Smart peptides and transformable nanoparticles for cancer immunotherapy

Abstract

To provide a compound of formula (I): A-B-C (I) (where, A is a hydrophobic moiety; B is a peptide, the peptide forming a β-sheet; and C is a hydrophilic targeting ligand, the hydrophilic targeting ligand being a LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, a LHRH peptide, a HER2 ligand, an EGFR ligand, or a toll-like receptor agonist CpG oligonucleotides).SOLUTION: The present invention also provides nanocarriers comprising compounds of the present invention, nanofibril formation from the nanocarriers, and methods of using the nanocarriers for treating diseases and imaging.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Nos. 62 / 886,698 and 62 / 886,718, both filed August 14, 2019, each of which is incorporated herein by reference in its entirety for all purposes. STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under grant numbers R01EB012569 and U01CA198880 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Background of the Invention Recent clinical successes in cancer immunotherapy have sparked intense interest in our fight against cancer. For example, immune checkpoint receptor pathway-blocking monoclonal antibodies, such as anti-PD-1, anti-PD-L1, and anti-CTLA-4, can reverse T effector cell (Teff) dysfunction and exhaustion, resulting in dramatic tumor shrinkage and even complete remission in some patients, even with late-stage metastatic disease. However, response rates vary widely across tumor types: up to 40% in melanoma, 25% in non-small cell lung cancer, but <10% in most other tumor types. To date, the US Food and Drug Administration (FDA) has approved seven immune checkpoint-blocking monoclonal antibodies (ICB-Abs): one CTLA-4 inhibitor (ipilimumab), three PD-1 inhibitors (nivolumab, pembrolizumab, and cemiplimab), and three PD-L1 inhibitors (atezolizumab, durvalumab, and avelumab) for use alone or in combination with other chemotherapy regimens against a variety of tumor types.

[0004] The tumor microenvironment (TME), consisting of immune and stromal cells, blood vessels, extracellular matrix, cytokines, chemokines, and growth factors, can have a profound effect on tumor response to immune checkpoint blockade (ICB) therapy. Emerging data suggest that defective Teff cell homing to the tumor site is a key factor in resistance to ICB therapy. Other mechanisms of ICB resistance include the presence of immunosuppressive regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and M2 macrophages at the tumor site. High levels of CCL5, CCL17, CCL22, CXCL8, and CXCL12 facilitate the recruitment of Tregs and MDSCs to the TME, resulting in ICB responses. In contrast, CXCL9 and CXCL10 promote homing of cytotoxic T cells (CTLs) to the tumor site and promote antitumor immune responses; transforming growth factor beta (TGF-β) is the opposite, so Tregs are also upregulated. VEGF upregulates inhibitory receptors for CTLs, contributing to their exhaustion. Upregulation of other immune checkpoint receptors, such as mucin domain-3 protein (TIM-3), lymphocyte activation gene 3 (LAG-3), B and T lymphocyte attenuator (BTLA), T cell immunoreceptor, tyrosine-based inhibitory motif domain (TIGIT), and V-domain immunoglobulin-containing inhibitor of T cell activation (VISTA), has been associated with ICB resistance. Coexpression of these checkpoint receptors can lead to T cell exhaustion. Oncogenic or tumor suppressor pathways, such as mitogen-activated protein kinase (MAPK) and PI3K-γ, in cancer cells also affect the TME by altering immune cell composition and cytokine profiles, contributing to ICB resistance. Inhibitors of these pathways have been shown to improve ICB responses.

[0005] In an attempt to overcome ICB resistance, a number of combination therapy strategies have been tried preclinically and clinically. These include the addition of the following drugs to an ICB-Ab: another ICB-Ab (antibodies against CTLA-4, PD-1, PD-L1, LAG-3, and TIM-3), chemotherapy drugs (paclitaxel, gemcitabine, and carboplatin), radiation therapy, targeted therapy (inhibitors against PI3K, VEGF, BRAF / MEK, IDO, A2AR, FGFR, EGFR, PARP, and mTOR), macrophage inhibitors (inhibitors against CSF1R and ARG1), cytokine / chemokine inhibitors (inhibitors against CXCR4, CXCR2, and TGF-β), epigenetic modulators (histone deacetylase inhibitors and hypomethylating agents), immunomodulatory drugs (antibodies against OX40, 41BB, GITR, CD40, and ICOS), adoptive cell transfer therapy (carT, TIL, and TCR), and modulation of the gut microbiota.

[0006] Advances and optimization of nanoimmunotherapy lie in the development of innovative approaches to enhance the specificity and control of immunotherapeutic interventions and target desired cell types in the TME. Advanced bionanomaterials or more controlled approaches may enhance immunotherapy efficacy by enhancing the accumulation and prolonging the residence of immunomodulatory and immune cell homing agents in the TME, while sparing normal tissues and organs, thereby mitigating off-target adverse effects such as systemic cytokine storms. In situ assembly of nanomaterials has been demonstrated to improve the performance of bioactive molecules. One plausible explanation is that T cell-targeting ligands and / or immunomodulatory agents incorporated into in situ fibril-tunable nanoplatforms create a nanofibrillar network in the TME, enhancing Teff cell homing to tumor sites and improving the efficacy of immunotherapy, with or without additional ICB therapy.

[0007] Human epidermal growth factor receptor 2 (HER2) is overexpressed in over 20% of breast cancers and, to a lesser extent, in gastric, colorectal, ovarian, and bladder cancers. Unlike those cancers caused by mutations or fusion oncogenes (e.g., EGFR in lung cancer and Bcr-Abl in chronic myeloid leukemia), which respond well to monotherapy, HER2-overexpressing cancers often require drug combinations. This latter group of tumors is driven by gene amplification or massive overexpression of HER2. HER2 is a receptor tyrosine kinase that is normally activated by dimerization induced by itself or its family members EGFR, HER3, or HER4. In HER2-positive tumors, HER2 is massively overexpressed and constitutively dimerized, leading to unabated activation of downstream proliferation and survival pathways and a malignant phenotype.

[0008] Due to the high expression levels of HER2, trastuzumab and pertuzumab, two anti-HER2 monoclonal antibodies, are ineffective as monotherapy for these tumors. They must be given in combination with other HER2-targeted therapies, chemotherapy, or hormonal therapy. Herein, several embodiments describe a novel HER2-mediated, peptide-based, and nontoxic tunable nanoagent that is highly effective as monotherapy for HER2+ breast cancer xenograft models. This receptor-mediated tunable nanotherapeutic (RMTN) consists of a peptide with a unique domain that enables self-assembly to form micelles under aqueous conditions and transformation into nanofibrils at the tumor site where HER2 is encountered. The resulting nanofibrillar network effectively inhibits HER2 dimerization and downstream signaling, facilitating tumor cell death.

[0009] Herein, smart supramolecular materials for cancer immunotherapy were constructed. Summary of the Invention

[0010] BRIEF SUMMARY OF THE INVENTION In one embodiment, the present invention provides a compound of formula (I): ABC(I), wherein A is a hydrophobic moiety; B is a peptide, wherein the peptide forms a β-sheet; and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting ligand is an LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, an LHRH peptide, a HER2 ligand, an EGFR ligand, or a toll-like receptor agonist CpG oligonucleotide.

[0011] In another embodiment, the present invention provides a compound of formula (I): ABC(I), wherein A is a hydrophobic moiety; B is a peptide, wherein the peptide forms a β-sheet; and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting ligand is an LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, an LHRH peptide, an HER2 ligand, an EGFR ligand, or a toll-like receptor agonist CpG oligonucleotide, and wherein when the hydrophobic moiety is bis-pyrene, C is an LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, an LHRH peptide, an EGFR ligand, or a toll-like receptor agonist CpG oligonucleotide.

[0012] In another embodiment, the present invention provides a nanocarrier having an interior and an exterior, the nanocarrier comprising a plurality of compounds of the present invention, wherein each compound self-assembles in an aqueous medium to form a nanocarrier such that a hydrophobic pocket is formed on the interior of the nanocarrier and hydrophilic groups self-assemble on the exterior of the nanocarrier.

[0013] In another embodiment, the invention provides a nanocarrier having an interior and an exterior, the nanocarrier comprising a plurality of first conjugates and second conjugates, wherein the first conjugate comprises Formula (I): ABC(I) and the second conjugate comprises Formula (II): A'-B'-C'(II) (wherein A and A' are each independently a hydrophobic moiety; B and B' are each independently a peptide, wherein each peptide independently forms a β-sheet; and C and C' are each independently a hydrophilic targeting ligand, wherein each hydrophilic targeting ligand is independently an LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, LHRH peptide, HER2 ligand, EGFR ligand, or radiometal chelator; and where A and A' are different hydrophobic moieties, and / or C and C' are different hydrophilic targeting ligands).

[0014] In another embodiment, the present invention provides a method of forming nanofibrils, comprising contacting a nanocarrier of the present invention with a cell surface or acellular components in a tumor microenvironment, wherein the nanocarrier undergoes in situ transformation to form a fibril structure, thereby forming nanofibrils.

[0015] In another embodiment, the present invention provides a method of treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of a nanocarrier of the present invention, wherein the nanocarrier forms nanofibrils in situ after binding to cell surfaces or acellular components in the tumor microenvironment, thereby treating the disease.

[0016] In another embodiment, the present invention provides a method of imaging, comprising administering to a subject to be imaged an effective amount of a nanocarrier of the present invention. [Brief explanation of the drawings]

[0017] [Figure 1] Figures 1A-1F show the assembly and fibril transformation of variable peptide monomer 1 (TPM1') BP-FFVLK-YCDGFYACYMDV. Figures 1A-1B show the changes in UV-vis absorption (Figure 1A) and fluorescence (Figure 1B) of NP1 upon the stepwise addition of water to DMSO solutions of NP1 at HO:DMSO ratios from 0:100 to 20:80, 40:60, 60:40, 80:20, 90:10, 98:2, and 99.5:0.5, Ex = 380 nm. Figure 1C shows TEM images of pristine NP1 and nanofibers (NF1) transformed by NP1 interaction with HER2 protein (molecular weight ≈ 72 kDa) at various time points (0.5, 6, and 24 h). Scale bar in d: 100 nm. Figures 1D-1F show the size distribution (Figure 1D), CD spectra (Figure 1E), and fluorescence signals (Figure 1F) of initial NP1 and NF1 at various time points. The molar ratio of HER2 peptide to HER2 protein was approximately 1000:1.

[0018] [Figure 2]Figures 2A-2H show the morphological characterization of fibril-mutable NP1 coculture with HER2-positive cancer cells. Figures 2A-2C show cytofluorescence distribution images of NP1 interaction with SKBR-3 cells (HER2+) (Figure 2A), BT474 cells (HER2+) (Figure 2B), and MCF-7 cells (HER2-) (Figure 2C) at 6 h. Scale bars in Figures 2A-2C: 50 μm. Figure 2D shows Western blot and quantitative analysis of relative HER2 protein expression in MCF-7 and MCF-7 / C6 cells. ***P<0.001. Figure 2E shows cytofluorescence distribution images of NP1 interaction with MCF-7 / C6 cells (HER2+) at various time points (0.5, 6, and 24 h). Scale bar in Figure 2E: 50 μm. Figure 2F shows the fluorescent binding distribution image of the nanofibrillar network of NP1 and HER2 antibody (29D8, rabbit, a different receptor binding site from the HER2 peptide of NP1) on the cell membrane of MCF-7 / C6 cells. The HER2 antibody was used to label the HER2 receptor. Figure 2G shows SEM images of untreated MCF-7 / C6 cells and cells treated with NP1 for 6 and 24 hours. Figure 2H shows TEM images of untreated MCF-7 / C6 cells and cells treated with NP1 for 24 hours. The red arrows indicate the fibrillar network. The concentration of NP1 was 50 μM.

[0019] [Figure 3]Figures 3A-3G show the extracellular and intracellular mechanisms of fibril-variable NP interaction using MCF-7 / C6 breast cancer cells. Figure 3A shows the cellular fluorescence distribution images of HER2 receptors bound to NP1, NP2, and HER2 antibodies (29D8, rabbit; different receptor binding sites for NP1 and NP2 with the HER2 peptide) in MCF-7 / C6 cells, respectively. HER2 antibodies were used to label the HER2 receptor. The concentrations of NP1 and NP2 were 50 μM. Scale bar in a: 20 μm. Figure 3B shows the viability of MCF-7 / C6 cells incubated with various concentrations of NP1-4 (n = 3). *P < 0.05, **P < 0.01. Figure 3C shows Western blot analysis of apoptosis-related proteins and total HER2 protein in MCF-7 / C6 cells treated with NP1 at various concentrations for 24 hours. Figures 3D-3E show Western blot analysis of the inhibition and dissociation mechanisms of HER2 protein dimers in MCF-7 / C6 cells treated with NP1 at various concentrations for 24 hours (Figure 3D) and at 50 μM for various time points (Figure 3E). Figure 3F shows Western blot analysis of the inhibition mechanisms of proliferation proteins in MCF-7 / C6 cells treated with NP1 at 50 μM for various time points and at various concentrations for 24 hours. Figure 3G shows Western blot analysis of the inhibition mechanisms of proliferation proteins in MCF-7 / C6 cells treated with NP1-4 and Herceptin (HP) for 36 hours. The concentrations of NP1-4 were 50 μM, and the concentration of Herceptin as a positive control was 15 μg / mL.

[0020] [Figure 4]Figures 4A–4F show the in vivo evaluation of fibril-modifiable NPs. Figure 4A shows time-dependent ex vivo fluorescence images, and Figure 4B shows quantitative analysis of tumor tissues and major tissues (heart, liver, spleen, lung, kidney, intestine, muscle, and skin) collected at 10, 24, 48, 72, and 168 h after NP1 injection. In Figure 4B, ***P<0.001, the fluorescence signal in tumor tissues at 72 and 168 h compared with other organs indicates tumor accumulation with a long retention time and in situ deformation of the fibril network; ***P<0.001, the fluorescence signal in liver and kidneys at 10 h compared with those at 72 and 168 h indicates that NP1 can be rapidly cleared from the liver and kidney. Figure 4C shows the fluorescence distribution and H&E images of NP1 in tumor tissues and normal skin tissues at 72 h after injection (green: NP1 BP; blue: DAPI; scale bar in c: 100 μm). Figure 4D shows time-dependent ex vivo fluorescence images of tumor tissues and major tissues collected 72 hours after NP2-4 injection. Figure 4E shows quantitative analysis of tumor tissues and liver collected 72 hours after NP1-4 injection. In Figure 4E, ***P<0.001, the fluorescence signal for tumor tissue in the NP1 group compared with that of the other control groups indicated that the fibril network in the NP1 group promoted long retention time at the tumor site. Figure 4F shows TEM images of the distribution of NP1-4 in tumor tissue and in situ fibril deformation 72 hours after IV injection and in the untreated group. The dose of NP1-4 was 8 mg / kg per injection. In Figure 4F, "C" refers to MCF-7 / C6 cells; "N" refers to cell nuclei.

