WO2023004364A2

WO2023004364A2 - Self-assembling nanomaterial for the detection, imaging, or treatment of cancer

Info

Publication number: WO2023004364A2
Application number: PCT/US2022/073964
Authority: WO
Inventors: Adem YILDIRIM; Jared FISCHER; Bruce Branchaud
Original assignee: Oregon Health and Science University
Current assignee: Oregon Health and Science University
Priority date: 2021-07-20
Filing date: 2022-07-20
Publication date: 2023-01-26
Anticipated expiration: 2024-01-20
Also published as: WO2023004364A8; WO2023004364A3

Abstract

Self-assembling nanomaterials and uses thereof in the detection, imaging, and treatment of cancer are described. 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 the hydrophilic motifs to remain in contact with the aqueous solution, thereby forming the self-assembled nanomaterial. Self-assembling components can further include a protease cleavable site and/or a functional molecule. The functional molecule can include a dye for detection and/or imaging or a drug for treatment of cancer.

Description

SELF-ASSEMBLING NANOMATERIAL FOR THE DETECTION, IMAGING, OR TREATMENT OF CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to US Provisional Patent Application No. 63/223,902 filed July 20, 2021 and US Provisional Patent Application No. 63/253,093 filed October 6, 2021 , the contents of both of which are incorporated by reference herein in their entirety.

REFERENCE TO SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 0046-0068PCT.XML. The text file is 72.9 KB, was created on July 18, 2022, and is being submitted electronically via EFS-Web.

FIELD OF THE DISCLOSURE

[0003] 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.

BACKGROUND OF THE DISCLOSURE

[0004] 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. However, current clinical imaging modalities are limited to >5 mm tumors and cannot accurately identify tumor margins. While small molecular probes can easily extravasate from circulation and penetrate deep into solid malignancies, they can rapidly be cleared from circulation and tissues. On the other hand, 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. However, tumor accumulation is usually low due to their larger sizes, limiting deep tissue penetration.

[0005] To this end, enzyme instructed self-assembly (EISA) of small molecules or nanoparticles has been used to improve the accumulation and retention of functional molecules. In EISA, 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. Up to date, 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). However, existing EISA methods require a self-assembling component that is cleavable by a protease, adding complexity to the EISA method.

SUMMARY OF THE DISCLOSURE

[0006] 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. In their simplest form, 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.

[0007] In particular embodiments, the self-assembling component further includes a functional molecule operatively connected to the self-assembling component. In particular embodiments, the functional molecule includes a dye for detection and/or imaging. In some embodiments, the functional molecule is a drug or a prodrug. In particular examples, the functional molecule is attached to the hydrophilic motif.

[0008] In some embodiments, the self-assembly motif is optionally operatively connected to the hydrophilic motif by a spacer. In particular embodiments, the spacer includes a linker and/or a substrate having a cleavage site. In particular embodiments, the linker includes a glycine, a glycine-histidine, or a proline-rich linker.

[0009] In some embodiments, the optional substrate having a cleavage site is cleaved by a protease. These embodiments can be used to detect protease activity or elicit an effect where protease activity is high (e.g., at a tumor site). For example, self-assembled nanomaterials with a functional molecule (e.g., a dye or drug) form a micellular structure that quenches or prevents release of the signal or effect of the functional molecule. Upon cleavage of the substrate by the protease, the hydrophilic motifs are disconnected and the functional molecule is released at the site where the target protease is present. [0010] Additional aspects and advantages of the disclosed self-assembling nanomaterials will be apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

[0011] Some of the drawings submitted herein may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0012] FIGs. 1A, 1B. Schematic showing an embodiment of a self-assembling component. FIG. 1A) 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. In this example, 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. If 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. If 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. As used herein, a “probe” refers to a self-assembled nanomaterial.

[0013] 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. In certain examples, 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.

[0014] 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, and FIG. 3F) NoSubs (No Substrate)

[0015] 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). [0016] FIG. 5. Structures of the self-assembling components used in the experimental examples. Lower case letters are symbols of d amino acids ami¹ indicates that the residue is amidated.

[0017] 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.

[0018] FIGs. 7A-7C. 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.

[0019] FIGs. 8A-8D. FIG. 8A) 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. 8D) A similar self-quenched probe was also designed for another protease, Cathepsin G, by simply changing the cleavable substrate portion of the self-assembling component. A linear increase in the signal with increasing protease concentration was also observed for this probe (CatG).

[0020] 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.

[0021] 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.

[0022] FIG. 11. Mice were IV injected with the Glu-Probe into Balb/c mice containing 4T1 breast cancer cells located in the mammary fat pad. 2 days after probe injection, mice were euthanized, and probe tissue distribution was determined using fluorescence. Tumors were between 2-5 mm in size. Tumors had significantly more fluorescent signal compared to normal tissue (n=3, p<0.001).

[0023] 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.

[0024] FIGs. 13A, 13B. FIG. 13A) Blood toxicity did not change for white blood cells (WBC), red blood cells (RBC), platelets (PLT), Hemoglobin (HGB) and hematocrit (HCT). FIG. 13B) In addition, there was no change in the liver toxicity measured by alanine serum transferase or creatinine levels.

[0025] FIG. 14A, 14B. Tumor accumulation (FIG. 14A) and tumor to background ratio (FIG. 14B) of different probes.

[0026] 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. To form xenografts, 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).

[0027] 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.

[0028] 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).

[0029] FIG. 18. Targeting transgenic mouse models of cancer. A transgenic model of breast cancer (mouse mammary tumor virus (MMTV) mouse model) was used and the Glu-Probe was injected IV (50 nmole) at varying times. Probe was imaged using the IVIS® (Xenogen Corporation, Hopkinton, MA) machine for fluorescence (red-yellow). (Top) The probe was able to predict the location of tumor before the tumor was visible or palpable (arrows). (Bottom) The signal was specific for the tumor compared to other normal tissues.

[0030] 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. [0031] 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.

[0032] FIG. 21. Glu-Probe accumulation in occult 4T1 tumors with submillimeter sizes.

[0033] 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). 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.

[0034] FIGs. 23A, 23B. FIG. 23A) To investigate the suitability of the Glu-Probe (sometimes referred to as Legumain probe) for image-guided surgery, 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.

[0035] 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).

[0036] 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. [0037] 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. MRI images were taken at different time points after probe injection (left and middle panels) and IVIS® (Xenogen Corporation, Hopkinton, MA) imaging of a brain slice (right panel). [0038] 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. [0039] FIG. 28. Comparison of performance of the Glu-Probe with commercially available products; MMPsense, 800CW-2DG, and cRGD-ICG using 4T1 mouse model.

[0040] 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).

[0041] 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.

[0042] 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).

[0043] 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.

[0044] FIGs. 33A, 33B. Time dependent absorbance (FIG. 33A) or fluorescence (3 FIG. 3B) spectra of different probes (10 pM) in 20% mouse plasma.

[0045] FIG. 34. Fluorescence quenching of BSA fluorescence in the presence different amounts of probes.

[0046] FIG. 35. Fluorescence spectra of Glu-Probe after incubating with different proteins. [0047] FIG. 36. Proposed mechanism of improved tumor accumulation for Glu-Probe and similar probes.

[0048] FIG. 37. Reducing angiogenesis decreases the tumor accumulation of the Glu-Probe in 4T1 tumors.

