted in micelle morphology, positively charged (Lys-Probe) or neutral (Zwitter-Probe), or polyethylene glycol (PEG-Probe)) motifs yielded short rod structures (FIG. 4B). Finally, changing the functional molecule did not affect the morphology of peptide conjugates (FIG. 4C). Peptide sequences and functional molecules of self-assembling components developed in this work were also summarized in FIG. 5.
  • the hydrolysis of the probes by a target protease can induce morphological transformation as a result of increased hydrophobicity.
  • the Glu- Probe was used, which can be cleaved by legumain protease.
  • TEM analysis was performed after incubating the Glu-Probe (50 mM) with legumain (2.5 pg/mL) for 2 h in assay buffer (pH 5.5 MES (2-(N-morpholino)ethanesulfonic acid) buffer).
  • TEM showed the formation of micrometer-sized aggregated networks (FIG. 6).
  • Hydrolysis of the probe (50 mM) was also studied using LC-MS (FIGs. 7A-7C), which showed that 30% of the probe was hydrolyzed after incubating with legumain (2.5 pg/mL) for 2 h.
  • FIGs. 8A-8D The changes in the optical properties of the Glu-Probe before and after incubation with different amounts of legumain was also investigated.
  • the fluorescence of ICG was found to be quenched due to its high concentration in micelle structures (i.e., aggregation-induced quenching) (FIG. 8A).
  • Incubation with 2.5 pg/mL legumain for 2 h resulted in a more than 100-fold increase in the fluorescence of the probe as a result of disassembly of the micelles upon hydrolysis (FIG. 8A).
  • the kinetics of probe hydrolysis was also studied by continuously monitoring the fluorescence of the probe up to 2 h in the presence of different amounts of legumain (FIG. 8B), which showed that peptide hydrolysis was mostly completed in 1 h.
  • a linear relationship was found between the legumain concentration and probe fluorescence (FIG. 8C), which can be used to detect low concentrations of legumain down to a few ng/ml_.
  • this method could be used to detect other proteases by using a spacer composed of the AGFSL (SEQ ID NO: 8) motif (a substrate for Cathepsin G protease).
  • a similar linear response was also obtained for this probe (CatG) (FIG. 8D).
  • FIG. 9A shows a representative in vivo fluorescence image of 4T1 tumor-bearing mice intravenously (IV) injected with the probe (50 nmole) 2 days after probe injection, showing the strong accumulation of the probe in the tumor region.
  • FIG. 9B shows the ICG signal in the tumor over time up to 11 days. It was found that tumor signal peaks 10 h after injection and slowly decays after this point. Remarkably, even after 11 days ICG fluorescence was still detectable.
  • FIG. 9C The tumor signal to normal ratio over time (FIG. 9C).
  • Glu-Probe demonstrated a significantly higher tumor to normal ratio compared to other tested probes, which peaked around 2-4 days after probe injection. Accordingly, 2 days were selected as an optimal imaging time point for the probes and used it throughout the experimental example unless otherwise specified.
  • FIG. 9D compares the total signal of different probes over the course of the experiment. Total accumulation of Glu-Probe was found to be 1.6x and 2.7x higher than NoSA and Lys-Probe, respectively.
  • the Glu-Probe was injected at different doses (2.5 to 50 nmole) into 4T1 tumor-bearing mice and measured the ICG fluorescence 2 days after probe injection using an IVIS® (Xenogen Corporation, Hopkinton, MA) system. It was observed that tumor ICG signal increased with increasing probe concentrations. Accordingly, 2 days were selected as an optimal imaging point and 50 nmole as the optimal dose and used these conditions throughout the experimental example unless otherwise stated.
  • the blood circulation time of the probe was studied in wild-type mice.
  • the Glu-Probe or free ICG 50 nmole was intravenously injected into wild type mice.
  • 20 pL of blood samples were collected retroorbitally and ICG fluorescence was detected using a microplate reader. While free ICG was rapidly cleared from the circulation in less than 12 h, 10% of the injected Glu-Probe dose was still present in the circulation at this time point (FIG. 12).
  • Glu-Probe was still at detectable levels even 7 days after injection.
  • Glu-Probe does not require protease activity to accumulate in solid tumors.
  • EISA enzyme-instructed self-assembly
  • the legumain substrate (AANG (SEQ ID NO: 3)) of the self-assembling component was replaced with AARG (SEQ ID NO: 6, probe is referred to as Matriptase) or PLGVR (SEQ ID NO: 7, probe is referred to as MMP) to target activities of matriptase or MMPs, respectively.
  • the matriptase substrate is very similar to the legumain substrate with only one amino acid difference, but it cannot be cleaved by legumain as it specifically cleaves after asparagine residues of peptides. Matriptase can cleave this peptide after arginine residue.