[0021] [Figure 5]Figures 5A-5K show the antitumor activity of NPs in Balb / c nude mice bearing HER2-positive breast tumors. Figure 5A shows a schematic diagram of the mouse tumor inoculation and treatment protocol. Figures 5B-5C show the observation of the tumor-inhibitory effect (Figure 5B) and the body weight change of the mice (Figure 5C) during 40 days of treatment in a subcutaneous tumor model (n = 8 per group; the dose of NPs 1-4 was 8 mg / kg per injection). **P<0.01, ***P<0.001. Figure 5D shows the cumulative survival rate of various treatment groups of mice bearing MCF-7 / C6 breast tumors. Figure 5E shows a schematic diagram of the three-treatment protocol for tumor tissue analysis. Figure 5F shows the fluorescence distribution image and H&E antitumor image in tumor tissue after three injections of NP1 (green: NP1 BP; blue: DAPI; scale bar in f: 100 μm). Figure 5G shows representative TEM images of delayed membrane rupture and cell death due to nanofibril networks after three injections of NP1. Red arrows indicate the fibril networks. Figure 5H shows Ki-67 staining images of tumor tissues treated with various groups after three injections. Scale bar in h: 25 μm. Figure 5I shows Western blot analysis of the mechanism of inhibition of HER2 protein and proliferation proteins in MCF-7 / C6 tumor tissues treated with various groups after three injections. Figures 5J–5K show observations on tumor inhibitory effects in subcutaneous tumor SKBR-3 (Figure 5J) and BT474 HER2-positive breast cancer (Figure 5K) models during 40 days of treatment (n = 8 per group, NP1 dose was 8 mg / kg per injection). ***P<0.001 compared to the PBS control group.

[0022] [Figure 6] FIG. 6 shows the chemical structure and MALDI-TOF mass spectrum of the variable peptide monomer 1 BP-FFVLK-YCDGFYACYMDV.

[0023] [Figure 7] FIG. 7 shows the chemical structure and MALDI-TOF mass spectrum of the variable peptide monomer 2BP-GGAAK-YCDGFYACYMDV.

[0024] [Figure 8] FIG. 8 shows the chemical structure and MALDI-TOF mass spectrum of the variable peptide monomer 3 BP-FFVLK-PEG.

[0025] [Figure 9] FIG. 9 shows the chemical structure and MALDI-TOF mass spectrum of the variable peptide monomer 4BP-GGAAK-PEG.

[0026] [Figure 10] Figure 10 shows the effect of the HER2 protein / peptide ligand ratio on fibril transformation. TEM images and particle size measurements of NP1 were obtained after incubation with soluble HER2 protein in PBS solution for 24 hours. The NP1 concentration was kept constant at 20 μM. The scale bar is 200 nm. The HER2 protein / peptide ligand ratio is labeled for each micrograph. The experiment was repeated three times.

[0027] [Figure 11] Figure 11A shows the observation of antitumor effects in subcutaneous SKBR-3 tumors during 40 days of treatment (n = 6 per group; NP1-4 dose was 8 mg / kg qod per injection; data are presented as mean ± sd). Statistical significance was calculated by one-way ANOVA with Tukey's post-hoc test. *P < 0.05. Figures 11B-11C show the body weights of mice bearing subcutaneous BT474 tumors (Figure 11B) and SKBR-3 tumors (Figure 11C) during 40 days of treatment (n = 6 per group; data are presented as mean ± sd). Red arrows indicate the respective single iv injections.

[0028] [Figure 12]Figure 12 shows that nanofibril networks promote T cell homing and reprogram the tumor microenvironment for enhanced immunotherapy. Schematic diagram of TPM self-assembly and fibril transformation, (I), (II), and (III) processes in tumor tissue: in situ fibril transformation of NPs, pro-LLP2A to LLP2A conversion, subsequent T cell binding and targeting, and TAM reeducation from an M2 to an M1 phenotype. TPM, NP, NF, M1-TAM, and M2-TAM represent variable peptide monomers, nanoparticles, nanofibrils, M1-like tumor-associated microphages, and M2-like tumor-associated microphages, respectively.

[0029] [Figure 13]Figures 13A-13H show the assembly and fibril transformation of the variable peptides TPM1 (LXY30-KLVFFK(Pa)) and TPM2 (proLLP2A-KLVFFK(R848)). Figure 13A shows a schematic diagram of the molecular structure and function of TPM1 and TPM2. Figure 13B shows the change in fluorescence (FL) of T-NPs after the stepwise addition of water (0-99%) to a solution of T-NPs in DMSO consisting of a 1:1 ratio of TPM1 and TPM2; excitation wavelength, 405 nm. Figure 13C shows TEM images of pristine T-NPs and T-NPs transformed into nanofibrils (T-NPs) after interaction with soluble α3β1 integrin protein for 24 h (99:1 HO to DMSO ratio). The concentration of T-NPs used in the experiments was 20 μM. The scale bar in c is 100 nm. Figure 13D shows the variation in the fluorescence signal for Pa during the fibril transformation process of T-NPs into T-NFs over time. Figure 13E shows TEM images of pristine T-NPs and T-NFs after interaction with esterase, soluble α4β1 integrin protein, or α4β1 integrin protein plus esterase for 24 hours (HO to DMSO ratio of 99:1). The concentration of T-NPs used in the experiments was 20 μM. The scale bar in Figure 13 is 100 nm. Figures 13F-3G show the differences in size distribution (Figure 13F) and circular dichroism spectra (Figure 13G) of pristine T-NPs and T-NFs under various conditions. Figure 13H shows the in vitro release profile of R848 from T-NFs over time. The molar ratio of α3β1 or α4β1 integrin protein to peptide ligand was approximately 1:1000. au, arbitrary units; mdeg, millidegrees.

[0030] [Figure 14] Figure 14 shows a DLS experiment to confirm the transformation of T-NP into T-NF. The peak at 20 nm gradually decreased in solution, while the peak around 700 nm increased.

[0031] [Figure 15]Figures 15A-15H show the morphological characterization of fibril-tunable nanoparticles after incubation with 4T1 mouse breast cancer cells. Figure 15A shows cytofluorescence distribution images of T-NP and UT-NP interactions with 4T1 cells over a 6-hour period. The scale bar is 10 μm. The experiment was repeated three times. Figure 15B shows cytofluorescence signal retention images of 4T1 cells after 6 hours of exposure to T-NP and UT-NP, followed by 18 hours of incubation in NP-free fresh medium. The scale bar is 10 μm. The experiment was repeated three times. Figure 15C shows representative TEM images of 4T1 cells treated with T-NP and UT-NP for 24 hours, showing abundant nanofibrils around the T-NP-treated cells. The scale bar is 200 nm. The experiment was repeated three times. The concentration of T-NP was 50 μM. Figure 15D shows a cellular fluorescence distribution image of Jurkat T lymphoma cells (GFP-labeled) after incubation with esterase-treated T-NPs. Jurkat cells were used to mimic T lymphocytes, and they also express α4β1 integrin. The scale bar is 10 μm. The experiment was repeated three times. Figure 15E shows representative SEM images of untreated 4T1 and Jurkat cells, as well as cells treated with T-NPs for 6 hours. The scale bar is 10 μm. The experiment was repeated three times. Figure 15F shows the experimental scheme and a cellular fluorescence distribution image of T-NPs (fluorescent red) after interaction with 4T1 and GFP-labeled Jurkat cells. It shows a nanofibril network covering the 4T1 cells, which in turn can attract and bind Jurkat malignant T cells. The scale bar is 10 μm. The experiment was repeated three times. Figure 15G shows representative SEM images of 4T1 and Jurkat cells after treatment with T-NP (see Figure 15F). The experiment was repeated three times. Figure 15H shows representative images of M2-like murine macrophages and subsequent re-education with T-NF, T-NF + esterase, or R848 at various time points. The scale bar is 20 μm. The experiment was repeated three times. Statistical significance was calculated using an unpaired two-tailed t-test. *P<0.05, **P<0.01, ***P<0.001.

[0032] [Figure 16]Figures 16A-16M show the in vivo evaluation of fibril-modifiable nanoparticles. Figures 16A-16B show time-dependent ex vivo fluorescence (FL) images (Figure 16A) and quantitative analysis (Figure 16B) of tumor tissues and major tissues (heart (H), liver (Li), spleen (Sp), lung (Lu), kidney (K), intestine (I), muscle (M), and skin (Sk)) harvested at 10, 24, 48, 72, 120, and 168 h after T-NP injection. Data are presented as mean ± sd, n = 3 independent experiments. Figure 16C shows time-dependent ex vivo fluorescence (FL) images of tumor tissues harvested at 10, 24, 48, 72, 120, and 168 h after UT-NP injection. Data are presented as mean ± sd, n = 3 independent experiments. Figure 16D shows fluorescence (FL) quantification of tumor tissues collected at 10, 24, 48, 72, 120, and 168 hours after T-NP and UT-NP injection. Figure 16E shows representative TEM images of tumor tissue distribution and in situ fibril deformation for T-NP, UT-NP, and untreated control groups 72 hours after injection. "N" indicates nucleus. Figure 16F shows fluorescence (FL) distribution images of T-NP in tumor tissues and normal skin tissues 72 hours after injection (red, T-NP Pa; blue, DAPI; scale bar, 50 μm). Figure 16G shows R484 distribution and retention in tumor tissues at various time points after T-NP and UT-NP injection. R484 dose: 0.94 mg kg-1; data were mean ± SD, n = 3 for each time point. Figure 16H shows the expression of CXCL10 chemokine in tumor tissue 3 days after treatment with T-NP, UT-NP, and saline (n = 3; data were mean ± SD). Figures 16I-16K show representative flow cytometry images showing CD45+CD3+ (Figure 16I), CD8+ / CD4+ (Figure 16J), and CD4+Foxp3+ (Figure 16K) T cells in 4T1 tumors excised from mice treated with T-NP, UT-NP, or saline control. Figure 16L shows immunohistochemistry (IHC) of tumors excised from mice after treatment with T-NP or UT-NP. Representative images are shown for IHC staining of T cells (CD8+, CD4+, Foxp3+) and macrophage markers (CD68, CD163). The scale bar is 100 μm.Figure 16M shows the expression levels (qPCR assay) of IFN-γ, TGF-β, IL12, IL10, Nos2, and Arg-1 in 4T1 tumors excised from mice 15 days after treatment with T-NPs or UT-NPs (n=3; data were mean ± SD). Statistical significance was calculated using an unpaired two-tailed t-test. *P<0.05, **P<0.01, ***P<0.001.

[0033] [Figure 17] Figures 17A-17G show the antitumor effects of fibril-determinable nanoparticles in Balb / c mice bearing 4T1 breast tumors. Figure 17A shows the experimental design: orthotopic tumor inoculation and treatment protocol; regimen 6 is a T-NP with all four key components. Figures 17B-17C show observations of tumor suppression (Figure 17B) and body weight changes (Figure 17C) in mice bearing orthotopic 4T1 tumors over 21 days after the start of treatment (n = 8 per group). Data are presented as mean ± s.d. Figure 17D shows the cumulative survival rates of various treatment groups of mice bearing 4T1 breast tumors. Figure 17E shows representative flow cytometry images of CD3+CD8+ T cells in 4T1 tumors excised from treated mice on day 21. Figure 17F shows H&E and IHC images of excised tumors. Representative images are shown for IHC staining of Ki67, T cell (CD8, Foxp3), and macrophage markers (CD68, CD163). Scale bars are 100 μm. Figure 17G shows the expression levels of IFN-γ, TNF-α, IL12, IL6, TGF-β, IL10, Nos2, and Arg-1 (analyzed by qPCR) in 4T1 tumors excised from mice on day 21 (data were mean ± SD). Statistical significance was calculated using an unpaired two-tailed t-test. *P<0.05, **P<0.01, ***P<0.001).

[0034] [Figure 18]Figures 18A-18L show the antitumor effects of fibril-determinable nanoparticles plus anti-PD-1 treatment in mice bearing 4T1 breast tumors or Lewis lung tumors. Figure 18A shows the experimental design: orthotopic tumor inoculation and treatment protocol (four treatment groups; regimens 4, 5, and 6 are the same as those shown in Figure 4a). Figure 18B shows tumor responses in mice bearing orthotopic 4T1 tumors over 21 days of treatment (n=8 per group). Data are presented as mean ± s.d. ***P<0.001. Figure 18C shows cumulative survival rates for the four treatment groups. Figure 18D shows the experimental design: mice pretreated with T-NPs (regimen 6) plus anti-PD-1 Ab were rechallenged by reinoculation with cancer cells on day 90, followed by three qod ip doses of anti-PD-1 Ab. Figure 18E shows that no antitumor immune memory effect was observed in age-matched naive mice. Figure 18F shows that an antitumor immune memory effect was observed in mice pretreated with T-NP and anti-PD-1 Ab. Figure 18G shows the cumulative survival rates of naive mice and mice pretreated with T-NP + anti-PD-1. Figures 18H-18I show IFN-γ (Figure 18H) and TNF-α (Figure 18I) levels in mouse serum 6 days after the mice were rechallenged with 4T1 tumor cells and 1 day after the last dose of anti-PD-1 Ab. Figures 18J-18K show the tumor suppression effect (Figure 18J) and body weight change (Figure 18K) in mice bearing subcutaneous Lewis lung tumors over a 21-day period after the start of treatment (n = 8 per group); the treatment protocol followed the experimental design in Figure 18A, for 5 cycles (iv regimens 4-6 and ip anti-PD-1). Data are presented as mean ± SD. Figure 18L shows the cumulative survival rate of various treatment groups in mice bearing murine Lewis lung tumors. Statistical significance was calculated using an unpaired two-tailed t-test; *P<0.05, **P<0.01, ***P<0.001.