[0049] FIG. 38. Tumor accumulation of the Glu-Probe in Matrigel® plugs. (Left panel) I VIS® (Xenogen Corporation, Hopkinton, MA) imaging of a nude mice with a Matrigel® plug. (Right panels) White light and IVIS® (Xenogen Corporation, Hopkinton, MA) images of removed Matrigel® plugs.

[0050] 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.

[0051] 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.

[0052] 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. These results suggest that the Glu-Probe-Cy5 is entering cells through endocytosis mediated by heparan sulfate proteoglycan (HSPG) binding.

[0053] FIG. 42. Table of probes with random spacers.

[0054] 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.

[0055] FIG. 44. Table of probes with only hydrophilic domain.

[0056] 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.

[0057] FIG. 46. Table of probes with different hydrophilic domains.

[0058] 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). [0059] 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. [0060] FIG. 49. Tumor signal of the probes with different self-assembly motifs in 4T1 mouse model 2 days after injection. Negatively charged Fmoc and Phe-Probe-1 demonstrated high tumor accumulation. Positively charged Phe-Probe-2 demonstrated low tumor accumulation. [0061] FIG. 50. Sequences of exemplary linkers, substrates, hydrophilic motifs, and peptide combinations.

[0062] FIG. 51. Table of self-assembling components and their associated self-assembly motif, linker, substrate, and hydrophilic motif.

[0063] FIG. 52. Table of functional molecules and exemplary self-assembling components.

DETAILED DESCRIPTION

[0064] The current disclosure provides self-assembling nanomaterials for the detection, imaging, and/or treatment of cancer. In particular embodiments, the self-assembled nanomaterials include a plurality of self-assembling components. In particular embodiments, 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. Upon dissolution in an aqueous solution, 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.

[0065] “Self-assembled” as used herein means a multi-subunit nanomaterial formed from subunit monomers that, under suitable conditions, form the multi-subunit nanomaterial.

[0066] 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. In some embodiments, the self-assembly motif includes a saturated hydrocarbon, an unsaturated hydrocarbon, an aromatic hydrocarbon, a fluorocarbon, a hydrophobic amino acid, or combinations thereof. In particular embodiments, the saturated hydrocarbon is a long chain saturated hydrocarbon. In some embodiments, 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). In particular embodiments, the saturated hydrocarbon includes lauroyl, tridecanoyl, myristoyl, pentadecenoyl, palmitoyl, heptadecanoyl, stearoyl, nonadecanoyl, heneicosanoyl, or behenoyl. In particular embodiments, hydrophobic amino acids include F, Y, A, V, I, L, or W, or combinations thereof. In particular embodiments, the self-assembly motif includes a peptide sequence. In particular embodiments, 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. In certain examples, 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. [0067] In particular embodiments, the self-assembly motif includes any hydrophobic moiety that can collocate upon dissolution in an aqueous solution including waxes, lipids, cholesterol, or steroid hormones.

[0068] The hydrophilic motif includes a hydrophilic moiety. In particular embodiments, the hydrophilic motif can include a peptide and/or a polyethylene glycol (PEG). In some embodiments, 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), EKEKEGCGKEKEK (SEQ ID NO: 33), EE, RR, ECE, EGEE (SEQ ID NO: 15), or CGEKEE (SEQ ID NO: 16). Lowercase amino acids indicate D-amino acids.

[0069] . In some embodiments, the hydrophilic motif includes polyethylene glycol (PEG). In certain examples, the hydrophilic motif includes 3-10 PEG molecules (e.g., 3, 4, 5, 6, 7, 8, 9 or 10). In certain examples, the hydrophilic motif includes PEG₆ or glycine (G)-PEG, for example, G-PEG3, G-PEG4, G-PEG5, G-PEG6, G-PEG7, G-PEGs, G-PEGg, or G-PEG10· In certain examples, the hydrophilic motif includes G-PEG_&

[0070] In some embodiments, the self-assembling component further includes a functional molecule operatively connected to the self-assembling component. In some embodiments, the functional molecule includes a dye for detection and/or imaging. In some embodiments, the dye is a near-infrared (NIR) dye. Near-infrared (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. In particular embodiments, 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. In particular embodiments, the NIR dye includes indocyanine green (ICG), sulfo-Cy7-maleimide (Cy7) dye, or IR-783.

[0071] In particular embodiments, the dye includes two functional molecules. In particular embodiments, the dye includes an NIR fluorescent dye attached to an MRI contrast agent to create and MRI/NRI Probe. In particular embodiments, an MRI/NRI Probe includes the functional molecules ICG and Gd-DOTA. In particular embodiments, dyes can be used to prepare a self-assembling component for PET/SPECT imaging.

[0072] As will be understood by one skilled in the art, 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. In particular embodiments, an MRI contrast agent is Gd-DOTA. [0073] In some embodiments, the functional molecule is a drug or prodrug. In particular embodiments, the drug is an anti-cancer drug. In particular embodiments, an anti-cancer drug includes alkylating agents, nitrosoureas, antimetabolites, anthracyclines, topoisomerase I inhibitors, topoisomerase II inhibitors, mitotic inhibitors, and corticosteroids. In particular embodiments, alkylating agents include altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, and trabectedin. In particular embodiments, nitrosoureas include carmustine, lomustine, and streptozocin. In particular embodiments, 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. In particular embodiments, anthracyclines include daunorubicin, doxorubicin, doxorubicin liposomal, epirubicin, idarubicin, and valrubicin. In particular embodiments, topoisomerase I inhibitors (also called camptothecins) include irinotecan, irinotecan liposomal, topotecan, and CL2-SN- 38. In particular embodiments, topoisomerase II inhibitors (also called epipodophyllotoxins) include etoposide (VP-16), mitoxantrone, and teniposide. In particular embodiments, mitotic inhibitors include cabazitaxel, docetaxel, Nab-paclitaxel, paclitaxel, vinblastine, vincristine, vincristine liposomal, and vinorelbine. In particular embodiments, corticosteroids include prednisone, methylprednisolone, and dexamethasone. In particular embodiments, 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. In particular embodiments, the drug is functionalized, for example, with an iodine. In some embodiments, a functionalized doxorubicin includes N-(lodoacetamido)-Doxorubicin (Dox). In particular embodiments, the drug includes CL2-SN-38 (contains the topoisomerase I inhibitor, SN-38).

[0074] In some embodiments, the self-assembly motif is operatively connected to the hydrophilic motif by a spacer. In particular embodiments, the spacer includes a linker and/or a substrate.

[0075] 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. In one aspect of this embodiment, 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. In some embodiments, the linker is from 1 to 50 amino acids in length or 1 to 30 amino acids in length. In some embodiments, 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).

[0076] Particular examples can utilize proline-rich linkers. In particular embodiments, a proline-rich linker is a peptide sequence having more proline residues than would be expected based on chance alone. In particular embodiments, 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. Particular examples of proline-rich linkers include fragments of proline-rich salivary proteins (PRPs). In particular embodiments, a proline-rich linker includes PPPP (SEQ ID NO: 21).