  • MMP can be cleaved by a wide range of MMPs, including MMP-2, MMP- 7, MMP-9, MMP-13, and it is the substrate that is used in the commercially available MMPsense probes. Both matriptase and MMPs were over-expressed in a wide range of tumors. Finally, a probe without a legumain substrate (NoSubs) was prepared. TEM analysis of the probes demonstrated that all of the self-assembling components formed micelle structures similar to the Glu-Probe when dispersed in PBS (FIG. 4A).
  • FIG. 14A shows the fluorescence intensity of Glu-Probe and control probes 2d after intravenous injection (50 nmole) to 4T 1 bearing mice. All of the probes demonstrated a similar tumor signal, and there was no statistically significant difference between the probes, suggesting that protease (legumain, matriptase, or MMPs) activity has little or no effect on the specific tumor accumulation of the Glu-Probe. The tumor signal to background ratio of all probes was also similar, with the exception of d-amino acid probe, which demonstrated a more intense background (FIG. 14B).
  • Glu-Probe accumulates in a broad range of solid tumors. Based on the findings above, Glu-Probe should accumulate in a broad range of solid tumors almost universally as the tumor accumulation of the probe does not rely on protease activity or any other active targeting mechanism. To test this hypothesis, the accumulation of Glu-Probe was investigated in a number of other xenograft models of pancreatic, colon, breast, skin, and brain cancers; HCT-116, MCF7, A375, LS174T, BxPC-3, RG2. In fact, it was found that the Glu-Probe could clearly visualize all of these tumors with a variety of sizes from a few millimeters to a centimeter in Balb/c or nude mice FIG. 15.
  • the cells were expressing luciferase, and the luciferin signal matched where the probe signal was (FIG. 15).
  • A375 cells also moved to the local lymph node, which could be visualized by the luciferin signal.
  • the Glu-Probe could also detect the lymph node invasion of A375 cells (FIG. 15).
  • a good correlation between HCT-116 tumor size and tumor signal of the Glu-Probe at 2 days was found (FIG. 16), suggesting that the probe can be applied to estimate the tumor size.
  • mice Mouse mammary tumor virus (MMTV) infected mice were allowed to accumulate breast tumors with time. Mice were then injected with probe at different time points and imaged 2 days later (FIG. 18). The probe specifically labelled the breast tumors and could even predict tumor location prior to any palpable tumor (FIG. 18, left panel). For instance, at day 60 there was a weak signal at the bottom left mammary gland (black arrow in FIG. 18, left panel), where a large tumor observed on day 71. Similarly, a small tumor was detected at day 71 at the left top mammary gland, which continued to grow until the experiment terminated at day 89. At the end of the experiment, organs were harvested and imaged, which also showed the strong accumulation of tumors in MMTV tumors (FIG. 18, right panel).
  • MMTV mouse mammary tumor virus
  • FIG. 19 shows the average Glu-Probe tumor signal generated for all of the mouse models tested in living mice.
  • probe accumulation was higher in breast, melanoma, and glioma models than in colon and pancreatic cancer models, with the lowest probe accumulation observed for xenografts of these cancer types; BxPC-3 and HCT-116.
  • the Glu-Probe can detect early disease and occult lesions.
  • the Glu-Probe accumulation was studied in APCmin mice, which is a transgenic model for colon cancer.
  • APCmin mice were injected with Glu-Probe (50 nmole) at 4 months of age and the intestines were analyzed 2 days later.
  • Small intestinal adenomas and colon polyps had higher fluorescent signal that surrounding normal intestine (FIG. 20).
  • FOG. 20 fluorescent signal that surrounding normal intestine
  • Glu-Probe can detect micrometastasis.
  • the potential of the probes in fluorescent detection of micrometastasis was evaluated using experimental metastasis models of 4T 1 and HCT-116 cells. Initially, mice were injected with HCT-116 cells intravenously to induce metastases in internal organs. There were 3 small metastatic lesions (1 mm) in 2 kidneys, all of which were specifically labeled with the Glu-Probe probe (FIG. 22A). HCT116 cancer cells containing luciferase to form metastases were also IV injected. Glu-Probe (50 nmole) was injected after the metastases were formed.
  • IVIS® Xenogen Corporation, Hopkinton, MA
  • IVIS® Xenogen Corporation, Hopkinton, MA
  • HCT-116 cells estimated a cell number of 7500 cells in the detected lesion (FIG. 22C).
  • 4T1 cells were also injected to develop lung metastases. The probe was also accumulated in these lesions (FIG. 22D).
  • Glu-Probe can be used for image-guided surgery.