[0035] [Figure 19]Figure 19A shows the structure of CPTNP (BP-klvffk-(r)8), where green indicates bispyrene, blue indicates hydrophobic binding motif, and red indicates cell-penetrating peptide. Figure 19B shows GG-CPTNP (BP-klvggk-(r)8) with the same coloring as in A, where the double phenylalanine motif is replaced with a double glycine motif. Figure 19C shows DLS of CPTNP (FF) and GG-CPTNP (GG) at various pH levels. Figure 19D shows the fluorescence of CPTNP nanoparticles and CPTNP monomers, where the AIEE effect of BP can be observed. Figure 19E shows the zeta potential of FF and GG CPTNP measured at 50 μM (a:b, p<0.0005). Figure 19F shows TEM images of CPTNP in various designated environments. The scale bar in each image is 100 μm.

[0036] [Figure 20] Figure 20 shows the chemical structures and MALDI-TOF mass spectra of the variable peptide monomers (TPMs) 1 LXY30-KLVFFK(Pa), 2 proLLP2A-KLVFFK(R848), 3 LXY30-KAAGGK(Pa), and 4 proLLP2A-KAAGGK(R848). Experiments were repeated three times.

[0037] [Figure 21]Figure 21A shows TEM images and size distributions of NPTPPM1, NPTPPM1, and T-NP in a 99:1 HO to DMSO ratio. The experiment was repeated three times. Figure 21B shows the critical aggregation concentration (CAC) of T-NP measured by using pyrene as a probe. The experiment was repeated three times. Figure 21C shows the nanoparticle stability of T-NP in serum and protease (PBS solution at pH 7.4 with or without 10% FBS and protease) measured by dynamic light scattering at 37 °C. Data are presented as mean ± SD, n = 3 independent experiments. Figure 21D shows TEM images of freshly prepared T-NP and T-NP after 24 hours in PBS solution. The experiment was repeated three times. Figure 21E shows the T-NP CAC measured by using pyrene as a probe. The experiment was repeated three times. The scale bars in all TEM images are 100 nm. The concentration of T-NP used in Figures 21A, 21C, and 21D was 20 μM.

[0038] [Figure 22] Figure 22 shows TEM images of initial UT-NPs and their interaction with α3β1 integrin protein over a 24-hour period. The molar ratio of α3β1 integrin protein to peptide ligand was approximately 1:1000. The scale bar is 100 nm. The concentration used in the experiment was 20 μM. The experiment was repeated three times.

[0039] [Figure 23] Figure 23 shows that incubation of 4T1 cells with biotinylated LXY30 peptide (blue curve) and negative control (red curve) was analyzed by flow cytometry. The experiment was repeated three times. 3 x 10 cells were incubated with 1 μM biotinylated LXY30 on ice for 30 minutes, washed with PBS, and then incubated with 1:500 streptavidin-PE (1 mg / mL) for 30 minutes. Flow cytometry was then performed.

[0040] [Figure 24] Figure 24 shows the viability of 4T1 cells after incubation with T-NP and UT-NP at various concentrations for 48 hours. Data are presented as mean ± sd, n = 3 independent experiments.

[0041] [Figure 25] Figure 25 shows blood test parameters for red blood cells (RBC), white blood cells (WBC), platelets, hemoglobin, lymphocytes, and total protein in healthy Balb / c mice after eight qod intravenous injections of T-NP and UT-NP (13 mg / kg per injection). Data are presented as mean ± SD, n = 3 independent experiments.

[0042] [Figure 26] Figure 26 shows blood test parameters for liver function creatinine, alanine transaminase, aspartate transaminase, albumin, alkaline phosphatase, and total bilirubin in healthy Balb / c mice after eight qod intravenous infusions of T-NP and UT-NP (13 mg / kg per infusion). Data are presented as mean ± SD, n = 3 independent experiments.

[0043] [Figure 27] Figure 27 shows the in vitro blood pharmacokinetics and parameters for T-NP and UT-NP (data presented as mean ± SD, n = 3 independent experiments). Cmax, AUC, and T (hours) were calculated by Kinetica 5.0. DETAILED DESCRIPTION OF THE INVENTION

[0044] Detailed Description of the Invention I. General The present invention provides compounds comprising a hydrophobic moiety, a β-sheet peptide, and a hydrophilic targeting ligand, which can form nanocarriers. The nanocarriers can contain multiple conjugates of one type or two distinct conjugates. The nanocarriers can transform in situ to form nanofibrils for disease treatment and imaging. II. Definition

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any methods or materials similar or equivalent to those described herein can be used in the practice of the present invention. For purposes of the present invention, the following terms are defined:

[0046] As used herein, "a," "an," or "the" includes not only embodiments with one member, but also embodiments with two or more members. For example, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "cell" includes a plurality of such cells, and a reference to an "agent" includes a reference to one or more agents known to those of skill in the art.

[0047] "Hydrophobic moiety" refers to a portion of a compound that is substantially insoluble in water. For example, when a compound containing hydrophobic and hydrophilic moieties exists, the hydrophobic moieties orient themselves in a manner that avoids and minimizes interactions with water molecules. The hydrophobicity of a moiety can be measured by those skilled in the art by using the octanol-water reference system to measure the logarithm of the distribution coefficient (logP value). A LogP value greater than 0 indicates that the compound is hydrophobic, with larger values ​​indicating greater hydrophobicity.

[0048] "Peptide" refers to a compound comprising two or more amino acids covalently linked by peptide bonds. As used herein, the term also includes amino acid chains of any length, including full-length proteins.

[0049] "β-sheet", also known as β-pleated sheet, refers to the secondary structure of a protein and contains β-strands stabilized by hydrogen bonds. The β-strands are stacked parallel or antiparallel to each other to form the β-sheet.

[0050] A "β-sheet peptide domain" refers to a domain within a protein structure that contains a β-sheet.

[0051] "β-amyloid peptide" refers to a peptide that forms amyloid plaques in the brain, which are found in subjects suffering from Alzheimer's disease.

[0052] A "hydrophilic targeting ligand" refers to a portion of a compound that can target a cell surface receptor, cell surface protein, or extracellular component and is hydrophilic. Hydrophilicity can be measured by measuring the logP value of the compound, where a value less than 0 indicates hydrophilicity. Lower values ​​indicate greater hydrophilicity. Targeting ligands can be used to target transmembrane receptors, such as, but not limited to, integrins and epidermal growth factor receptors, and to deliver compounds, drugs, or components of interest to the cellular or extracellular environment. Hydrophilic targeting ligands include, but are not limited to, peptides.

[0053] A "prodrug" refers to a compound that is biologically inactive but becomes biologically active after being metabolized in situ. The prodrug is metabolized by natural reactions or enzymes within a mammal and yields the active compound. Functional groups useful in prodrugs include, but are not limited to, esters, amides, carbamates, oximes, imines, ethers, phosphates, or beta-aminoketones.

[0054] "LLP2A," "LXY30," and "LXW64" refer to compounds capable of binding to integrin proteins, the structures of the three individual compounds being known to those skilled in the art.

[0055] "DUPA" refers to a glutamic acid urea compound and can be used to deliver cytotoxic drugs to prostate cancer cells. DUPA, 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid, has the following structure: [ka] It has.

[0056] "LHRH peptide" refers to luteinizing hormone-releasing hormone peptide, which is commercially available. LHRH peptide can be used to target ovarian and prostate cancer cells.

[0057] "HER2 ligand" refers to a ligand capable of binding to the HER2 protein. Examples include, but are not limited to, anti-HER2 monoclonal antibodies, such as, but not limited to, trastuzumab and pertuzumab, and the EGFR ligands listed below.

[0058] "EGFR ligand" refers to a ligand capable of binding to the EGFR protein. Examples include, but are not limited to, EGF, TGF-α, HB-EGF, amphiregulin, betacellulin, epigen, epiregulin, neuregulin 1, neuregulin 2, neuregulin 3, and neuregulin 4.

[0059] "Toll-like receptor agonists" refer to compounds that bind to Toll-like receptors on cells, which play an important role in the immune system. Binding to the receptor activates the receptor, which can produce a biological response. Examples of Toll-like receptor agonists include, but are not limited to, CpG oligonucleotides.

[0060] "CpG oligonucleotide," also known as CpG ODN, refers to a cytosine-guanosine dinucleotide motif, in which the two nucleotides can be linked by a phosphodiester linker or a modified phosphorothioate linker.

[0061] "Dye" or "fluorescent dye" refers to a chemical molecule that emits light, typically in the 300-700 nm range, after excitation of the chemical molecule. Upon absorption of transferred light energy (e.g., a photon), the dye molecule reaches an excited state. When the molecule exits the excited state, it emits light energy in the form of a lower energy photon (e.g., fluorescent emission), and the dye molecule returns to its ground state. Dyes can be natural or synthetic chemicals. Dyes include, but are not limited to, cyanines, porphyrins, and bis-pyrenes.

[0062] "Porphyrin" refers to the following porphyrin core: [ka] where the porphine core is substituted or unsubstituted.

[0063] "Bis-pyrene" refers to a compound comprising two pyrene subunits covalently linked to each other. The two pyrene subunits can be linked directly or through a linker. The linker can be any linker known to those skilled in the art, including, but not limited to, alkylene, alkenylene, alkynylene, aryl, heteroaryl, aryl ketone, ketone, amine, amide, and urea, where the linker can be substituted.

[0064] A "radiometal chelator" refers to a multidentate ligand that binds to a central metal atom or ion. The metal atom or ion may be a radioisotope of the metal. Radiometal chelators include, but are not limited to, Gd(III) chelators, DOTA chelators, and NOTA chelators. Gd(III) chelators include, but are not limited to, gadopentetic acid, gadoteric acid, gadodiamide, gadobenic acid, gadoteridol, gadoversetamide, and gadobutrol.

[0065] "Cyanine" or "cyanine dye" refers to a family of synthetic dyes belonging to the polymethine group. Cyanines can be used as fluorescent dyes for biomedical imaging. Cyanines can be streptocyanines (also known as open-chain cyanines), hemicyanines, and closed-chain cyanines. Closed-chain cyanines each have a nitrogen that is independently part of a heteroaromatic moiety.

[0066] "Drug" refers to a substance capable of treating and / or ameliorating a condition or disease. A drug may be a hydrophobic drug, which is any drug that repels water. Hydrophobic drugs useful in the present invention include, but are not limited to, deoxycholic acid, taxanes, doxorubicin, etoposide, irinotecan, SN-38, cyclosporine A, podophyllotoxin, carmustine, amphotericin, ixabepilone, patupilone (epoterone class), rapamycin, and platinum drugs. Other drugs include nonsteroidal anti-inflammatory drugs and vinca alkaloids, such as vinblastine and vincristine. Drugs of the present invention also include prodrug forms. Those skilled in the art will recognize that other drugs are useful in the present invention.

[0067] "Chemotherapeutic agent" refers to a chemical agent that can be used to treat diseases such as, but not limited to, cancer, tumors, and neoplasms. In some embodiments, the chemotherapeutic agent can be in the form of a prodrug that can be activated to a cytotoxic form. Chemotherapeutic agents commonly known by those skilled in the art can be used in the present invention. Chemotherapeutic agents include, but are not limited to, resiquimod, gardikimod, and imiquimod.

[0068] "Immunomodulators" refer to a class of drugs that modify the immune response by stimulating or suppressing the immune system. Immunomodulators include, but are not limited to, resiquimod, gardikimod, and imiquimod.

[0069] "Anti-HER2 rhumAb 4D5" refers to a type of HER2 antibody, also known as trastuzumab. Trastuzumab is commonly used to treat breast and stomach cancer and is commercially available. Trastuzumab contains at least 50% peptide sequence identity to SEQ ID NO: 4. The peptide sequence of trastuzumab is described as "rationally designed anti-HER2 / neu peptidomimetics neutralize the P185 HER2 / neu tyrosine kinase in vitro and in vivo" (Park et al. Nat Biotechnol. 2000 Feb;18(2):194-8).

[0070] "CDR-H3 loop" refers to the internal region of a HER2 antibody involved in antigen binding.

[0071] "Nanocarriers" or "nanoparticles" refer to micelles resulting from the aggregation of compounds of the invention. Nanocarriers of the invention may have a hydrophobic core and a hydrophilic exterior.

[0072] "Nanofibrils" refer to tubular, rod-like fibrils having diameters ranging from tens to hundreds of nanometers. Nanofibrils can have high length-to-diameter ratios. Nanofibrils of the invention can be formed by in situ deformation of nanoparticles after binding at a targeted site.

[0073] "Fibril structure" refers to straight, rod-like fibrils with diameters on the order of nanometers to micrometers and a high length-to-diameter ratio. The fibril structure may contain biopolymers. Fibril structures include, but are not limited to, nanofibrils and microfibrils.

[0074] "Cell surface" refers to the plasma membrane, which separates the interior of the cell from the extracellular space. The cell surface comprises a lipid bilayer, proteins, and carbohydrates.

[0075] "Acellular components" refers to the extracellular environment of a cell and includes, but is not limited to, the extracellular matrix, extracellular vesicles, and pericellular cytokines. The extracellular matrix includes collagen, fibronectin, and other matrix proteins. Ligands and compounds can interact with the acellular components of cancer cells to affect cancer cell proliferation.

[0076] The "tumor microenvironment" refers to tumor cells and the acellular environment surrounding them, and includes, but is not limited to, the extracellular matrix, signaling molecules, immune cells, stromal cells, vasculature, blood vessels, cytokines, chemokines, growth factors, and fibroblasts. Tumors interact with surrounding cells in the microenvironment through the lymphatic and circulatory systems to influence the growth and development of cancer cells.

[0077] "Treat," "treating," and "treatment" mean any indicator of success in treating or ameliorating an injury, condition, state, or symptom (e.g., pain), including any objective or subjective parameter, such as remission; relief; lessening of symptoms or making the symptom, injury, condition, or state more tolerable to the patient; reducing the frequency or duration of the symptom or state; or, in some cases, preventing the symptom from occurring at all. Treatment or amelioration of symptoms can be based on any objective or subjective parameter, including, for example, the results of a physical examination.