[0077] In particular embodiments, the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (Gly_xSer_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). Particular examples include (Gly Ser)_n (SEQ ID NO: 57), (Gly₃Ser)_n(Gly₄Ser)_n (SEQ ID NO: 58), (Gly₃Ser)_n(Gly2Ser)_n (SEQ ID NO: 59), or (Gly3Ser)_n(Gly₄Ser)1 (SEQ ID NO: 60), wherein n is an integer including, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more. In particular embodiments, the linker is (Gly Ser)₄ (SEQ ID NO: 61), (Gly Ser) ₃ (SEQ ID NO: 62), (Gly₄Ser)₂ (SEQ ID NO: 63), (Gly₄Ser)_! (SEQ ID NO: 64), (Gly₃Ser)₂ (SEQ ID NO: 65), (Gly₃Ser)_! (SEQ ID NO: 66), (Gly₂Ser)₂ (SEQ ID NO: 67) or (Gly₂Ser)i, GGSGGGSGGSG (SEQ ID NO: 68), GGSGGGSGSG (SEQ ID NO: 69), GGSGGGSG (SEQ ID NO: 70), or GGSGGSGG (SEQ ID NO: 17).

[0078] In particular embodiments, 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.

[0079] Additional exemplary, peptide linkers include AAEPKSS (SEQ ID NO: 72), AAEPKSSDKTHTCPPCP (SEQ ID NO: 73), or GGGGDKTHTCPPCP (SEQ ID NO: 74). Alternatively, a variety of 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.

[0080] In some embodiments, the spacer includes a substrate. The substrate refers to a peptide sequence having a cleavage site. In some embodiments, the cleavage site results in cleavage of the self-assembling component by a protease. [0081] In particular embodiments, 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.

[0082] In particular embodiments, 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.

[0083] 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. However, upon cleavage of the substrate of self-assembling components by proteases, disconnection of the hydrophilic motifs from the self-assembling components results in the effects of the functional molecule to no longer be quenched or for the functional molecule to be released. Because proteases are highly expressed in tumor environments, substrates are more frequently cleaved in those environments.

[0084] In particular embodiments, “released” means that the functional molecule is no longer trapped by the self-assembled nanomaterial but is able to have activity within its environment. In particular embodiments, 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).

[0085] In particular embodiments, the functional molecule is a dye (e.g., NIR dye). Upon cleavage of the substrate of the self-assembling components by proteases, disconnection of the hydrophilic motifs from the self-assembling components frees the dye from its quenched state. In particular embodiments, the dye is able to generate and convey a fluorescent signal. In particular embodiments, aggregation of the cleaved self-assembling components generates and conveys a stronger fluorescent signal at tumor sites (FIG. 1 B).

[0086] In particular embodiments, the functional molecule is a drug. Upon cleavage of the substrate by a protease, 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.

[0087] In some embodiments, the self-assembling component includes a self-assembly motif, a hydrophilic motif, and a functional molecule. In some embodiments the functional molecule is a fluorescent dye such as ICG or Cy7 or a drug such as doxorubicin or a drug such as taxol. In particular embodiments, the self-assembling component includes a spacer. In particular embodiments, the spacer includes a linker and/or a substrate that can be cleaved by a protease. In some embodiments, 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.

[0088] 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. In particular embodiments, “probes” refer to self-assembled nanomaterials in a form suitable for an intended use. In particular embodiments, “probes” refer to self-assembled nanomaterials in a form suitable for an intended use and in a form suitable for administration to a subject.

[0089] 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. Moreover, 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. [0090] In particular embodiments, 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.

[0091] In particular embodiments, 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.

[0092] The 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.

[0093] Also provided herein, are kits. Kits can include components to practice, for example, the methods described herein. In particular embodiments, kits can include self-assembling components (or components thereof (e.g., PEG) wherein the self-assembling components are not linked to a functional molecule. In particular embodiments, 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. In particular embodiments, kits can include self-assembling components that include a functional molecule linked to the self-assembling components. In particular embodiments, 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.

[0094] In particular embodiments, the kit can be tailored to include materials necessary for detection, imaging, or treatment. In some embodiments, the kit for imaging includes materials for high-resolution fluorescence imaging, NIR fluorescence imaging, photoacoustic imaging, and/or image-guided surgery imaging.

[0095] “Photoacoustic imaging” as used herein 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.

[0096] There are numerous uses for the self-assembling nanomaterials disclosed herein. Certain examples include detecting, imaging, or treating cancer within a subject. 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).

[0097] 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.

[0098] 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. In particular embodiments, a condition includes cancer expressing high protease activity. [0099] 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.

[0100] 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.

[0101] 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.

[0102] In particular embodiments, 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. [0103] A “tumor” is a swelling or lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells). A “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.

[0104] The self-assembled nanomaterials can similarly be used to deliver drugs to a tumor site. In particular embodiments, 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. Thus, in some embodiments, the accumulated self-assembled nanomaterials can release loaded drugs slowly and specifically to the tumors for extended periods.

[0105] In particular embodiments, 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. [0106] 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. In certain examples, the tumors have high protease activity (e.g., tumor environments). In particular embodiments, detection is for research, diagnostic, and/or prognostic uses. In particular embodiments, methods of detection include administering an effective amount of a composition disclosed herein having a dye as the functional molecule.

[0107] In particular embodiments, 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. The term “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.

[0108] In particular embodiments, 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).

[0109] In particular embodiments, methods for imaging 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 image the high protease activity environment (e.g., tumor site). By conjugating different functional molecules to the self-assembling components (e.g., radiolabels), different imaging modalities can be performed. 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.

[0110] While the disclosure is presented in the field of cancer imaging, detection, and treatment, the self-assembled nanomaterials can be used in other contexts, particularly in the presence of specific protease activity.

[0111] Following administration of the composition to a subject, and after a time sufficient for protease cleavage, the biodistribution of the composition can be visualized. The term “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.

[0112] In some embodiments, upon cleavage by a protease, the cleaved self-assembling components 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.

[0113] In particular embodiments, self-assembled nanomaterials accumulate in tumors, facilitating the detection, imaging, and/or treatment of cancer. For detection, 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). In particular embodiments, 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. In particular embodiments, 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. In particular embodiments, 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.

[0114] For administration, therapeutically effective amounts (also referred to herein as doses) 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.

[0115] Useful doses can range from 0.1 nmoles to 20 pmoles. In particular embodiments, a dose can range from 2.5 nmoles to 50 nmoles. In particular embodiments, 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. In particular embodiments, a dose can include 200 nmoles. In particular embodiments, a dose can include 10 pmoles.

[0116] Useful doses can range from 0.1 to 5 pg/kg or from 0.5 to 1 pg /kg. In other examples, 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. In other examples, 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.

[0117] 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). In particular embodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA-approved treatment protocol.

[0118] The 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.

[0119] In some embodiments, self-assembling components can be operably to solid supports (or “solid phase”) in order to form the micellar structure. Skilled persons will understand that examples of solid supports include microbeads, nanoparticles, dendrimers, surfaces, and membranes.

[0120] As used herein, the phrase “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.

[0121] Embodiments disclosed herein can have 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to sequences disclosed herein.

[0122] Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, 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.

[0123] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p. 224). 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 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (non-polar): Proline (Pro), Ala, Val, Leu, lie, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu, and lie; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0124] In making such changes, 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). These values are: lie (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glutamate (-3.5); Gin (-3.5); aspartate (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0125] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.

[0126] As detailed in US 4,554,101 , the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0127] As outlined above, 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. As indicated elsewhere, 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.