  • a custom-made fluorescence imaging setup was used. Initially, 4T 1 tumor bearing mice were injected with Glu-Probe or free ICG (both 50 nmole) and tumors were harvested 2 days after injection and imaged using a clinically relevant exposure time (500 ms). For the Glu-Probe injected mouse, a bright fluorescent signal was observed only in the tumor area (FIG. 23A). For free ICG injected tumor, there was no detectable signal under the same experimental conditions (FIG. 23A).
  • mice were injected with 4T1 cells intravenously to induce metastases in the lungs and injected the Glu-Probe1 day before harvesting the lungs.
  • One of the mice developed a small lesion in one of the lungs, which was barely visible under white light (FIG. 23B).
  • IVIS® (Xenogen Corporation, Hopkinton, MA) imaging detected this lesion (FIG. 23B).
  • the same lesion was also clearly visible under the fluorescence imaging setup (FIG. 23B) with significantly higher fluorescence intensity compared with the surrounding healthy tissue.
  • Glu-Probe accumulates in orthotopic brain tumors in rats.
  • RG2 cells were injected orthotopically into the rat brain. Gliomas were established, then the Glu-Probe (500 nmole) was injected IV. 2 days later, Gadolinium was injected IV and imaged 5 minutes after to visualize the tumor with MRI. Then, rats were sacrificed, brains were harvested, and ICG fluorescence was imaged using IVIS® (Xenogen Corporation, Hopkinton, MA) and fluorescence-guided surgery device using brain sections. Bright-field image shows the brain slice used for fluorescence imaging. It was found that MRI and ICG signals overlapped perfectly (FIG. 24), and the ICG signal is present in the glioma. Tissue sections were also imaged under a fluorescence microscope to visualize the probe and tumor and normal cells, which showed high probe accumulation in cancer cells.
  • Probes can be used for photoacoustic imaging.
  • two peptide dye conjugates were prepared: Glu-Probe-Cy7 and PA-ICG probes (see FIG. 5 for their structure).
  • Photoacoustic imaging was performed on wild type mice containing 4T1 breast tumors. Tumors were imaged before probe injection and 1 day after probe injection (200 nmole). Mock injected showed no change in photoacoustic properties, but Cy7 and ICG containing probes showed a 100-200% increase in photoacoustic signal (FIG. 25).
  • RG2 rat glioma model described above was also used in these studies.
  • peptides were modified with ICG and an MRI contrast agent; DOTA chelated Gadolinium (Gd-DOTA). See FIG. 5 for the molecular structure of the peptide.
  • Gd- DOTA DOTA chelated Gadolinium
  • the peptide was dialyzed against water and PBS to remove excess Gd.
  • the probe was injected IV (10 pmole). At 5 minutes post injection there was no MRI T1 contrast. But at 20 hours post injection there was both MRI T1 contrast and ICG fluorescence as measured in the IVIS® (Xenogen Corporation, Hopkinton, MA) (red-yellow) (FIG. 26).
  • Probe can detect cancer in the presence of inflammation background. For image- guided surgery applications, it is important that the probes can differentiate between cancer and inflammation.
  • Glu-Probe outperforms other ICG conjugates or commercial products.
  • the performance of Glu-Probe was compared with several other commercial NIR imaging probes using 4T1 tumor bearing mice (FIG. 28).
  • Commercially available products used in this experimental example were; MMP-sense (a Forster resonance energy transfer (FRET) probe that targets a broad range of MMPs), 800CW-2DG (2-deoxyglucose conjugated NIR dye which accumulates in solid tumors through glycolysis), and cRGD-ICG (ICG conjugated cyclic RGD peptide which can bind to integrins).
  • FRET Forster resonance energy transfer
  • hydrophobic molecules like the peptides described here, are introduced into circulation, they can quickly bind to the hydrophobic domains of serum proteins such as albumin and lipoproteins. It can be considered that hydrophobic molecules mainly bind to albumin as it is the most abundant protein in serum, and it has multiple hydrophobic binding pockets. As an example, the Glu-Probe has two hydrophobic domains that can potentially bind to albumin (or others); ICG and n-terminal palmitoyl (C16) modification. Thus, experiments were performed (FIGs. 32A and 32B) to understand the interaction between the probes with albumin and other blood proteins.
  • the albumin-binding properties of three ICG conjugated probes were investigated: Glu-Probe, NoSA, Lys-Probe, and free ICG.
  • Glu-Probe (10 mM) demonstrated a broad adsorption band, and its fluorescence was almost completely quenched as a result of the close packing of ICG molecules in the micelle structures. While some aggregation was observed for free ICG (10 mM) it was mostly solubilized in PBS with a slightly broadened absorption band and a fairly intense fluorescence spectrum.