[0078] "Administering" means administering to a subject orally, as a suppository, by topical contact, parenterally, intravenously, intraperitoneally, intramuscularly, intralesionally, intranasally or subcutaneously, intrathecally, intralymphatically, by inhalation of microdroplets, or by implantation of a sustained-release device, e.g., a mini-osmotic pump.

[0079] By "subject" is meant an animal such as a mammal, including, but not limited to, a primate (e.g., a human), cow, sheep, goat, horse, dog, cat, rabbit, rat, mouse, etc. In certain embodiments, the subject is a human.

[0080] "Therapeutically effective amount" or "therapeutically sufficient amount" or "effective or sufficient amount" refers to a dose that produces the therapeutic effect for which it is administered. The exact dose will depend on the purpose of the treatment and will be ascertainable by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, the therapeutically effective dose will often be lower than the usual therapeutically effective dose for non-sensitized cells.

[0081] "Cancer" refers to a disease involving abnormal cell growth and uncontrolled division. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. The term is also intended to include any disease of an organ or tissue characterized by poorly controlled or uncontrolled growth of normal or abnormal cells within that tissue, and its effects on the whole body.

[0082] "Imaging" refers to the use of a device outside the subject to measure the location of an imaging agent such as the compound of the present invention. Examples of imaging tools include, but are not limited to, fluorescence microscopy, positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound, single-photon emission computed tomography (SPECT), and X-ray computed tomography (CT). Positron emission tomography detects radiation from the emission of positrons by the imaging agent. III. Compound

[0083] In some embodiments, the present invention provides compounds of formula (I): ABC(I), where A is a hydrophobic moiety; B is a peptide, wherein the peptide forms a β-sheet; and C is a hydrophilic targeting ligand, which may include a HER2 ligand and any other suitable targeting ligand.

[0084] In some embodiments, the present invention provides a compound of formula (I): ABC(I), where A is a hydrophobic moiety; B is a peptide, wherein the peptide forms a β-sheet; and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting ligand is an LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, an LHRH peptide, a HER2 ligand, an EGFR ligand, or a toll-like receptor agonist CpG oligonucleotide.

[0085] In some embodiments, the present invention provides a compound of formula (I), wherein A is bis-pyrene; B is a peptide, wherein the peptide forms a β-sheet; and C is a HER2 ligand.

[0086] In some embodiments, the present invention provides a compound of formula (I): ABC(I), where A is a hydrophobic moiety; B is a peptide, wherein the peptide forms a β-sheet; and C is a hydrophilic targeting ligand, wherein when the hydrophobic moiety is bis-pyrene, C is other than a HER2 ligand.

[0087] In some embodiments, the present invention provides a compound of formula (I): ABC(I), where A is a hydrophobic moiety; B is a peptide, wherein the peptide forms a β-sheet; and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting ligand is an LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, LHRH peptide, HER2 ligand, EGFR ligand, or Toll-like receptor agonist CpG oligonucleotide; and wherein when the hydrophobic moiety is bis-pyrene, C is an LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, LHRH peptide, EGFR ligand, or Toll-like receptor agonist CpG oligonucleotide.

[0088] In some embodiments, the present invention provides a compound of formula (I): ABC(I), where A is a hydrophobic moiety; B is a peptide, wherein the peptide forms a β-sheet; and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting ligand is an LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, an LHRH peptide, an HER2 ligand, an EGFR ligand, or a Toll-like receptor agonist CpG oligonucleotide; and wherein when the hydrophobic moiety is bis-pyrene, C is an LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, an LHRH peptide, an EGFR ligand, or a Toll-like receptor agonist CpG oligonucleotide.

[0089] In some embodiments, the present invention provides a compound of formula (I): ABC(I), where A is a hydrophobic moiety; B is a peptide, wherein the peptide forms a β-sheet; and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting ligand is an LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, LHRH peptide, HER2 ligand, EGFR ligand, or Toll-like receptor agonist CpG oligonucleotide; and wherein when the hydrophobic moiety is bis-pyrene, C is an LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, LHRH peptide, EGFR ligand, or Toll-like receptor agonist CpG oligonucleotide.

[0090] Hydrophobic moieties useful in the present invention include any suitable hydrophobic moiety known to those of skill in the art. Hydrophobicity and hydrophilicity are generally measured by the logP value of a compound using the octane-water reference system. Values ​​below 0 indicate hydrophobicity, while values ​​above 0 indicate hydrophilicity. Hydrophobic moieties useful in the present invention include moieties having a logP value of less than 1. In some embodiments, hydrophobic moieties useful in the present invention have a logP value of at least 1.5. In some embodiments, hydrophobic moieties useful in the present invention have a logP value of 1.5 to 15. Hydrophobic moieties include, but are not limited to, cholesterol, vitamin D, vitamin D derivatives, vitamin E, vitamin E derivatives, dyes, drugs, and radioactive metal chealators. In some embodiments, the hydrophobic moiety is cholesterol, vitamin D, vitamin D derivatives, vitamin E, vitamin E derivatives, dyes, or drugs. In some embodiments, the hydrophobic moiety is cholesterol, vitamin D, vitamin E, dyes, or drugs. In some embodiments, the hydrophobic moiety is cholesterol, vitamin D, vitamin E, dyes, or drugs. In some embodiments, the hydrophobic moiety is cholesterol, vitamin D, or vitamin E. In some embodiments, the hydrophobic moiety is a dye or a drug.

[0091] Dyes useful in the present invention include, but are not limited to, any of the dyes described in Johnson, I., Histochemical Journal, 20:123-140 (1998) and The Molecular Probes® Handbook, 11th Edition, ed. Johnson and Spence, Life Technologies, Carlsbad, CA, 2010. The dyes can be fluorescent dyes, triarylmethane dyes, cyanine dyes, benzylidene imidazolinone dyes, indigo dyes, bis-pyrenes, and porphyrins. In some embodiments, the hydrophobic moiety is a dye. In some embodiments, the hydrophobic moiety is a fluorescent dye, a porphyrin, or a bis-pyrenes. In some embodiments, the hydrophobic moiety is a cyanine dye, a porphyrin, or a bis-pyrenes.

[0092] Drugs useful in the present invention include chemotherapeutic drugs and immunomodulatory drugs. For example, the drug may be, but is not limited to, deoxycholic acid or deoxycholate salt form, pembrolizumab, nivolumab, cemiplimab, taxanes (e.g., paclitaxel, docetaxel, cabazitaxel, baccatin III, 10-deacetylbaccatin, Hongdoushan A, Hongdoushan B, or Hongdoushan C), doxorubicin, etoposide, irinotecan, SN-38, cyclosporin A, podophyllotoxin, carmustine, amphotericin, ixabepilone, patupilone (epoterone class), rapamycin, and platinum drugs. Other drugs include nonsteroidal anti-inflammatory drugs and vinca alkaloids, such as vinblastine and vincristine. In some embodiments, the drug is paclitaxel, resiquimod, gardikimod, or deoxycholate.

[0093] In some embodiments, the hydrophobic moiety is a chemotherapeutic agent, a fluorescent dye, an immunomodulatory agent, a toll-like receptor agonist, a small molecule agonist of stimulator of interferon genes (STING), a porphyrin, deoxycholate, cholesterol, vitamin D, or vitamin E. In some embodiments, the hydrophobic moiety is a chemotherapeutic agent, a fluorescent dye, an immunomodulatory agent, a small molecule agonist of stimulator of interferon genes (STING), a porphyrin, cholesterol, vitamin D, or vitamin E. In some embodiments, the hydrophobic moiety is a chemotherapeutic agent, a fluorescent dye, an immunomodulatory agent, a small molecule agonist of stimulator of interferon genes (STING), a porphyrin, or deoxycholate. In some embodiments, the hydrophobic moiety is a chemotherapeutic agent, a fluorescent dye, an immunomodulatory agent, a porphyrin, or deoxycholate. In some embodiments, the hydrophobic moiety is paclitaxel, bis-pyrene, a cyanine dye, resiquimod, gardikimod, amidobenzimidazole, a porphyrin, or deoxycholate. In some embodiments, the hydrophobic moiety is paclitaxel, bis-pyrene, a cyanine dye, resiquimod, gardikimod, a porphyrin, or deoxycholate. In some embodiments, the hydrophobic moiety is resiquimod or a porphyrin.

[0094] Porphyrins useful in the present invention include any porphyrin known to those skilled in the art. In some embodiments, the porphyrin is substituted or unsubstituted porphine, protoporphyrin IX, octaethylporphyrin, tetraphenylporphyrin, pyropheophorbide-a, pheophorbide, chlorin e6, purpurin, or purpurinimide. In some embodiments, the porphyrin is pyropheophorbide-a, pheophorbide, chlorin e6, purpurin, or purpurinimide. In some embodiments, the porphyrin is pheophorbide-a. In some embodiments, the porphyrin has the following structure: [ka] It has.

[0095] In some embodiments, the hydrophobic moiety is bis-pyrene. Bis-pyrenes useful in the present invention include any bis-pyrene known to those skilled in the art. In some embodiments, the bis-pyrene is a bis-pyrene having the following moiety: [ka] In some embodiments, the bis-pyrene includes: [ka] In some embodiments, the bis-pyrene has the following structure: [ka] It has.

[0096] Peptides useful in the present invention can be any suitable peptide and have any suitable peptide sequence length known to those of skill in the art. In some embodiments, the peptide is a peptide sequence between 5 and 50 amino acids in length. In some embodiments, the peptide is a peptide sequence between 5 and 40 amino acids in length. In some embodiments, the peptide is a peptide sequence between 5 and 30 amino acids in length. In some embodiments, the peptide is a peptide sequence between 5 and 25 amino acids in length. In some embodiments, the peptide is a peptide sequence between 5 and 20 amino acids in length. In some embodiments, the peptide is a peptide sequence between 5 and 15 amino acids in length. In some embodiments, the peptide is a peptide sequence between about 5 and 10 amino acids in length.

[0097] Adjacent β-strand peptides form hydrogen bonds between each strand, resulting in a β-sheet peptide. The β-sheet peptide sequence useful in the present invention can be any suitable peptide sequence known to those skilled in the art. For example, commonly known β-sheet peptides are described in "Branched KLVFF tetramers strongly potentiate inhibition of β-amyloid aggregation" (Chafekar et al., Chembiochem. 2007 Oct 15;8(15):1857-64). In some embodiments, the peptide comprises a peptide sequence from a β-sheet peptide domain of green fluorescent protein, interleukin, immunoglobulin, or β-amyloid peptide. In some embodiments, the peptide comprises a peptide sequence from a β-sheet peptide domain of β-amyloid peptide. In some embodiments, the β-amyloid peptide is β-amyloid 40 or β-amyloid 42. In some embodiments, the β-amyloid peptide is β-amyloid 40.

[0098] In some embodiments, the peptide comprises at least 40% sequence identity to SEQ ID NO: 1. In some embodiments, the peptide comprises at least 50% sequence identity to SEQ ID NO: 1. In some embodiments, the peptide comprises at least 60% sequence identity to SEQ ID NO: 1. In some embodiments, the peptide comprises at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the peptide comprises SEQ ID NO: 1.

[0099] In some embodiments, the peptide comprises at least 40% sequence identity to SEQ ID NO: 2. In some embodiments, the peptide comprises at least 50% sequence identity to SEQ ID NO: 2. In some embodiments, the peptide comprises at least 60% sequence identity to SEQ ID NO: 2. In some embodiments, the peptide comprises at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the peptide comprises SEQ ID NO: 2.

[0100] In some embodiments, the peptide comprises at least 40% sequence identity to SEQ ID NO: 3. In some embodiments, the peptide comprises at least 50% sequence identity to SEQ ID NO: 3. In some embodiments, the peptide comprises at least 60% sequence identity to SEQ ID NO: 3. In some embodiments, the peptide comprises at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, the peptide comprises SEQ ID NO: 3.

[0101] Hydrophilic targeting ligands useful in the present invention can target receptors on cell surfaces or acellular components of the tumor microenvironment. Hydrophilicity and hydrophobicity are commonly measured by the logP value of a compound using the octane-water reference system. Values ​​below 0 indicate hydrophobicity, whereas values ​​above 0 indicate hydrophilicity. In some embodiments, hydrophilic targeting ligands include peptides that target cell surface receptors or acellular components within the tumor microenvironment, including, but not limited to, immune cells such as macrophages, T cells, and B cells. In some embodiments, hydrophilic targeting ligands target cell surface receptors, including, but not limited to, integrins and epidermal growth factor receptors. In some embodiments, hydrophilic targeting ligands target integrins, epidermal growth factor receptors, and toll-like receptors.

[0102] In some embodiments, the hydrophilic targeting ligand is a HER2 ligand, a prodrug of a HER2 ligand, a receptor tyrosine protein kinase targeting ligand, an integrin targeting ligand, an epidermal growth factor receptor targeting ligand, an ovarian cancer cell targeting ligand, or a prostate cancer cell targeting ligand. In some embodiments, the hydrophilic targeting ligand is a HER2 ligand, a prodrug of a HER2 ligand, an integrin targeting ligand, an epidermal growth factor receptor targeting ligand, an ovarian cancer cell targeting ligand, or a prostate cancer cell targeting ligand.

[0103] In some embodiments, the hydrophilic targeting ligand is a HER2 ligand. In some embodiments, the HER2 ligand is an anti-HER2 antibody peptide. In some embodiments, the hydrophilic targeting ligand is a HER2 ligand, wherein the HER2 ligand is an anti-HER2 antibody peptidomimetic derived from the primary sequence of the CDR-H3 loop of anti-HER2 rhumAb 4D5. In some embodiments, the HER2 ligand is described as "rationally designed anti-HER2 / neu peptidomimetics neutralize P185 HER2 / neu tyrosine kinase in vitro and in vivo" (Park et al. Nat Biotechnol. 2000 Feb;18(2):194-8.).

[0104] In some embodiments, the HER2 ligand has at least 40% sequence identity to SEQ ID NO: 4. In some embodiments, the HER2 ligand has at least 50% sequence identity to SEQ ID NO: 4. In some embodiments, the HER2 ligand has at least 60% sequence identity to SEQ ID NO: 4. In some embodiments, the HER2 ligand has at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, the HER2 ligand is SEQ ID NO: 4.