[0128] “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.

[0129] When linked together, different amino acid-based components of the self-assembling components disclosed herein form fusion proteins. As used herein, 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. As used herein, 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). For example, the amino acid sequences of bacterial enzymes such as B. stearothermophilus dihydrolipoyl acyltransferase (E2p) and the amino acid sequences of HIV- 1 gp120 or gp41 glycoproteins are not normally found joined together via a peptide bond. [0130] When a non-amino acid component is operatively connected to a peptide, the selfassembling components can be referred to as fusion molecules.

[0131] As used herein, the term “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. Thus, as used herein, 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. The term further includes 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. Conventional nomenclature exists in the art for polynucleotide and polypeptide structures.

[0132] For example, 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). Amino acid residues described herein are preferred to be in the “L” isomeric form. However, residues in the “D” isomeric form may be substituted for any L- amino acid residue provided the desired properties of the polypeptide are retained. For example as disclosed herein, 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, and a “G-PEG6 peptide” is a Glycine bound to six repeating polyethylene glycol linkers. [0133] As used herein, “polymer” means a chemical compound or mixture of compounds formed by polymerization and including repeating structural units. Polymers may be constructed in multiple forms and compositions or combinations of compositions.

[0134] As used herein, “operatively connected,” “operatively linked,” or “operably linked” refers to two distinct molecules connected by a spacer (e.g., a linker) or that are chemically bound to each other covalently.

[0135] The Exemplary Embodiments and Example below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0136] Exemplary Embodiments.

1. 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.

2. The plurality of self-assembling components of embodiment 1 , wherein the 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.

3. The plurality of self-assembling components of embodiment 2, wherein the saturated hydrocarbon includes 10-20 carbon atoms.

4. The plurality of self-assembling components of embodiments 2 or 3, wherein the saturated hydrocarbon includes palmitoyl.

5. The plurality of self-assembling components of embodiment 2, wherein the aromatic hydrocarbon includes Fmoc. 6. The plurality of self-assembling components of any of embodiments 1-5, wherein the hydrophobic self-assembly motif comprises a peptide sequence selected from GGGH (SEQ ID NO: 1), GGGh (SEQ ID NO: 2), PPPP (SEQ ID NO: 21), VFFC (SEQ ID NO: 39), or FFY.

7. The plurality of self-assembling components of any of embodiments 1-6, wherein the 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.

8. The plurality of self-assembling components of any of embodiments 1-7, wherein the hydrophilic motif of each of the plurality or a subset thereof includes a hydrophilic peptide and/or a polyethylene glycol (PEG).

9. The plurality of self-assembling components of embodiment 8, wherein the 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: 16).

10. The plurality of self-assembling components of embodiment 8, wherein the PEG is PEG₃-

10.

11. The plurality of self-assembling components of embodiments 8 or 10, wherein the PEG is PEGe.

12. The plurality of self-assembling components of embodiment 8, wherein the PEG is G- PEG3-10·

13. The plurality of self-assembling components of embodiments 8 or 12, wherein the PEG is G-PEGa

14. The plurality of self-assembling components of any of embodiments 1-13, wherein the hydrophilic motif of each of the plurality or a subset thereof includes a hydrophilic peptide and a PEG.

15. The plurality of self-assembling components of any of embodiments 1-14, wherein the hydrophobic self-assembly motif of each of the plurality or a subset thereof is operatively connected to hydrophilic motif via a spacer.

16. The plurality of self-assembling components of embodiment 15, wherein the spacer is a linker and/or a substrate.

17. The plurality of self-assembling components of embodiment 16, wherein the linker has the sequence G, AGQGLR (SEQ ID NO: 18), FGSGKD (SEQ ID NO: 19), or TGGYPVE (SEQ ID NO: 20. 18. The plurality of self-assembling components of embodiments 16 or 17, wherein in the linker includes a glycine-serine linker.

19. The plurality of self-assembling components of embodiment 18, wherein in glycine-serine linker is GGSGGSGG (SEQ ID NO: 17).

20. The plurality of self-assembling components of any of embodiments 16-19, wherein the linker includes a polyethylene glycol (PEG) linker.

21. The plurality of self-assembling components of any of embodiments 16-20, wherein the linker includes a polypropylene glycol linker.

22. The plurality of self-assembling components of any of embodiments 16-21, wherein the linker includes a polyoxyalkylene linker.

23. The plurality of self-assembling components of any of embodiments 16-22, wherein the linker is a copolymer of polyethylene glycol and polypropylene glycol.

24. The plurality of self-assembling components of any of embodiments 16-23, wherein the substrate includes a cleavage site that is cleaved by a protease.

25. The plurality of self-assembling components of embodiment 24, wherein the substrate has 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.

26. The plurality of self-assembling components of embodiments 24 or 25, wherein 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 7, Caspase 10, pyroglutamate aminopeptidase, acylamino-acid-releasing enzyme, Cathepsin C, carboxypeptidase, clostripain, subtilisin, proteinase K, or pancreatin.

27. The plurality of self-assembling components of any of embodiments 24-26, wherein the protease includes legumain, CathepsinG, or Matriptase.

28. The plurality of self-assembling components of any of embodiments 24-27, wherein the protease includes legumain.

29. The plurality of self-assembling components of any of embodiments 15-28, wherein the spacer includes a linker and a substrate.

30. The plurality of self-assembling components of embodiment 29, wherein the 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.

31. The plurality of self-assembling components of any of embodiments 15-30, wherein 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). 32. The plurality of self-assembling components of any of embodiments 1 -31 , further including a functional molecule operatively connected to each of the plurality or a subset thereof.

33. The plurality of self-assembling components of embodiment 32, wherein the functional molecule includes a dye.

34. The plurality of self-assembling components of embodiment 33, wherein the dye includes a fluorophore, affinity tag, radiolabel, and/or contrast agent.

35. The plurality of self-assembling components of embodiment 34, wherein the fluorophore includes an NIR dye.

36. The plurality of self-assembling components of embodiment 35, wherein 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.

37. The plurality of self-assembling components of any of embodiments 32-36, wherein the functional molecule is a drug or prodrug.

38. The plurality of self-assembling components of embodiment 37, wherein the drug is an anti-cancer drug.

39. The plurality of self-assembling components of embodiment 38, wherein the 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.

40. The plurality of self-assembling components of embodiment 39, wherein the alkylating agent includes altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, or trabectedin.

41. The plurality of self-assembling components of embodiment 39, wherein the nitrosourea includes carmustine, lomustine, or streptozocin.

42. The plurality of self-assembling components of embodiment 39, wherein 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, thioguanine, or trifluridine/tipiracil combination.

43. The plurality of self-assembling components of embodiment 39, wherein the anthracycline includes daunorubicin, doxorubicin, doxorubicin liposomal, epirubicin, idarubicin, orvalrubicin.

44. The plurality of self-assembling components of embodiment 39, wherein the topoisomerase I inhibitor includes irinotecan, irinotecan liposomal, topotecan, or CL2-SN-38.

45. The plurality of self-assembling components of embodiment 39, wherein the topoisomerase II inhibitor includes etoposide (VP-16), mitoxantrone, and teniposide. 46. The plurality of self-assembling components of embodiment 39, wherein the mitotic inhibitors include cabazitaxel, docetaxel, Nab-paclitaxel, paclitaxel, vinblastine, vincristine, vincristine liposomal, or vinorelbine.