  • NoSA probe (10 mM) a monomeric absorption peak and bright fluorescence were detected in PBS due to the good solubility of this probe.
  • the Glu-Probe was also incubated with other proteins, immunoglobulins and fibrinogen and measured its fluorescence using a plate reader (FIG. 35). While the increase in the fluorescence was lower compared to BSA, a significant increase in the fluorescence for these proteins was observed. This result suggests that Glu-Probe can also bind other proteins in circulation.
  • FIGs. 32, 33, 34, and 35 suggest that upon introduction into circulation, self-assembled structures formed by Glu-Probe (or other probes) can disassociate through probe binding to the hydrophobic domains of the proteins present in the blood including albumin, lipoproteins, IgG, and fibrinogen. Protein binding provides the probes prolonged blood circulation (FIG. 12) and can improve their tumor accumulation (FIG 36).
  • Positive correlation with angiogenesis 4T1 cells were injected into wildtype mouse mammary fat pad. Mice were then treated intratumorally with axitinib which is a VEGF inhibitor to suppress angiogenesis. After 3 treatments with axitinib, mice were injected IV with the Glu- Probe and fluorescent signal was measured. Axitinib reduced probe targeting to the tumor with a positive correlation with angiogenesis (CD31) (FIG. 37).
  • Probe accumulates at sites of wound healing. A small incision was created on the skin of the mouse and sutured back together. A week later the probe was injected IV and strongly went to the healing wound. Once the wound was completely healed, the probe no longer went to the site of the wound.
  • each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component.
  • the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.”
  • the transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts.
  • the transitional phrase “consisting of” excludes any element, step, ingredient or component not specified.
  • the transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant decrease in detection of tumors less than 1 mm in size.
  • the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ⁇ 20% of the stated value; ⁇ 19% of the stated value; ⁇ 18% of the stated value; ⁇ 17% of the stated value; ⁇ 16% of the stated value; ⁇ 15% of the stated value; ⁇ 14% of the stated value; ⁇ 13% of the stated value; ⁇ 12% of the stated value; ⁇ 11% of the stated value; ⁇ 10% of the stated value; ⁇ 9% of the stated value; ⁇ 8% of the stated value; ⁇ 7% of the stated value; ⁇ 6% of the stated value; ⁇ 5% of the stated value; ⁇ 4% of the stated value; ⁇ 3% of the stated value; ⁇ 2% of the stated value; or ⁇ 1% of the stated value.

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Abstract

L'invention concerne des nanomatériaux à auto-assemblage et leurs utilisations dans la détection, l'imagerie et le traitement du cancer. Les nanomatériaux à auto-assemblage comprennent une pluralité de composants à auto-assemblage. Chaque composant à auto-assemblage est amphiphile, y compris un motif d'auto-assemblage hydrophobe fonctionnellement relié à un motif hydrophile, ce par quoi, en cas de dissolution dans une solution aqueuse, les composants à auto-assemblage forment une structure micellaire et orientent généralement les motifs hydrophiles pour rester en contact avec la solution aqueuse, formant ainsi le nanomatériau à auto-assemblage. Les composants à auto-assemblage peuvent en outre comprendre un site clivable par protéase et/ou une molécule fonctionnelle. La molécule fonctionnelle peut comprendre un colorant pour la détection et/ou l'imagerie ou un médicament pour le traitement du cancer.
PCT/US2022/073964 2021-07-20 2022-07-20 Nanomatériau à auto-assemblage pour la détection, l'imagerie ou le traitement du cancer Ceased WO2023004364A2 (fr)

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WO2025080800A1 (fr) * 2023-10-10 2025-04-17 Oregon Health & Science University Sondes de détection de l'activité de protéases pour la détection et le pronostic du cancer

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EP2550529B1 (fr) * 2010-03-23 2021-11-17 Iogenetics, LLC. Procédés bioinformatiques pour déterminer la liaison de peptides
EP2678042B1 (fr) * 2011-02-23 2018-05-09 The Board of Trustees of the University of Illionis Dendron-hélices amphiphiles, micelles de ceux-ci et utilisations
WO2014130846A1 (fr) * 2013-02-22 2014-08-28 Seungpyo Hong Administration d'un médicament par voie transdermique faisant appel à des micelles amphiphiles « dendron-coil »
US10869939B2 (en) * 2015-08-03 2020-12-22 Ramot At Tel-Aviv University Ltd. Delivery system in micellar form having modular spectral response based on enzyme-responsive amphiphilic PEG-dendron hybrid polymers

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WO2025080800A1 (fr) * 2023-10-10 2025-04-17 Oregon Health & Science University Sondes de détection de l'activité de protéases pour la détection et le pronostic du cancer

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