[0105] In some embodiments, the hydrophilic targeting ligand is an integrin targeting ligand, an epidermal growth factor receptor targeting ligand, an ovarian cancer cell targeting ligand, or a prostate cancer cell targeting ligand. In some embodiments, the hydrophilic targeting ligand is a prodrug of an integrin targeting ligand, an epidermal growth factor receptor targeting ligand, an ovarian cancer cell targeting ligand, or a prostate cancer cell targeting ligand.

[0106] In some embodiments, the hydrophilic targeting ligand is an LLP2A prodrug, LLP2A, LXY30, DUPA, folate, an LHRH peptide, or an EGFR ligand. Any one of the carboxylic acid groups of the DUPA structure can be used to link to a β-sheet peptide. LHRH analog peptides include the following peptide sequences: H-Glp-His-Trp-Ser-Thr-Lys-Leu-Arg-Pro-Gly-NH2 or H-Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2. The NH2 group of the Lys side chain of the LHRH peptide can be used to link to the β-peptide sheet. In some embodiments, the NH2 group is used for covalent linkage to the β-peptide sheet.

[0107] EGFR ligands useful in the present invention include any EGFR ligand known to those skilled in the art, in some embodiments, the EGFR ligand can be EGF, TGF-α, HB-EGF, amphiregulin, betacellulin, epigen, epiregulin, neuregulin 1, neuregulin 2, neuregulin 3, and neuregulin 4.

[0108] In some embodiments, the hydrophilic targeting ligand is an LLP2A prodrug, LLP2A, or LXY30. LLP2A prodrugs can include any cleavable functional group known to those skilled in the art that is metabolized in situ. In some embodiments, the LLP2A prodrug comprises an ester, amide, carbamate, oxime, imine, ether, phosphate, or β-amino-ketone functional group. In some embodiments, the LLP2A prodrug comprises an ester, amide, carbamate, ether, or phosphate functional group. In some embodiments, the LLP2A prodrug comprises an ester, amide, carbamate, or phosphate functional group. In some embodiments, the LLP2A prodrug comprises an ester group.

[0109] In some embodiments, the hydrophilic targeting ligand has the following structure: [ka] In some embodiments, the hydrophilic targeting ligand is an LLP2A prodrug having the following structure: [ka] In some embodiments, the hydrophilic targeting ligand is LLP2A having the following structure: [ka] The LXY30 has the following characteristics.

[0110] In some embodiments, the compound of the present invention has the following structure: [ka] It has.

[0111] In some embodiments, the compound of the present invention has the following structure: [ka] It has.

[0112] In some embodiments, the compound of the present invention has the following structure: [ka] It has.

[0113] In some embodiments, the compound of the present invention has the following structure: [ka] It has.

[0114] In some embodiments, the compound of the present invention has the following structure: [ka] It has.

[0115] In some embodiments, the compound of the present invention has the following structure: [ka] It has. IV. Nanocarriers

[0116] In some embodiments, the invention provides nanocarriers having an interior and an exterior, the nanocarriers comprising a plurality of compounds of the invention, wherein each compound self-assembles in an aqueous medium to form a nanocarrier such that a hydrophobic pocket is formed on the interior of the nanocarrier and hydrophilic groups self-assemble on the exterior of the nanocarrier.

[0117] The diameter of nanocarriers of the present invention can be any suitable size known to those of skill in the art. In some embodiments, nanocarriers have a diameter of 5-100 nm. In some embodiments, nanocarriers have a diameter of 10-100 nm. In some embodiments, nanocarriers have a diameter of 15-80 nm. In some embodiments, nanocarriers have a diameter of 25-60 nm. In some embodiments, nanocarriers have a diameter of about 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, or about 70 nm. In some embodiments, nanocarriers have a diameter of about 20 nm or about 30 nm. In some embodiments, nanocarriers have a diameter of about 20 nm. In some embodiments, nanocarriers have a diameter of about 30 nm.

[0118] The exterior of the nanocarrier can be used for cell targeting. The nanocarriers of the present invention can target cell membrane surface receptors and proteins such as, but not limited to, integrins, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptors, and G protein-coupled receptors. In some embodiments, the nanocarriers can target integrins and HER2.

[0119] The nanocarriers can deform in situ to form nanofibrillar structures after binding to cell surface receptors or proteins, hi some embodiments, the nanocarriers can deform in situ after binding to HER2 on the cell surface.

[0120] In some embodiments, the nanocarrier further comprises a hydrophobic drug or imaging agent sequestered within the hydrophobic pocket of the nanocarrier.

[0121] The hydrophobic drug useful in the present invention may be any hydrophobic drug known to those skilled in the art. Hydrophobic drugs useful in the present invention include, but are not limited to, deoxycholic acid, deoxycholate, resiquimod, gardquimod, imiquimod, taxanes (e.g., paclitaxel, docetaxel, cabazitaxel, baccatin III, 10-deacetylbaccatin, Hongdoushan A, Hongdoushan B, or Hongdoushan C), doxorubicin, etoposide, irinotecan, SN-38, cyclosporin A, podophyllotoxin, carmustine, amphotericin, ixabepilone, patupilone (epoterone class), rapamycin, and platinum drugs. Other drugs include nonsteroidal anti-inflammatory drugs and vinca alkaloids such as vinblastine and vincristine.

[0122] Contrast agents useful in the present invention can be any of those known to those skilled in the art. Contrast agents include, but are not limited to, paramagnetic agents, optical probes, and radionuclides. Paramagnetic agents are contrast agents that become magnetic under externally applied conditions. Examples of paramagnetic agents include, but are not limited to, iron particles, including nanoparticles. Optical probes are fluorescent compounds that can be detected by excitation at one emission wavelength and detection at a second, different emission wavelength. Optical probes useful in the present invention include, but are not limited to, Cy5.5, Alexa 680, Cy5, DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate), and DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide). Other optical probes include quantum dots. Radionuclides refer to elements that undergo radioactive decay. Radionuclides useful in the present invention include, but are not limited to: 3 H, 11 C. 13 N, 18 F, 19 F, 60 Co, 64 Cu, 67 Cu,68 Ga, 82 Rb, 90 Sr, 90 Y, 99 Tc, 99m Tc, 111 In, 123 I, 124 I, 125 I, 129 I, 131 I, 137 Cs, 177 Lu, 186 Re, 188 Re, 211 At, Rn, Ra, Th, U, Pu and 241 Am is one example.

[0123] A nanocarrier can comprise a plurality of conjugates. For example, a nanocarrier can comprise a plurality of 2, 3, 4, 5, 6, or more distinct conjugates. In some embodiments, a nanocarrier comprises a plurality of 2 distinct conjugates. In some embodiments, a nanocarrier comprises a plurality of 3 distinct conjugates. In some embodiments, a nanocarrier comprises a plurality of 4 distinct conjugates.

[0124] In some embodiments, the invention provides nanocarriers having an interior and an exterior, the nanocarriers comprising a plurality of first conjugates and second conjugates, wherein the first conjugates comprise Formula (I): ABC(I) and the second conjugates comprise Formula (II): A'-B'-C'(II), where A and A' are each independently a hydrophobic moiety; B and B' are each independently a peptide, wherein each peptide independently forms a β-sheet; and C and C' are each independently a hydrophilic targeting ligand, wherein each hydrophilic targeting ligand is independently an LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, an LHRH peptide, a HER2 ligand, an EGFR ligand, or a radiometal chelator; and where A and A' are different hydrophobic moieties, and / or C and C' are different hydrophilic targeting ligands.

[0125] In some embodiments, the nanocarrier comprises a plurality of first and second conjugates as described above, and further comprises Formula (III): A'''-B'''-C'''(III), where A'' is a hydrophobic moiety, B'' is a peptide, wherein the peptide forms a β-sheet, and C'' is a hydrophilic targeting ligand, and where A, A', and A'' are different hydrophobic moieties and / or C, C', and C'' are different hydrophilic targeting ligands. In some embodiments, the nanocarrier further comprises a fourth, fifth, or sixth conjugate, where each additional conjugate is independent of Formula III.

[0126] Nanocarriers of the present invention can comprise a plurality of two distinct conjugates. Nanocarriers comprising a plurality of two distinct conjugates can have the diameters described above. Nanocarriers comprising a plurality of two distinct conjugates can have similar targeting and tunable properties as described above.

[0127] Suitable hydrophobic moieties for nanocarriers of the present invention are described above. In some embodiments, each hydrophobic moiety is independently a dye, a drug, or a radioactive metal chelator. In some embodiments, each hydrophobic moiety is independently a bis-pyrene, a porphyrin, resiquimod, or gardikimod.

[0128] In some embodiments, each hydrophobic moiety is independently a porphyrin or resiquimod. In some embodiments, the porphyrin is pyropheophorbide-a, pheophorbide, chlorin e6, purpurin, or purpurinimide. In some embodiments, the porphyrin is pheophorbide-a. In some embodiments, the porphyrin has the following structure: [ka] It has.

[0129] In some embodiments, resiquimod has the following structure: [ka] It has.

[0130] Radioactive metal chelators useful in the present invention include any radioactive metal chelator known to those skilled in the art. In some embodiments, the radioactive metal chelator is a Gd(III) chelator, diethylenetriaminepentaacetic anhydride (DTPA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), or 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). In some embodiments, the radioactive metal chelator is a Gd(III) chelator, a DOTA chelator, or a NOTA chelator.

[0131] Suitable peptide sequence lengths for nanocarriers of the present invention are described above. In some embodiments, each peptide is independently a peptide sequence between 5 and 30 amino acids in length. In some embodiments, each peptide is independently a peptide sequence between 5 and 25 amino acids in length. In some embodiments, each peptide is independently a peptide sequence between 5 and 20 amino acids in length.

[0132] Suitable peptide sequences for the nanocarriers of the present invention are described above. In some embodiments, each peptide independently comprises a peptide sequence from the β-sheet peptide domain of β-amyloid peptide. In some embodiments, the β-amyloid peptide is β-amyloid 40 or β-amyloid 42. In some embodiments, the β-amyloid peptide is β-amyloid 40.

[0133] In some embodiments, each peptide independently comprises at least 40% sequence identity to SEQ ID NO: 1. In some embodiments, each peptide independently comprises at least 50% sequence identity to SEQ ID NO: 1. In some embodiments, each peptide independently comprises at least 60% sequence identity to SEQ ID NO: 1. In some embodiments, each peptide independently comprises at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, each peptide independently comprises SEQ ID NO: 1.

[0134] In some embodiments, each peptide independently comprises at least 40% sequence identity to SEQ ID NO: 2. In some embodiments, each peptide independently comprises at least 50% sequence identity to SEQ ID NO: 2. In some embodiments, each peptide independently comprises at least 60% sequence identity to SEQ ID NO: 2. In some embodiments, each peptide independently comprises at least 80% sequence identity to SEQ ID NO: 2.

[0135] In some embodiments, suitable hydrophilic targeting ligands for the nanocarriers of the present invention are described above. In some embodiments, each hydrophilic targeting ligand is independently an LLP2A prodrug, LLP2A, LXY30, folate, an LHRH peptide, a HER2 ligand, an EGFR ligand, a Gd(III) chelator, a DOTA chelator, or a NOTA chelator. In some embodiments, each hydrophilic targeting ligand is independently an LLP2A prodrug, LLP2A, LXY30, an LHRH peptide, a HER2 ligand, an EGFR ligand, a DOTA chelator, or a NOTA chelator. In some embodiments, each hydrophilic targeting ligand is independently an LLP2A prodrug, LLP2A, or LXY30.

[0136] In some embodiments, each hydrophilic targeting ligand independently has the following structure: [ka] is an LLP2A prodrug having the formula:

[0137] In some embodiments, each hydrophilic targeting ligand independently has the following structure: [ka] LLP2A has the following.

[0138] In some embodiments, each hydrophilic targeting ligand independently has the following structure: [ka] The LXY30 has the following characteristics.

[0139] In some embodiments, the first conjugate has the following structure: [ka] It has.

[0140] In some embodiments, the second conjugate has the following structure: [ka] It has.

[0141] In some embodiments, the second conjugate has the following structure: [ka] is converted in situ into

[0142] The ratio of the first conjugate to the second conjugate of the nanocarriers of the present invention can be any suitable ratio known to one of skill in the art and is reported as a molar ratio. In some embodiments, the ratio of the first conjugate to the second conjugate is about 25:1 to 1:25. In some embodiments, the ratio of the first conjugate to the second conjugate is about 25:1 to 1:10. In some embodiments, the ratio of the first conjugate to the second conjugate is about 10:1 to about 1:10. In some embodiments, the ratio of the first conjugate to the second conjugate is about 10:1, 8:1, 5:1, 3:1, or 1:1. In some embodiments, the ratio of the first conjugate to the second conjugate is about 1:1. V. Nanofibrils

[0143] In some embodiments, the present invention provides a method of forming nanofibrils, comprising contacting a nanocarrier of the present invention with a cell surface or acellular components in a tumor microenvironment, wherein the nanocarrier undergoes deformation in situ to form a fibril structure, thereby forming nanofibrils.

[0144] When the nanocarriers of the present invention bind to cell surfaces or acellular components in the tumor microenvironment, they undergo in situ deformation to form nanofibrils, which perturb the cells and / or the tumor microenvironment. Deformation of the nanocarriers occurs when the hydrophilic targeting ligands of the nanocarriers bind to the cell surface or acellular components of interest and cause the formation of fibril structures that form nanofibrils.

[0145] The tumor microenvironment includes tumor cells and the surrounding environment, including, but not limited to, extracellular matrix, infiltrating host cells, secreted factors, signaling molecules, immune cells, stromal cells, dendritic cells, T cells, myeloid-derived suppressor cells, vasculature, blood cells, cytokines, chemokines, growth factors, fibroblasts, and macrophages, any of which the nanocarriers of the present invention interact with to form nanofibrils.

[0146] The nanocarriers of the present invention can form highly ordered β-sheet fibril structures of nanofibrils. Without being bound by any particular theory, one possible explanation for the formation of β-sheet fibril structures is that the β-sheet forming peptide in the conjugate influences the formation of β-sheet fibril structures of nanofibrils.