47. The plurality of self-assembling components of embodiment 39, wherein the corticosteroid includes prednisone, methylprednisolone, or dexamethasone.

48. The plurality of self-assembling components of embodiment 38, wherein the 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.

49. The plurality of self-assembling components of any of embodiments 37-48, wherein the drug is functionalized with an iodine.

50. The plurality of self-assembling components of any of embodiments 37-49, wherein the drug is N-(lodoacetamido)-Doxorubicin (Dox).

51. The plurality of self-assembling components of any of embodiments 1-50, wherein the 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), EKEKEGCGKEKEK (SEQ ID NO: 33), EE, RR, ECE, EGEE (SEQ ID NO: 15), or CGEKEE (SEQ ID NO: 16).

52. The plurality of self-assembling components of any of embodiments 15-51, wherein of each of the plurality or a subset thereof further includes a spacer including the sequence GGGH (SEQ ID NO: 1).

53. The plurality of self-assembling components of any of embodiments 15-52, wherein the hydrophobic self-assembly motif of each of the plurality or a subset thereof includes a palmitoyl molecule and a peptide including the sequence GGGH (SEQ ID NO: 1), and the hydrophilic motif includes an ECEE (SEQ ID NO: 10) peptide.

54. The plurality of self-assembling components of embodiment 15-52, wherein the hydrophobic self-assembly motif of each of the plurality or a subset thereof includes a palmitoyl molecule and a peptide including the sequence GGGH (SEQ ID NO: 1), and the hydrophilic motif includes a DCDD (SEQ ID NO: 11) peptide.

55. The plurality of self-assembling components of any of embodiments 15-52, wherein the hydrophobic self-assembly motif of each of the plurality or a subset thereof includes a palmitoyl molecule and a peptide including the sequence GGGH (SEQ ID NO: 1), and the hydrophilic motif includes an KCKK (SEQ ID NO: 12) peptide. 56. The plurality of self-assembling components of any of embodiments 15-52, wherein the hydrophobic self-assembly motif of each of the plurality or a subset thereof includes a palmitoyl molecule and a peptide including the sequence GGGH (SEQ ID NO: 1), and the hydrophilic motif includes a KCEK (SEQ ID NO: 13) peptide.

57. The plurality of self-assembling components of any of embodiments 15-50, wherein the hydrophobic self-assembly motif of each of the plurality or a subset thereof includes a palmitoyl molecule and a peptide including the sequence GGGh (SEQ ID NO: 2), and the hydrophilic motif includes a ecee (SEQ ID NO: 14) peptide.

58. The plurality of self-assembling components of any of embodiments 15-52, wherein the hydrophobic self-assembly motif of each of the plurality or a subset thereof includes a palmitoyl molecule and a peptide including the sequence GGGH (SEQ ID NO: 1), and the hydrophilic motif includes PEG₆ or G-PEG₆.

59. The plurality of self-assembling components of any of embodiments 15-52, wherein the hydrophobic self-assembly motif of each of the plurality or a subset thereof includes a palmitoyl molecule and a peptide including the sequence GGGH (SEQ ID NO: 1), and the hydrophilic motif includes a G-PEG₆.

60. A self-assembled nanomaterial formed from the plurality of self-assembling components of any of embodiments 1-59.

61. The self-assembled nanomaterial of embodiment 60, wherein the self-assembled nanomaterial has a micellar structure 5-10 nm in diameter.

62. A self-assembled nanomaterial formed from the plurality of self-assembling components of any of embodiments 33-36.

63. The self-assembled nanomaterial of embodiment 62, wherein the self-assembling components include a cleavage site that is cleaved by a protease between the self-assembly motif and the hydrophilic motif.

64. A pharmaceutical composition including the self-assembled nanomaterial of embodiments 62 or 63 and a pharmaceutically acceptable carrier.

65. A self-assembled nanomaterial formed from the plurality of self-assembling components of any of embodiments 37-50.

66. The self-assembled nanomaterial of embodiment 65, wherein the self-assembling components include a cleavage site that is cleaved by a protease between the self-assembly motif and the hydrophilic motif.

67. A pharmaceutical composition including the self-assembled nanomaterial of embodiments 65 or 66 and a pharmaceutically acceptable carrier.

68. 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. 69. The method of embodiment 68, wherein the tumor is less than 5 mm in diameter.

70. The method of embodiment 69, wherein the tumor is less than 1 mm in diameter.

71. The method of any of embodiments 68-70, wherein 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.

72. The method of any of embodiments 68-71 , wherein 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.

73. The method of any of embodiments 68-72, further including detecting protease activity.

74. 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.

75. The method of embodiment 74, wherein the cancer is colon cancer, pancreatic cancer, melanoma, breast cancer, kidney cancer, lung cancer, or glioma.

76. The method of embodiments 74 or 75, further including detecting protease activity.

77. The method of any of embodiments 74-76, wherein the administering is through intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, oral, intrapulmonary, subcutaneous, or sublingual administering.

[0137] Experimental Example.

[0138] Introduction. Self-assembling peptides with a myriad of morphologies and functionalities have been developed in recent years by exploiting the rich chemistry of amino acid side chains and synthetic and natural peptide modifications. Among their many potential uses, these naturally biodegradable and biocompatible materials are especially promising for cancer theranostics with their finely tunable properties. Thus, they have been extensively explored as delivery vehicles or contrast agents to develop better targeted cancer therapies and molecular imaging methods. A common strategy to enable their tumor-specific accumulation is targeting the cell surface receptors such as HER-2, EGFR, PSMA, or integrins. While molecular targeting showed promise in clinical trials, such materials can only target a subpopulation of patients whose tumors express the targeted biomarkers at a sufficiently high level. For example, EGFR is only overexpressed in 50% of glioblastoma tumors. Similarly, HER-2 positivity of primary tumors and metastasis are low; 30% and 55%, respectively. Enzyme-instructed self-assembly (EISA) of small peptides is another tumor targeting approach for 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. However, 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.

[0139] Here, 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. The cancer-specific accumulation of the self-assembled nanoprobe in vivo in several xenografts, syngeneic orthografts, experimental metastatic disease, transgenic models, and patient-derived xenografts of breast, colon, skin, and pancreatic cancers was demonstrated. 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. In addition, the self-assembled nanomaterial outperformed several commercially available tumor imaging products such as MMPsense™ 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.

[0140] 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.

[0141] In addition, it can be used to deliver chemotherapeutic drugs with better efficiency and reduced side effects. Here, it was also shown that the probe could be conjugated to a chemotherapy drug, doxorubicin, to significantly reduce its side effects without reducing its efficacy.

[0142] Finally, it was shown that the peptide probes developed here could also accumulate in wounds and senescent cells, showing the application potential of the materials developed here in other applications beyond cancer.

[0143] Results.

[0144] Design of the Probes. The overall design of the peptide probes is shown in FIGs. 1A, 1B, and 2. 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.)· In addition, a spacer motif (randomly selected or rationally designed amino acid sequences) can be added between hydrophilic and self-assembly motifs. One function of the spacer is that it can be specifically cleaved by a protease, such as legumain, matriptase, or matrix metalloproteinases (MMPs). The cleavage of the substrate can induce aggregation of the self-assembling component and improve tumor accumulation. It can also help to release the attached drug specifically in the site of tumors.

[0145] 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. For maleimide coupling, peptides and maleimide functionalized molecules (such as ICG-maleimide) are mixed (usually at a 1 :1 molar ratio, but functional molecules can be used in excess) in dimethyl sulfoxide (DMSO) or a buffer solution with a pH of 6.5-7. For NHS coupling, peptides and NHS functionalized molecules (such as ICG-NHS) 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. N-(lodoacetamido)-Doxorubicin and IR-783 are two examples of such molecules. Then, 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. The successful conjugation reaction is verified using liquid chromatography-mass spectrometry (LC-MS) measurements. Morphology of the peptides is investigated using transmission electron microscopy (TEM). 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.

[0146] 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). On the other hand, it was found that the 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.

[0147] Effect of proteolytic on the morphology and optical properties of peptide probes.

The hydrolysis of the probes by a target protease (or another enzyme) can induce morphological transformation as a result of increased hydrophobicity. To explore this, 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.

[0148] The changes in the optical properties of the Glu-Probe before and after incubation with different amounts of legumain was also investigated (FIGs. 8A-8D). When the Glu-Probe was dispersed in PBS, 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_. Finally, it was shown that 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).

[0149] Tumor imaging in living mice. In initial studies, the accumulation of Glu-Probe was tested in 4T1 syngeneic breast tumor-bearing Balb/c mice as this model is known to be expressing high levels of legumain and has been widely used to validate legumain responsive materials. 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. The accumulation of different probes were also studied to investigate the effects of different motifs on the performance of the probes. First, a peptide without a self- assembly motif (NoSA) was prepared. This peptide also accumulated strongly in the tumor; however, it demonstrated different kinetics. The NoSA probe peaked at an earlier time point and decayed more quickly than the Glu-Probe, suggesting the importance of self-assembly motifs for better tumor retention. A probe with a different hydrophilic motif (KCKK (SEQ ID NO: 12)), referred to as Lys-Probe, was also tested. This positively charged peptide forms short nanorods instead of micelles, as discussed above (FIG. 4B). It was found that the tumor accumulation of this probe was significantly lower than Glu-Probe and NoSA indicating the importance of negative charge and/or micelle morphology for high tumor accumulation. Finally, the tumor accumulation of free ICG was tested as control which showed very little accumulation as expected. 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.

[0150] To identify an optimal dose for further studies, 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.

[0151] Pharmacokinetics of the Glu-Probe. The organ distribution of the Glu-Probe in Balb/c mice containing 4T 1 tumors in the mammary fat pad were studied. Two days after probe injection, organs were collected, and probe distribution was determined using an IVIS® (Xenogen Corporation, Hopkinton, MA) system (FIG. 11). Even though the tumor sizes used in this experimental example were small (2-5 mm), the tumor signal was still very strong compared with other organs. There was a lower signal (at least 4 fold) in the liver, kidney, and lung. The ICG signal in other organs was below the detection limit. There was signal in the stool but not in urine 2 days after probe injection, indicating that the probe was mainly eliminated from the body through bile ducts, which is also the predominant clearance mechanism reported for free ICG.

[0152] Next, the blood circulation time of the probe was studied in wild-type mice. For this experiment, the Glu-Probe or free ICG (50 nmole) was intravenously injected into wild type mice. At different time points, 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). In addition, Glu-Probe was still at detectable levels even 7 days after injection.

[0153] The toxicity of the probe in wild type mice was also studied. Mice were injected with a high dose of Glu-Probe (0.4 pmole). Even at this very high dose, blood toxicity results did not change for white blood cells (WBC), red blood cells (RBC), platelets (PLT), Hemoglobin (HGB) and hematocrit (HCT) (FIG. 13A). In addition, there was not change in the liver toxicity measured by alanine serum transferase or creatinine levels (FIG. 13B). These results indicate the good biocompatibility of the probes developed here.

[0154] Glu-Probe does not require protease activity to accumulate in solid tumors.

Several previous studies have reported that enzymatic activity-induced self-assembly of peptide probes could increase their accumulation in solid tumors. This process, usually called as enzyme-instructed self-assembly (EISA), has been shown to be an effective strategy for improved tumor imaging and drug delivery. To investigate the contribution of the EISA process on the specific tumor accumulation of legumain responsive Glu-Probe, several control probes were tested. First, Glu-Probe with the same amino sequence but composed of d-amino acids (d-amino) was prepared. Two probes were also prepared with different substrates (spacers). In these probes, 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. The latter (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).

[0155] 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).

[0156] 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. For the A375 tumor model, 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. Remarkably, the Glu-Probe could also detect the lymph node invasion of A375 cells (FIG. 15). [0157] In addition, 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.

[0158] To further evaluate the tumor accumulation of Glu-Probe in a broad range of tumors, patient-derived colon or pancreatic cancer xenografts were used. The Glu-Probe also demonstrated a strong accumulation in the patient-derived tumors (FIG. 17).

[0159] Encouraged by the above results, the Glu-Probe accumulation was explored in a transgenic mouse model. 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).

[0160] FIG. 19 shows the average Glu-Probe tumor signal generated for all of the mouse models tested in living mice. In general, 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.

[0161] The Glu-Probe can detect early disease and occult lesions. Next, 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). 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. [0162] To investigate the potential of the Glu-Probe in detecting occult lesions, 10⁵ red fluorescent protein (RFP) expressing 4T1 cells were injected into the fat pad of wild type mice. 1 day after cell injection, Glu-Probe (50 nmole) was injected IV, and mice were sacrificed 1 day after probe injection. While the tumor was not visually observable, IVIS® (Xenogen Corporation, Hopkinton, MA) imaging showed that the RFP fluorescence of 4T1 cells overlapped with the ICG signal of the probe (FIG. 21). This result indicates that the Glu-Probe can detect small occult tumors at an early stage.

[0163] 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. While there were no visible metastatic lesions in the lung, IVIS® (Xenogen Corporation, Hopkinton, MA) imaging found that the ICG signal and luciferase signal overlapped nicely in a very small lesion, showing that the probe can detect occult lesions (FIG. 22B). IVIS® (Xenogen Corporation, Hopkinton, MA) measurements with cultured HCT-116 cells estimated a cell number of 7500 cells in the detected lesion (FIG. 22C). Finally, 4T1 cells were also injected to develop lung metastases. The probe was also accumulated in these lesions (FIG. 22D).

[0164] Glu-Probe can be used for image-guided surgery. To explore the applicability of Glu-Probe in 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). Next, 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.

[0165] Glu-Probe accumulates in orthotopic brain tumors in rats. To develop rat gliomas, 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.

[0166] Probes can be used for photoacoustic imaging. To test the ability of probes to increase the contrast of photoacoustic images, 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).

[0167] MFtl/NIFt Fluorescence dual-mode imaging of orthotopic brain tumors in rats.

RG2 rat glioma model described above was also used in these studies. For dual-mode imaging 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. To attach Gd- DOTA, first DOTA-maleimide was conjugated to the cysteine residue of the peptide as described above. Then, ICG-NHS was conjugated to the lysine residue of the peptide as described above. Gd was loaded to the peptide by incubating the ICG and DOTA conjugated peptide with Gd (1.5x excess Gd) in water overnight. Finally, 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).