[0147] The nanofibrils of the present invention can have any suitable diameter known to those of skill in the art. In some embodiments, the diameter of the nanofibrils is 5 to 50 nm. In some embodiments, the diameter of the nanofibrils is 5 to 30 nm. In some embodiments, the diameter of the nanofibrils is 5 to 15 nm. In some embodiments, the diameter of the nanofibrils is 5 to 10 nm. In some embodiments, the diameter of the nanofibrils is about 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, or about 12 nm.

[0148] The transformation of nanocarriers into nanofibrils can be measured by imaging techniques known to those skilled in the art and by measuring the particle size of the nanocarriers. For example, the transformation of nanocarriers into nanofibrils can be measured using TEM imaging, where the round nanocarrier shape transforms into a nanofibrillar structure following binding of the nanocarriers to cell surfaces or acellular components in the tumor microenvironment. In another example, nanocarrier size can be measured using dynamic light scattering (DLS). In DLS studies, as nanocarriers transform into nanofibrils, a peak around the diameter of the nanocarrier, e.g., 10-100 nm, increases over time, as does a peak around approximately 500 nm-1000 nm, indicating the formation of nanofibrils. VI. Treatment and Imaging Methods

[0149] In some embodiments, the present invention provides a method of treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of a nanocarrier of the present invention, wherein the nanocarrier forms nanofibrils in situ after binding to cell surfaces or acellular components in the tumor microenvironment, thereby treating the disease.

[0150] Binding to cell surfaces or acellular components can be measured by those skilled in the art using a fluorescence microscope. Binding to cell surfaces or acellular components can be measured when the nanocarrier contains a conjugate with a fluorescent dye as the hydrophobic moiety and the cells are labeled with any fluorescent dye known to those skilled in the art. Those skilled in the art can select an appropriate dye to use based on which fluorescent dye is used as the hydrophobic moiety. For example, when the nanocarrier contains a conjugate containing bis-pyrene, whose hydrophobic moiety is a green fluorescent dye, the cells can be labeled with a non-green fluorescent dye, such as, but not limited to, a red fluorescent dye or a blue fluorescent dye. In another example, when the hydrophobic moiety contains a red fluorescent dye, such as, but not limited to, a porphyrin, those skilled in the art can select a non-red fluorescent dye, such as, for example, a green fluorescent dye or a blue fluorescent dye.

[0151] The tumor microenvironment comprises tumor cells and the surrounding environment, including, but not limited to, extracellular matrix, infiltrating host cells, secreted factors, signaling molecules, immune cells, stromal cells, dendritic cells, T cells, myeloid-derived suppressor cells, vasculature, blood cells, cytokines, chemokines, growth factors, fibroblasts, and macrophages. Tumor growth and progression can be influenced by the interaction of cancer cells with the microenvironment, which can result in the eradication of cancer cells, the metastasis of cancer cells, or the establishment of a dormant state of micrometastatic cancer cells. The tumor microenvironment can be targeted for treatment responses.

[0152] Binding to acellular components in the tumor microenvironment includes, but is not limited to, binding to proteins within the extracellular matrix, as well as other ligands, compounds, or dendritic cells that attach directly to tumor cells or surrounding cells.

[0153] The nanocarriers of the present invention can be administered to a subject for the treatment of hyperproliferative disorders, including, for example, but not limited to, cancers such as carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostate cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, cancer of the esophagus, stomach cancer, pancreatic cancer, hepatobiliary cancer, cancer of the gallbladder, cancer of the small intestine, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, cervical cancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastoma, multiple myeloma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma (for other cancers, see CANCER: PRINCIPLES AND PRACTICE). (See DeVita, VT et al. eds 2008).

[0154] Other diseases that may be treated by the nanocarriers of the present invention include: (I) inflammatory or allergic diseases such as systemic anaphylaxis or hypersensitivity reactions, drug allergies, insect sting allergies; inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, ileitis, and enteritis; vaginitis; psoriasis and inflammatory skin diseases such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis; spondyloarthropathy; scleroderma; respiratory allergic diseases such as asthma, allergic rhinitis, and hypersensitivity pulmonary disease; and (2) arthritis (rheumatoid and psoriatic), osteoarthritis, and the like. (3) autoimmune diseases such as inflammatory bowel disease, multiple sclerosis, systemic lupus erythematosus, diabetes mellitus, and glomerulonephritis; (4) transplant rejection (including allograft rejection and graft-versus-host disease); and (5) other diseases in which unwanted inflammatory responses should be inhibited (e.g., atherosclerosis, myositis, neurological conditions such as stroke and closed head injury, neurodegenerative diseases, Alzheimer's disease, encephalitis, meningitis, osteoporosis, gout, hepatitis, nephritis, sepsis, sarcoidosis, conjunctivitis, otitis, chronic obstructive pulmonary disease, sinusitis, and Behcet's syndrome).

[0155] In some embodiments, the disease is cancer. In some embodiments, the disease is selected from the group consisting of bladder cancer, brain cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, intestinal cancer, head and neck cancer, leukemia, liver cancer, lung cancer, melanoma, myeloma, ovarian cancer, pancreatic cancer, and uterine cancer. In some embodiments, the disease is selected from the group consisting of bladder cancer, breast cancer, colorectal cancer, esophageal cancer, glioblastoma, head and neck cancer, leukemia, lung cancer, myeloma, ovarian cancer, and pancreatic cancer.

[0156] In some embodiments, the nanocarriers of the present invention can be used in combination therapy. In some embodiments, the combination therapy comprises the nanocarriers of the present invention and at least one checkpoint inhibitor. Representative checkpoint inhibitors include, but are not limited to, anti-CTLA-4 therapy, anti-PD-1 therapy, or anti-PD-L1 therapy. Examples include ipilimumab, nivolumab, pembrolizumab, pidilizumab, atezolizumab, ipilimumab, and / or tremelimumab, and may also include combination therapies such as nivolumab plus ipilimumab.

[0157] In some embodiments, the present invention provides a method of imaging, comprising administering to a subject to be imaged an effective amount of a nanocarrier of the present invention.

[0158] Suitable imaging agents for the nanocarriers of the present invention have been described above. For example, imaging agents include, but are not limited to, paramagnetic agents, optical probes, and radionuclides. Optical probes include, but are not limited to, fluorescent dyes such as cyanine dyes, bis-pyrenes, and porphyrins. [Example]

[0159] VII. Working Examples Example 1: Nanocarrier of BP-FFVLK-YCDGFYACYMDV This example describes the design and synthesis of a high-performance supramolecular peptide, BP-FFVLK-YCDGFYACYMDV, which can (1) assemble into nanoparticles (NPs) in the blood circulation under aqueous conditions and (2) transform in situ into nanofibrillar (NF) structures upon binding to cell-surface HER2 at tumor sites. This variable peptide monomer (TPM) supramolecular material consists of three distinct functional domains: (1) a bis-pyrene (BP) moiety as a hydrophobic core that possesses aggregation-induced emission (AIE) properties for a fluorescent reporter and triggers the formation of micellar NPs; (2) a KLVFF β-sheet-forming peptide domain derived from the β-amyloid (Aβ) peptide; and (3) a YCDGFYACYMDV disulfide cyclic peptide HER2-binding domain, an anti-HER2 / neu antibody peptidomimetic derived from the primary sequence of the CDR-H3 loop of the anti-HER2 rhumAb 4D5. Under aqueous conditions, the supramolecular peptides would self-assemble into spherical NPs, in which the BP and KLVFF domains constituted the hydrophobic core, and the YCDGFYACYMDV peptide constituted the negatively charged hydrophilic corona. NPs injected intravenously (iv) into mice bearing HER2+ tumors were found to preferentially accumulate at the tumor site. Upon interaction with HER2 displayed on the tumor cell surface, the NPs would undergo in situ transformation into a fibril structural network with a long retention time. Such a HER2-bound extracellular fibril network was found to strongly inhibit HER2 dimerization and prevent downstream cell signaling and expression of proliferation and survival genes in the nucleus. These structural transformation-based supramolecular peptides represent a novel class of receptor-mediated targeted therapeutics for cancer. Materials and Methods

[0160] Preparation of variable peptide monomers (TPMS) 1'–4'. The hydrophobic bis-pyrene unit (BP-COOH) was synthesized as previously reported (Qiao, S.-L. et al. Thermo-Controlled in Situ Phase Transition of Polymer-Peptides on Cell Surfaces for High-Performance Proliferative Inhibition. ACS Appl. Mater. Interfaces 8, 17016–17022 (2016)). TPMs 1'–4' were synthesized using standard solid-phase peptide synthesis techniques. BP-COOH, as the hydrophobic moiety, was linked to the TPM 1'–4' chain. For TPMs 3' and 4', PEG1000, as the hydrophilic unit, was linked to the peptide, replacing the HER2 ligand in molecules 1 and 2. The molecular structures of the BP dye and peptides were confirmed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (ESI and MALDI-TOF mass spectrometry, Bruker Daltonics).

[0161] Preparation and Characterization of NP Self-Assembled Formation. TPM1'-4' were dissolved in DMSO to form respective solutions. The peptide solutions (5 μL) were further diluted with DMSO (995, 795, 595, 395, 195, 95, 15, and 0 μL) and mixed with deionized water (0, 200, 400, 600, 800, 900, 980, and 995 μL), respectively. UV-vis absorption and fluorescence spectra (Thermo Scientific, Waltham, MA) of the mixtures with various water contents were measured to confirm the authenticity of NP formation. Fresh NPs (99% water content, 20 μM) were used as the initial state for the measurements. Morphological transformation of the NPs into NFs was induced by adding HER2 extracellular receptor protein (expressed in HEK293 cells, Sigma-Aldrich) and incubating at 37°C for several hours. At various time points (0.5, 6, and 24 h), the solutions were used for size / zeta potential (Microtrac, America), CD (JASCO Inc, Easton, MD, USA), and TEM measurements (Philips CM-120 TEM, America). TEM samples were stained with uranyl acetate.

[0162] Stability of NP1 in human plasma. The stability of NP1 was investigated in 10% (v / v) plasma from healthy human volunteers. The mixture was incubated at physiological body temperature (37°C) followed by size measurements at scheduled time intervals up to 168 hours.

[0163] MCF-7 / C6 cell derivation process. The derivation method for MCF-7 / C6 cells was obtained from Professor Jian Jian Li's laboratory (Department of Radiation Oncology, University of California, Davis). The MCF-7 / C6 radioresistant cell line survived 25 fractionated ionizing radiation doses using γ-rays with a total dose of 50 Gy (2 Gy per fraction, 5 times per week).

[0164] CLSM and SEM validation of NP structural modifications on the cell surface. Cells were cultured in glass-bottom dishes for 12 h. NPs 1–4 (50 μM) were incubated with cells in DMEM for 0.5, 6, and 24 h at 37°C, respectively. For confocal laser scanning microscope (CLSM, Zeiss LSM710, Jena, Germany) imaging, specimens were solidified with glutaraldehyde (4%) for 10 min, washed three times with PBS, and then examined with a 40× or 63× immersion objective using a 405 nm laser. To further validate the binding of NP1 to HER2, rabbit anti-HER2 (29D8) monoclonal antibody (MAb) (Sigma-Aldrich, USA) was used to detect the extracellular domain of HER2 on the surface of MCF-7 / C6 cells. For SEM (Philips XL30 TMP, FEI Company, Hillsboro), cells were fixed overnight with glutaraldehyde (4%) and then coated with gold for 2 min.

[0165] In vitro cytotoxicity assay. MCF-7 / C6, MCF-7, SKBR-3, and BT474 cells were used to evaluate the cytotoxicity of NP1–4. Cells per well (n=3) were seeded into 96-well plates and cultured at 37°C in a humidified atmosphere containing 5% CO2 using DMEM supplemented with 10% FBS and 1% penicillin. DMSO solutions of NP1–4 were diluted in DMEM (1.5, 7.5, 15, 75, 150, and 300 μM) and then added to each well and incubated with the cells. After 48 hours of incubation, MTS reagent was added to each well. Relative cell viability was measured using a microplate reader (SpectraMax M2). The percentage of cell viability represented the drug efficacy, and 100% meant that all cells survived. Cell viability was calculated using the following equation: Cell viability (%) = (OD490nm of treatment / OD490nm of blank control) x 100%.

[0166] Western blot analysis. MCF-7 / C6 cells were treated with various conditions, then harvested by centrifugation at 14,000 rpm for 10 minutes and lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl) containing 1% (v / v) Triton X-100 and protease inhibitors. Total cellular protein was estimated using a BCA kit (Applygen). Each sample (50 μg of protein) was subjected to SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% (wt / v) nonfat dry milk in blotting solution (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) at room temperature for 2 hours, the membrane was incubated with primary antibodies overnight at 4°C. The membrane was then washed with TBST solution (3 x 5 min) and incubated with secondary antibodies for 2 h at room temperature. Signals were visualized by chemiluminescence using a Typhoon Trio Variable Mode Imager. Band densities were calculated using NIH Image J software.

[0167] For HER2 dimer Western blot analysis, MCF-7 / C6 cells were treated using the indicated protocol and then lysed with a buffer containing 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO, 1% Triton X-100, and a protease inhibitor cocktail (Sigma-Aldrich). The lysis supernatant was collected after centrifugation at 12,000 rpm for 15 minutes. 0.2% glutaraldehyde was added to the lysis supernatant for 10 minutes at 37°C. The lysate was collected for Western blot analysis.

[0168] Animal model. All animal experiments were performed according to protocol No. 19724, approved by the Animal Use and Care Administrative Advisory Committee at the University of California, Davis. Female BALB / c nude mice were 6-8 weeks old (weight 22 ± 2 g) and purchased from Harlan (Livermore, CA, USA). MCF-7 / C6 cells (5 × 10 per mouse) were cultured. 6 Female BALB / c nude mice were subcutaneously inoculated with NPs (8 mg / kg) into the flanks. Approximately 10 days later, NPs 1–4 (8 mg / kg) were injected via the tail vein. Ex vivo images of the tumor, heart, liver, spleen, lungs, kidneys, intestine, muscle, and skin were collected at 10, 24, 48, 72, and 168 hours after injection. Images were collected using an in vivo fluorescence imaging system (Carestream In-Vivo Imaging System FXPRO, USA). Tumors and major organs (heart, liver, spleen, lungs, kidneys, and brain) were harvested and solidified with glutaraldehyde (4%) at 72 hours after NP injection for TEM imaging.