[0168] 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. The experiments with 4T1 tumors developed on inflamed mammary glands in wild type mice showed that the Glu-Probe could detect these tumors in the presence of an inflammation background.

[0169] 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). All probes (50 nmole) were intravenously injected into 4T1 tumor bearing mice and imaged using an IVIS® (Xenogen Corporation, Hopkinton, MA) system. At day 2, the average tumor intensity was significantly higher for Glu-Probe than all of the other probes tested (FIG. 28).

[0170] Protein binding properties of the peptides. When 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. In PBS, 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. For the NoSA probe (10 mM), a monomeric absorption peak and bright fluorescence were detected in PBS due to the good solubility of this probe. The addition of mouse plasma (20%) or bovine serum albumin (BSA, 10 mg/ml_) resulted in substantial changes in the optical properties of all molecules (FIGs. 32A and 32B). After 2 h of incubation in plasma or BSA solution, a red-shifted monomeric ICG absorption peak was observed, and fluorescence enhancement was observed for all of the probes, indicating the binding of these molecules to albumin or other proteins. The evaluation of absorbance and fluorescence spectra of the probes over time in mouse plasma and BSA solution was also investigated (FIGs. 33A and 33B). While the red-shift or fluorescence increase for NoSA probe and free ICG was almost immediately observed after plasma addition, for Glu-Probe, the red- shifted monomeric ICG absorption peak was gradually evaluated over 80 min. Similarly, its fluorescence was increased slowly. The slower binding kinetics observed for Glu-Probe indicates that in the presence of proteins, micelles of Glu-Probe slowly dissociate through gradual protein binding. Interestingly, the optical properties of the Lys-Probe probe did not change significantly after 2 h of incubation, suggesting a stronger intramolecular interaction for the rod-shaped self-assemblies of this probe. This result also shows that protein binding is required for high tumor accumulation of peptide probes (FIG. 9D).

[0171] To further investigate the protein binding properties of the probes, fluorescence quenching experiments were performed utilizing the intrinsic fluorescence of tyrosine moieties of albumin, which can be quenched upon binding of aromatic molecules (such as ICG) to the hydrophobic pockets of albumin. For these studies, probes (0-25 mM) were incubated with BSA (2 mM), and fluorescence of tyrosine was detected using a fluorimeter. It was found that the Glu-Probe probe can more strongly bind to albumin than NoSA and Lys-Probe (FIG. 34), further showing that protein binding is necessary for strong tumor accumulation.

[0172] 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.

[0173] The results presented in 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). [0174] 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).

[0175] Nude mice were injected subcutaneously with Matrigel®. The Matrigel® was allowed to induce angiogenesis. Then the Glu-Probe probe was injected IV and mice were imaged for signal. Signal was localized to the Matrigel® plug (FIG. 38 left). The plug was removed and there was a positive correlation between angiogenesis and fluorescence (FIG. 38 right).

[0176] 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.

[0177] Closing Paragraphs.

[0178] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, 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.

[0179] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, 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.

[0180] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0181] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0182] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0183] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0184] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.

[0185] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0186] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. [0187] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the following examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

CLAIMS What is claimed is:

1. A plurality of self-assembling components to form a self-assembled nanomaterial, each of the plurality of self-assembling components comprising: a hydrophobic self-assembly motif operatively connected to a hydrophilic motif.

2. The plurality of self-assembling components of claim 1 , wherein the hydrophobic self- assembly motif of each of the plurality or a subset thereof comprises a saturated hydrocarbon, an unsaturated hydrocarbon, an aromatic hydrocarbon, a fluorocarbon, a hydrophobic amino acid, or combinations thereof.

3. The plurality of self-assembling components of claim 2, wherein the saturated hydrocarbon comprises 10-20 carbon atoms.

4. The plurality of self-assembling components of claim 2, wherein the saturated hydrocarbon comprises palmitoyl.

5. The plurality of self-assembling components of claim 2, wherein the aromatic hydrocarbon comprises Fmoc.

6. The plurality of self-assembling components of claim 1 , wherein the hydrophobic self- assembly motif comprises a peptide sequence selected from GGGH (SEQ ID NO: 1), GGGh (SEQ ID NO: 2), PPPP (SEQ ID NO: 21), VFFC (SEQ ID NO: 39), or FFY.

7. The plurality of self-assembling components of claim 1 , wherein the hydrophobic self- assembly motif comprises palmitoyl-GGGH (SEQ ID NO: 1), palmitoyl-GGGh (SEQ ID NO: 2), palmitoyl-PPPP (SEQ ID NO: 21), or Fmoc-FFY.

8. The plurality of self-assembling components of claim 1 , wherein the hydrophilic motif of each of the plurality or a subset thereof comprises a hydrophilic peptide and/or a polyethylene glycol (PEG).

9. The plurality of self-assembling components of claim 8, wherein the hydrophilic peptide comprises 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: 16).

10. The plurality of self-assembling components of claim 8, wherein the PEG is PEG3-10.

11. The plurality of self-assembling components of claim 8, wherein the PEG is PEG6.

12. The plurality of self-assembling components of claim 8, wherein the PEG is G-PEG3- 10

13. The plurality of self-assembling components of claim 8, wherein the PEG is G-PEG6.

14. The plurality of self-assembling components of claim 1 , wherein the hydrophilic motif of each of the plurality or a subset thereof comprises a hydrophilic peptide and a PEG.

15. The plurality of self-assembling components of claim 1 , wherein the hydrophobic self- assembly motif of each of the plurality or a subset thereof is operatively connected to hydrophilic motif via a spacer.

16. The plurality of self-assembling components of claim 15, wherein the spacer is a linker and/or a substrate.

17. The plurality of self-assembling components of claim 16, wherein the linker has the sequence G, AGQGLR (SEQ ID NO: 18), FGSGKD (SEQ ID NO: 19), or TGGYPVE (SEQ ID NO: 20).

18. The plurality of self-assembling components of claim 16, wherein in the linker comprises a glycine-serine linker.

19. The plurality of self-assembling components of claim 18, wherein in glycine-serine linker is GGSGGSGG (SEQ ID NO: 17.

20. The plurality of self-assembling components of claim 16, wherein the linker comprises a polyethylene glycol (PEG) linker.

21. The plurality of self-assembling components of claim 16, wherein the linker comprises a polypropylene glycol linker.

22. The plurality of self-assembling components of claim 16, wherein the linker comprises a polyoxyalkylene linker.

23. The plurality of self-assembling components of claim 16, wherein the linker is a copolymer of polyethylene glycol and polypropylene glycol.

24. The plurality of self-assembling components of claim 16, wherein the substrate comprises a cleavage site that is cleaved by a protease.

25. The plurality of self-assembling components of claim 24, wherein the substrate has 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.

26. The plurality of self-assembling components of claim 24, wherein the protease comprises 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.

27. The plurality of self-assembling components of claim 24, wherein the protease comprises legumain, CathepsinG, or Matriptase.

28. The plurality of self-assembling components of claim 24, wherein the protease comprises legumain.

29. The plurality of self-assembling components of claim 15, wherein the spacer comprises a linker and a substrate.

30. The plurality of self-assembling components of claim 29, wherein the 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.

31. The plurality of self-assembling components of claim 15, wherein the spacer comprises 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).

32. The plurality of self-assembling components of claim 1 , further comprising a functional molecule operatively connected to each of the plurality or a subset thereof.

33. The plurality of self-assembling components of claim 32, wherein the functional molecule comprises a dye.

34. The plurality of self-assembling components of claim 33, wherein the dye comprises a fluorophore, affinity tag, radiolabel, and/or contrast agent.

35. The plurality of self-assembling components of claim 34, wherein the fluorophore comprises an NIR dye.

36. The plurality of self-assembling components of claim 35, wherein the NIR dye comprises 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.

37. The plurality of self-assembling components of claim 32, wherein the functional molecule is a drug or prodrug.

38. The plurality of self-assembling components of claim 37, wherein the drug is an anticancer drug.

39. The plurality of self-assembling components of claim 38, wherein the anti-cancer drug comprises an alkylating agent, a nitrosourea, an antimetabolite, an anthracycline, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a mitotic inhibitor, or a corticosteroid.

40. The plurality of self-assembling components of claim 39, wherein the alkylating agent comprises altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, or trabectedin.

41. The plurality of self-assembling components of claim 39, wherein the nitrosourea comprises carmustine, lomustine, or streptozocin.

42. The plurality of self-assembling components of claim 39, wherein the antimetabolite comprises 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.

43. The plurality of self-assembling components of claim 39, wherein the anthracycline comprises daunorubicin, doxorubicin, doxorubicin liposomal, epirubicin, idarubicin, or valrubicin.

44. The plurality of self-assembling components of claim 39, wherein the topoisomerase I inhibitor comprises irinotecan, irinotecan liposomal, topotecan, or CL2-SN-38.

45. The plurality of self-assembling components of claim 39, wherein the topoisomerase II inhibitor comprises etoposide (VP-16), mitoxantrone, and teniposide.

46. The plurality of self-assembling components of claim 39, wherein the mitotic inhibitors comprise cabazitaxel, docetaxel, Nab-paclitaxel, paclitaxel, vinblastine, vincristine, vincristine liposomal, or vinorelbine.

47. The plurality of self-assembling components of claim 39, wherein the corticosteroid comprises prednisone, methylprednisolone, or dexamethasone.

48. The plurality of self-assembling components of claim 38, wherein the anti-cancer drug comprises all-trans-retinoic acid, arsenic trioxide, asparaginase, bleomycin, dactinomycin, mitomycin-C, eribulin, hydroxyurea, ixabepilone, mitotane, omacetaxine, pegaspargase, procarbazine, romidepsin, taxol, or vorinostat.

49. The plurality of self-assembling components of claim 37, wherein the drug is functionalized with an iodine.

50. The plurality of self-assembling components of claim 37, wherein the drug is N- (lodoacetamido)-Doxorubicin (Dox).

51. The plurality of self-assembling components of claim 1 , wherein the hydrophobic self- assembly motif of each of the plurality or a subset thereof comprises a palmitoyl molecule and the hydrophilic motif comprises 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), EKEKEGCGKEKEK (SEQ ID NO: 33), EE, RR, ECE, EGEE (SEQ ID NO: 15), or CGEKEE (SEQ ID NO: 16).

52. The plurality of self-assembling components of claim 15, wherein of each of the plurality or a subset thereof further comprises a spacer including the sequence GGGH (SEQ ID NO: 1).

53. The plurality of self-assembling components of claim 15, wherein the hydrophobic self- assembly motif of each of the plurality or a subset thereof comprises a palmitoyl molecule and a peptide including the sequence GGGH (SEQ ID NO: 1 ), and the hydrophilic motif comprises an ECEE (SEQ ID NO: 10) peptide.

54. The plurality of self-assembling components of claim 15, wherein the hydrophobic self- assembly motif of each of the plurality or a subset thereof comprises a palmitoyl molecule and a peptide including the sequence GGGH (SEQ ID NO: 1 ), and the hydrophilic motif comprises a DCDD (SEQ ID NO: 11) peptide.

55. The plurality of self-assembling components of claim 15, wherein the hydrophobic self- assembly motif of each of the plurality or a subset thereof comprises a palmitoyl molecule and a peptide including the sequence GGGH (SEQ ID NO: 1 ), and the hydrophilic motif comprises an KCKK (SEQ ID NO: 12) peptide.

56. The plurality of self-assembling components of claim 15, wherein the hydrophobic self- assembly motif of each of the plurality or a subset thereof comprises a palmitoyl molecule and a peptide including the sequence GGGH (SEQ ID NO: 1 ), and the hydrophilic motif comprises a KCEK (SEQ ID NO: 13) peptide.

57. The plurality of self-assembling components of claim 15, wherein the hydrophobic self- assembly motif of each of the plurality or a subset thereof comprises a palmitoyl molecule and a peptide including the sequence GGGh (SEQ ID NO: 2), and the hydrophilic motif comprises an ecee (SEQ ID NO: 14) peptide.

58. The plurality of self-assembling components of claim 15, wherein the hydrophobic self- assembly motif of each of the plurality or a subset thereof comprises a palmitoyl molecule and a peptide including the sequence GGGH (SEQ ID NO: 1 ), and the hydrophilic motif comprises PEG6 or G-PEG6.

59. The plurality of self-assembling components of claim 15, wherein the hydrophobic self- assembly motif of each of the plurality or a subset thereof comprises a palmitoyl molecule and a peptide including the sequence GGGH (SEQ ID NO: 1 ), and the hydrophilic motif comprises a G-PEG6.

60. A self-assembled nanomaterial formed from the plurality of self-assembling components of claim 1.

61. The self-assembled nanomaterial of claim 60, wherein the self-assembled nanomaterial has a micellar structure 5-10 nm in diameter.

62. A self-assembled nanomaterial formed from the plurality of self-assembling components of claim 33.

63. The self-assembled nanomaterial of claim 62, wherein the self-assembling components comprise a cleavage site that is cleaved by a protease between the self-assembly motif and the hydrophilic motif.

64. A pharmaceutical composition comprising the self-assembled nanomaterial of claim 63 and a pharmaceutically acceptable carrier.

65. A self-assembled nanomaterial formed from the plurality of self-assembling components of claim 37.

66. The self-assembled nanomaterial of claim 65, wherein the self-assembling components comprise a cleavage site that is cleaved by a protease between the self-assembly motif and the hydrophilic motif.

67. A pharmaceutical composition comprising the self-assembled nanomaterial of claim 66 and a pharmaceutically acceptable carrier.

68. A method for detecting a tumor in a subject comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 64 to the subject, and detecting the dye, thereby detecting the tumor in the subject.

69. The method of claim 68, wherein the tumor is less than 5 mm in diameter.

70. The method of claim 69, wherein the tumor is less than 1 mm in diameter.

71. The method of claim 68, wherein 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.

72. The method of claim 68, wherein the detecting comprises 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.

73. The method of claim 68, further comprising detecting protease activity.

74. A method of treating cancer in a subject in need thereof comprising: administering a therapeutically effective amount of the pharmaceutical composition of claim 67 to the subject thereby treating cancer in the subject in need thereof.

75. The method of claim 74, wherein the cancer is colon cancer, pancreatic cancer, melanoma, breast cancer, kidney cancer, lung cancer, or glioma.

76. The method of claim 74, further comprising detecting protease activity.

77. The method of claim 74, wherein the administering is through intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, oral, intrapulmonary, subcutaneous, or sublingual administering.