[0169] Effect of treatment in vivo. MCF-7 / C6 cells (5 × 10 per mouse) were inoculated subcutaneously in the flank. 6 BALB / c nude mice bearing tumors of 1000 cells were used in our experiments. Ten days after tumor inoculation, the mice were randomly assigned to five groups. Each of them was treated with PBS, NP1, NP2, NP3, or NP4 by intravenous administration every 48 hours. During the treatment process (40 days), tumor volume and body weight were measured twice weekly. In parallel, the therapeutic effect of NP1 was confirmed in mice bearing SKBR-3 and BT474 tumors using similar experimental methods. MCF-7 / C6 tumor-bearing mice were sacrificed after three treatments, and tumor tissues were collected for hematoxylin and eosin (H&E) staining and Ki-67 assays.

[0170] Statistical analysis. Data are presented as mean ± standard deviation (SD). Comparisons between groups were analyzed using Student's t-test (two-tailed). One-way analysis of variance (ANOVA) was used for multigroup analysis. Significance levels were defined as *p<0.05, **p<0.01, and ***p<0.001. All statistical tests were two-tailed. Results and Discussion

[0171] Self-Assembly and Fibrillar Deformation of Supramolecular Materials. Variable peptide monomer 1 (TPM1'), BP-FFVLK-YCDGFYACYMDV, was prepared by standard solid-phase peptide synthesis techniques, followed by N-terminal capping with bis-pyrene, and its identity was confirmed by MALDI-TOF-MS (Figure 6). For comparison purposes, TPM2' (BP-GGAAK-YCDGFYACYMDV), TPM3' (BP-FFVLK-PEG1000), and TPM4' (BP-GGAAK-PEG1000) were synthesized as negative controls (Table 1 and Figures 7-9). As the water content in the mixed solvent (water and DMSO) of TPM1' solution increased, there was a gradual decrease in the absorption peak (250-450 nm), reflecting the gradual formation of nanoparticles NP1 via self-assembly, caused by π-π interactions and the strong hydrophobicity of the BP and β-sheet-forming peptide sequences (Figure 1A). Concomitantly, the fluorescence peak at 520 nm was found to be dramatically enhanced due to the AIE fluorescence properties of the BP dye (Figure 1B). TPM2', TPM3', and TPM4' all exhibited similar self-assembly properties. Nanoparticles (NP1, NP2, NP3, and NP4) assembled from the four TPMs by rapid aqueous dilution were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM) (Figure 1C). The diameters of NP1-NP4 were found to be approximately 20 nm, 30 nm, 25-60 nm, and 20 nm, respectively. [Table 1]

[0172] To investigate the interaction between HER2 and NP1 in vitro, we selected the soluble extracellular domain of HER2 protein as a transformation inducer. As shown by the TEM image in Figure 1C, NP1 was found to maintain a globular structure of approximately 20 nm before interaction with HER2. After only 30 min of incubation with HER2 protein at room temperature (HER2 peptide / HER2 protein molar ratio ≈1000:1), a small number of granular nanofibrillar structures (NF1, approximately 10 nm in diameter) became apparent; more NF1 was detected at 6 h. At 24 h, a fibrillar network with a broad size distribution was clearly detected, indicating that the transformation process was receptor-mediated and time-dependent. No transformation was observed in NP1 preparations without the addition of HER2 protein, even after 24 h. The structural transformation of NP1 to NF1 was also confirmed in solution by DLS, with a gradual increase in the 20 nm peak and a corresponding decrease in the 100–1000 nm peak over time (Figure 1D). In contrast, similar treatment of NP2, NP3, and NP4 solutions with HER2 did not reveal any significant changes over 24 h. A common feature of the TPMs that formed these three negative control NPs was the simultaneous absence of two essential domains for receptor-mediated transformation in NP1: the HER2 ligand and the KLVFF β-sheet-forming peptide. Circular dichroism (CD) spectroscopy was used to observe the conformation and secondary structure of TPM1' upon transformation (Figure 1E). During the initial rapid self-assembly to form NP1, no clear secondary structure was observed, likely because the hydrophobic interactions induced by BP were too rapid to form any intermolecular hydrogen bonds. When NP1 begins to transform into NF1 over a 24-hour period in the presence of HER2, a negative CD signal at 216 nm and a positive CD signal at 195 nm gradually develop over time, indicating β-sheet formation via hydrogen bond formation. In addition to CD, we utilized the unique AIE fluorescence properties of BP to observe the kinetics of TPM1' transformation.As shown in Figure 1F, the fluorescence intensity of BP in NP1 decreased by approximately 10% 30 min after the addition of HER2, but recovered and increased as the transformation to NF1 progressed, ultimately reaching an approximately 50% increase by 24 h. One plausible explanation for this intriguing observation is that the packing density of BP or TPM1 within the fibril network (NF1 at 24 h) was significantly higher than that of the initial spherical structure (NP1). However, when spherical NP1 was exposed to HER2, there was a transient relaxation of packing density during the initial transformation process before reorganization into a more densely packed nanofibril network. We also demonstrated that the particle size of NP1 in PBS without HER2 remained unchanged over 7 days at 37 °C, regardless of the presence or absence of 10% fetal bovine serum (FBS).

[0173] Morphological Characterization of NP Fibril Transformation. To further characterize the interaction of the transforming peptide with cell surface receptors in live cells, HER2+ breast cancer cell lines (SKBR-3 and BT474 cells) were incubated with NP1 and then confocal laser scanning microscopy (CLSM) was used to track the fluorescent green signal emitted by the NP1 (Figure 2A-2B). After 6 hours of incubation of NP1 with these two cell lines, the green fluorescent signal was observed on the cell surface rather than intracellularly. In contrast, for MCF-7 breast cancer cells, which have low expression levels of HER2, the majority of the fluorescent signal was found to reside intracellularly after 6-24 hours, indicating that cell surface presentation of HER2 protein was required for the transformation of NP1 into a nanofibrillar network near the cells (Figure 2C).

[0174] Radiation treatment is commonly used in the management of breast cancer patients. It has previously been reported that prolonged fractionated ionizing radiation (FIR) induces HER2 expression in both clinical and experimental models. The HER2+ MCF-7 / C6 tumor cell line used was derived from a HER2-negative human breast cancer MCF-7 cell line that underwent 30 days of FIR induction, followed by colony formation and clonal isolation. MCF-7 / C6 cells exhibit characteristics of radiation resistance, high HER2 expression levels, a more aggressive phenotype, and high levels of cancer stem cell properties. The relative expression level of HER2 protein, as measured by Western blot, was found to be 5-fold higher in MCF-7 / C6 cells than in MCF-7 cells (Figure 2D). After 30 minutes of incubation of MCF-7 / C6 cells with NP1 (100 μM), green fluorescent dots were observed on the cell membrane (Figure 2E). By 24 hours, a highly abundant green fluorescent layer was observed surrounding the entire cell.

[0175] To further confirm the binding of NP1 to HER2, we used a rabbit anti-HER2 (29D8) monoclonal antibody (MAb) to detect the extracellular domain of HER2 on the surface of MCF-7 / C6 cells. The anti-HER2 MAb was fluorescently labeled red by a secondary Ab. NP1 and the transformed nanofibrillar network (NF1) were fluorescently labeled green by the intrinsic optical properties of BP. As shown in Figure 2F, the green fluorescence completely overlapped with the red fluorescence around the outer edges of the two cells. The merged image showed an overlap of green and red (forming a yellow color) around the cell surface, except for the adhesion interface between the two cells, which was stained only by the anti-HER2 MAb (red fluorescence), but not by NP1. This data was consistent with our concept that the transformation of NP1 to NF1 is caused by interaction with cell surface HER2 receptors exposed to the medium. The cellular distribution of negative control NPs (NP2, NP3, and NP4) was also investigated in MCF-7 / C6 cells. After 24 hours of incubation, the majority of the fluorescent signal was found intracellularly instead of on the cell surface. Scanning electron microscopy (SEM) confirmed the presence of a nanofibrillar network (NF1) on the surface of NP1-treated MCF-7 / C6 cells, but not on untreated cells (Figure 2G). In contrast, nanofibrillar structures were not detected on the surface of cells treated with NP2, NP3, or NP4. Transmission electron microscopy (TEM) was used to better define the ultrastructure of the nanofibrillar network. Similar to the results obtained by SEM, abundant bundles of nanofibrils were detected on the surface of C6 and MCF-7 / C6 cells for 24 hours after incubation with NP1. No nanofibrillar structures were detected in untreated MCF-7 / C6 cells or cells treated with three negative control NPs for 24 hours. In a separate negative control experiment in which the MCF-7 cell line, which has low levels of HER2 expression, was incubated with NP1 for 24 hours, only minimal nanofibrils were detected on the cell membrane.

[0176] Extracellular and Intracellular Mechanisms of Fibril Transformation. It is conceivable that HER2-mediated transformation of nanoparticles (NP1) into nanofibrillar networks (NF1) may attenuate HER2 dimerization, leading to the inhibition of downstream signal transduction. To demonstrate this plausible mechanism, MCF-7 / C6 cells were incubated with NP1, NP2, or PBS for 8 hours (Figure 3A). For NP1-treated cells, the majority of the green fluorescent signal (BP) colocalized with the red fluorescent signal (anti-HER2), indicating that the nanofibrillar network was closely associated with the HER2 receptor displayed on the cell surface. For cells treated with NP2, in which the HER2 ligand was present but the β-sheet-forming peptide was mutated, the green fluorescence on the cell surface was weak. Furthermore, the green / red fluorescent signal on the membrane of NP1-treated cells appeared significantly stronger and more discontinuous, suggesting clustering of nanofibrillar structures and possibly even membrane disruption.

[0177] The cytotoxic effects of NP1 and three negative control NPs on MCF-7 / C6 cells after 48 hours of incubation were measured using an MTS assay. As shown in Figure 3B, treatment with NP1 resulted in significant cell death in a dose-dependent manner, with cell viabilities of 37% and 13% at 150 μM and 300 μM, respectively. Similar results were obtained for two other HER2+ breast cancer cell lines, SKBR-3 and BT474. However, when MCF-7 cells, which have low levels of HER2 expression, were treated with these four NPs, no obvious cytotoxicity was observed, even at the highest concentration of 300 μM. This is consistent with our concept that nanotransformation and the resulting cytotoxicity of NP1 are HER2-mediated. To explore the mechanism by which NP1 induces apoptosis, the expression levels of various pro- and anti-apoptotic proteins were assessed by Western blot. As shown in Figure 3C, treatment of MCF-7 / C6 cells with NP1 resulted in a dose-dependent downregulation of the anti-apoptotic protein Bcl-2 and an upregulation of the apoptotic protein Bax. To investigate the effect of NP1 on HER2 dimerization, we used a simple chemical cross-linking method using 0.2% glutaraldehyde followed by Western blot analysis using an anti-HER2 antibody. This method allowed us to distinguish dimeric HER2 from its monomeric form. It was evident from Figures 3D and 3E that NP1 could inhibit HER2 dimerization in a dose-dependent manner. A time-course study showed that NP1 (50 μM) could not only inhibit HER2 dimerization but also promote the conversion of HER2 from the dimeric to the monomeric form. The effect of NP1 on the MAPK pathway was also investigated by Western blot. A significant decrease in pErk, pMek, and pRaf-1 levels over time was observed when cells were treated with 50 μM NP1; this inhibitory effect was dose-dependent (Figure 3F). For comparison, MCF-7 / C6 cells were incubated with 50 μM of each NP for 36 h, and Herceptin was used as a positive control (Figure 3G).Like Herceptin, NP1 could potently inhibit the phosphorylation of Erk, Mek, and Raf-1. In contrast, the three negative control NPs did not significantly alter the phosphorylation levels of Erk, Mek, and Raf-1. Collectively, these data strongly support that the transformation of NP1 into a nanofibrillar network on the surface of HER2+ tumor cells leads to the inhibition of HER2 dimerization and the conversion of HER2 dimers to monomers, leading to the inhibition of downstream proliferation, survival cell signaling, and cell death.

[0178] In vivo evaluation of fibril transformation. NP1 was found to be nontoxic; blood counts, platelets, total protein, creatinine, and liver function tests obtained from normal Balb / c mice treated with eight consecutive qod doses of NP1 were within normal ranges. For biodistribution studies, NP1 was administered intravenously to mice bearing MCF-7 / C6 tumors; at 10, 24, 48, 72, and 168 hours, major organs were harvested for ex vivo fluorescence imaging studies (Figures 4A-4B). Fluorescence uptake by tumors and normal organs, such as the liver, lungs, and kidneys, was high at 10 hours. The fluorescence signal persisted within the tumor for more than 3 days, with significant residual signal even after 7 days. In contrast, the fluorescence signal in normal organs began to decline after 10 hours and was barely detectable in major organs at 72 hours. At 72 hours, the tumor and overlying skin were excised for fluorescence microscopy. Compared with the very strong fluorescent signal in the tumor, negligible signal was detected in normal skin (Figure 4C). Histological examination of excised normal organs did not reveal any pathology. Similar in vivo biodistribution studies for NP2, NP3, and NP4 were also performed in the same tumor model system. At 72 h, the fluorescent signal in tumors from mice treated with NP1 was found to be 2-3 times higher than that in mice treated with NP2-4 (Figures 4D-4E). The prolonged retention of the fluorescent signal in NP1-treated mice, even after 7 days, can be attributed to the in situ receptor-mediated transformation of NP1 into the NF1 network within the tumor microenvironment. TEM examination of excised tumors 72 h after iv administration showed extensive entrapment of nanofibrils within the extracellular matrix of the tumor compartment. Such nanofibrils were not observed in negative control NP-treated and untreated mice (Figure 4F). In addition, many cells in tumors excised from NP1-treated mice appeared dead with large intercellular spaces. TEM images of other organs (heart, liver, spleen, lungs, kidneys, and brain) excised from the same mice were found to be normal with no signs of nanofibril networks, which was consistent with the results of optical imaging and histopathological examination.

[0179] Antitumor activity of fibril-variable NPs. Treatment efficacy tests of NP1, NP2, NP3, and NP4 were performed in MCF-7 / C6 HER2+ breast cancer-bearing mice (Figure 5A). The tumor volumes of the mice were approximately 50-80 mm. 3 When the NPs reached 100 mg / kg / day, they were injected via the tail vein for 8 consecutive qods (days 1, 3, 5, 7, 9, 11, 13, and 15) and observed for 40 days. As shown in Figure 5B, The tumor volume of NP1-treated mice gradually shrank and was completely eliminated after treatment without any signs of recurrence. In contrast, none of the other three negative control groups (NP2, NP3, and NP4) elicited any significant tumor response. Mice in this treatment study did not exhibit any symptoms of dehydration or significant weight loss throughout the 40-day treatment study (Figure 5C). The survival curve correlated well with the tumor growth results (Figure 5D). Seven of eight mice treated with NP1 survived for 150 days without any signs of tumor recurrence. One of these eight mice died around day 60 for unknown reasons, not due to a detectable tumor. In contrast, all mice in the PBS, NP2, NP3, and NP4-treated groups died within 51, 63, 57, and 60 days, respectively. The results are highly encouraging and clearly demonstrate the clinical potential of receptor-mediated tunable supramolecular nanotherapeutics (e.g., NP1) for solid tumors in general, and for HER2+ tumors more specifically.

[0180] To better understand the in vivo antitumor mechanism of NP1, mice were sacrificed, and residual tumors were harvested for biochemical and morphological evaluation after three consecutive qod injections of NP1 (Figure 5E). Frozen sections were obtained for fluorescence microscopy and hematoxylin-eosin (H&E) staining (Figure 5F). The extent of cell killing was found to correlate well with the degree of fluorescence intensity; necrosis was detected in tumor areas with strong fluorescence intensity. To understand how the nanofibril network kills HER2+ tumor cells, high-magnification TEM was performed on tumors from NP1-treated mice. TEM images of necrotic or necroptotic cells in Figure 5G revealed disrupted plasma membranes and the presence of abundant fibril nanostructures inside the damaged cells. Some nanofibril inclusions were seen adjacent to the outer nuclear membrane of the nucleus. No significant cell killing was detected in tumors from mice treated with PBS, NP2, NP3, or NP4. Tissue sections stained for the Ki-67 marker are a good tool for assessing the antiproliferative effects of NP1 in vivo. After three treatments with NP1, the expression level of Ki-67 in tumor tissues was significantly reduced compared with tumors from mice treated with the negative control NP (Figure 5H).

[0181] We previously demonstrated that NP1 can inhibit HER2 dimerization in cell culture and the phosphorylation of Erk, Mek, and Raf-1 in HER2+ cell lines. Here, similar Western blot studies were performed on tumors excised from mice that received three consecutive qod treatments of NP1. As shown in Figure 5I, total HER2 levels remained unchanged, whereas the phosphorylation of Erk, Mek, and Raf-1 was found to be significantly reduced compared with other negative control groups. Taken together, the data clearly demonstrated that the receptor-mediated tunable supramolecular nanotherapeutic NP1 is highly effective in suppressing downstream proliferative and survival cell signaling at the tumor tissue level. To better explore the generality of NP1 as an effective therapeutic agent for HER2+ tumors, two other human HER2+ breast cancer xenograft models (SKBR-3 and BT474) were selected for our study. As shown in Figures 5J-5K, the tumor volumes of mice treated with NP1 were very favorable, with complete elimination of SKBR-3 tumors and near-complete elimination of BT474 tumors by day 40. In contrast, tumor volumes in the PBS control group were 1200-1500 mm at day 40. 3 It proliferated to.

[0182] One of the known side effects of Herceptin is cardiac toxicity. It cannot be given to patients with cardiotoxic drugs such as doxorubicin. To date, no cardiovascular effects have been observed in our xenograft studies using NP1. No uptake of NP1 in the myocardium was detected. This is not surprising, as coronary vessels are expected to be intact and 20 nm of NP1 cannot reach the myocardium. The fact that NP1 was highly effective against three different HER2+ tumors warrants further preclinical and clinical development of NP1 against HER2+ breast, ovarian, gastric, and bladder cancers. There is good clinical evidence that some originally HER2-negative breast cancers can induce HER2 expression after prolonged fractionated ionizing radiation (FIR). This further expands the patient population that can benefit from this novel receptor-mediated tunable nanotherapy (RMTN).

[0183] Eight consecutive qod doses of NP1 alone as monotherapy were relatively small (≤100 mm 3 ) demonstrated efficacy in curing the majority of mice bearing HER2+ breast cancer xenografts. Example 2: Multiple nanocarriers containing two distinct conjugates

[0184] Immune checkpoint blockade (ICB) therapy has revolutionized clinical oncology. One of the major factors behind ICB resistance is the lack of Teff cell homing to tumor sites. This example describes 28 nm nontoxic peptide micellar nanoparticles, LXY30, an α3β1 integrin-targeting ligand. Upon interaction with α3β1 integrin, which is overexpressed in many epithelial cancers, these nanoparticles will undergo in situ transformation into a nanofibrillar structural network within the tumor microenvironment (TME). The nanofibrillar network will then direct cytotoxic CD8 T cells to the tumor site. + Not only did they promote T cell homing and macrophage reeducation at tumor sites, but they also enabled the sustained release of a TLR7 / 8 immune agonist (resiquimod) via esterases in the TME, leading to clearance of syngeneic 4T1 breast cancer and Lewis lung cancer models in mice when given with an anti-PD-1 antibody. These structurally modified supramolecular peptides represent an innovative class of receptor-mediated targeted immunotherapies for cancer by promoting T cell tumor homing and reprogramming in the TME.

[0185] This example describes a ligand-receptor-mediated, peptide-based, nontoxic, biligand fibril-tunable nanoplatform that can confer a systemic anti-immune response against cancer. This nanoplatform, initially in nanoparticle form, is self-assembled from two highly efficient tunable peptide monomers, TPM1 and TPM2. TPM1, LXY30-KLVFFK(Pa), consists of three distinct functional domains: (1) a high-affinity and high-specificity LXY30 cyclic peptide (cdG-Phe(3,5-diF)-G-Hyp-NcR) ligand that targets the α3β1 integrin heterodimeric transmembrane receptor expressed by many solid tumors; (2) a KLVFF β-sheet-forming peptide domain derived from the β-amyloid (Aβ) peptide; and (3) a fluorescent pheophorbide-α(Pa) moiety that serves as a hydrophobic core, triggering the formation of micellar nanoparticles. TPM2, proLLP2A-KLVFFK(R848), also contains three distinct functional domains: (1) proLLP2A, the "parent ligand" version of LLP2A, which is a high-affinity and high-specificity peptidomimetic ligand for the activating α4β1 integrin on lymphocytes; (2) the same KLVFF β-sheet-forming peptide domain; and (3) R848 (resiquimod), a hydrophobic Toll-like receptor (TLR) 7 / 8 agonist, linked to the TPM2 backbone via an ester bond. In proLLP2A, the carboxyl group of LLP2A is esterified with 3-methoxy-1-propanol to prevent it from interacting with normal lymphocytes and mesenchymal stem cells during circulation. In the TME, which has abundant esterases, proLLP2A transforms into LLP2A, promoting immune cell homing to tumor sites. Similarly, esterase-responsive release of R848 may occur in the TME to activate antigen-presenting cells (APCs), promote immune cells to generate antitumor response factors, and reverse the macrophage phenotype from M2 to M1.

[0186] Under aqueous conditions and in the blood circulation, TPM1 and TPM2 would self-assemble into a spherical, tunable nanoparticle (T-NP) at a 1:1 ratio, in which the KLVFFK(Pa) and KLVFFK(R848) domains constituted the hydrophobic core, and the LXY30 and pro-LLP2A ligand peptides constituted the hydrophilic corona. Upon interaction with the α3β1 integrin receptor protein displayed on tumor cell membranes, T-NPs would undergo in situ transformation into nanofibrillar (T-NF) structural networks on the surface of tumor cells and within the TME, where tumor-associated exosomes are abundant, thereby maintaining the prolonged residence of the nanofibrillar network at tumor sites (for at least 7 days). In this case, the more hydrophilic pro-LLP2A peptide ligand would be displayed on the outer surface of the fibrils, while the hydrophobic Pa and R848 would be sequestered in the fibrillar core. Due to the high esterase activity in the TME and on tumor cells, pro-LLP2A would be rapidly transformed into LLP2A (a T cell ligand) for activated α4β1 integrin. LLP2A displayed on fibrils would be expressed by Teff cells (e.g., CD8) in the TME and adjacent to tumor cells. + It would facilitate the homing and retention of activated immune cells, such as T cells. It would also enhance the interaction of the T cell receptor (TCR) of Teff with the major histocompatibility complex (MHC) of tumor cells. The addition of anti-PD-1 ICB therapy would activate cytotoxic T cells and T eff This will further enhance the antitumor immune response by halting the dysfunction and exhaustion of R848. In addition, the sustained release of R848 from the nanofibrillar network as a result of high esterase activity at the tumor site will halt the immunosuppressive TME. These structural variant-based supramolecular peptides represent an innovative class of receptor-mediated targeted immunotherapies for cancer by enhancing T cell homing to tumors and by improving the TME from an immunosuppressive state to a persistently immunoactive state (Figure 12).

[0187] Self-assembly and fibril transformation of nanoplatforms. Two variable peptide monomers (TPM1: LXY30-KLVFFK(Pa); TPM2: proLLP2A-KLVFFK(R848)) were synthesized and characterized (Figures 13A and 20). As the water content in the mixed solvent (water and DMSO) of TPM1 and TPM2 (1:1 ratio) increased, there was a gradual decrease in the fluorescence peak at 675 nm due to the ACQ characteristic of the Pa dye (Figure 13B), reflecting the gradual formation of variable NPs (termed T-NPs) via self-assembly. Simultaneously, there was a slight decrease in the absorption peaks at both 405 and 680 nm. The nanoparticles were analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). TPM1 and TPM2, alone, self-assembled into spherical nanoparticles (NPs) of 18 and 55 nm, respectively. TPM1 and NP TPM2 T-NPs assembled from a 1:1 mixture of TPM1 and TPM2 resulted in spherical structures of approximately 28 nm, which were then used to form NPs. TPM1 and NP TPM2 (Figure 21A). The critical aggregation concentration (CAC) of T-NP was determined to be 8 μM (Figure 21B). It was also demonstrated that T-NP can maintain good serum and proteolytic stability for 7 days at 37°C (Figure 21C).

[0188] To verify the receptor-mediated fibril transformation process of T-NPs in vitro, soluble α3β1 integrin protein (a receptor for LXY30) was added to the T-NP solution. After 24 h of incubation at room temperature, a fibril network (T-NF, approximately 8 nm in diameter) with a broad size distribution was clearly detected (Figures 13C and 13F). Even after 24 h, no transformation was observed in T-NP preparations without the addition of α3β1 integrin protein (Figure 21D). The CAC of T-NF was determined to be 5 μM, which was lower than that of T-NPs (8 μM), indicating that T-NF has a higher tendency to form nanostructures than T-NPs (Figure 21E). Fluorescence of Pa was also used to observe the fibril transformation process of T-NPs (Figure 13D). The addition of α3β1 integrin protein to the T-NP solution resulted in a gradual decrease in the fluorescence intensity of Pa and a significant shift in the fluorescence peak from 680 nm to 725 nm toward the red region within the first 2 h, consistent with a change in the aggregation structure of Pa from a spherical structure to a fibrillar shape during that period. The responsiveness of pro-LLP2A and LLP2A displayed on the surface of T-NPs to soluble α4β1 integrin protein in the presence or absence of esterase was investigated (Figures 13E–13F). Soluble α4β1 integrin protein alone was unable to alter the spherical structure of T-NPs displaying pro-LLP2A, even after 24 h of incubation. In contrast, the sequential addition of esterase followed by soluble α4β1 integrin protein was able to induce the conversion of spherical T-NPs into a fibrillar network after 24 h of incubation. This result confirmed that the esterase can transform the parent ligand proLLP2A into the ligand LLP2A, which in turn can induce the receptor-mediated transformation of T-NF into T-NF.Circular dichroism (CD) spectroscopy analysis of the transformation process of T-NPs showed a gradual progression of a negative signal at 216 nm and a positive signal at 195 nm upon incubation with α3β1 integrin protein or the esterase / α4β1 integrin protein combination, indicative of β-sheet formation (Figure 2G), consistent with the TEM results shown in Figures 13C and 13E. The in vitro release behavior of R848 from T-NPs was investigated at pH 6.5 with the addition of esterase to simulate TME conditions. As shown in Figure 13H, approximately 45% of R848 was released in the first 24 h, after which the release rate gradually slowed, with a cumulative release of approximately 86% observed by 168 h, indicating that prolonged and sustained R848 release may occur in the TME. To demonstrate the unique tunable properties of T-NPs, related control nontunable nanoparticles (UT-NPs) were formed by assembly of two TPMs without the β-sheet-forming KLVFF peptide sequence at a 1:1 ratio (TPM3:LXY30-KAAGGK(Pa) and TPM4:proLLP2A-KAAGGK(R848)). As expected, the α3β1 integrin protein was unable to transform UT-NPs into fibrillar structures even after 24 h, indicating that the β-sheet peptide is required for T-NP transformation into T-NF (Figures 20 and 21).

[0189] In vitro evaluation of nanoparticle fibril transformation and T effector cell homing to tumor sites. To further characterize the interaction between tunable nanoparticles and α3β1 integrin receptors on the surface of live cells, α3β1 integrin-expressing 4T1 mouse breast cancer cells were selected. Flow cytometry analysis confirmed that LXY30 (a high-affinity α3β1 integrin ligand) bound to 4T1 tumor cells (Figure 23). It was also found that T-NPs were slightly cytotoxic to 4T1 cells, with 85% cell viability at 50 μM (Figure 24). The distribution of NPs was investigated by tracking the red fluorescent signal emitted by Pa using confocal laser scanning microscopy (CLSM). After 6 hours of incubation of 4T1 cells with T-NPs, a strong red fluorescent signal was observed at and near the cell surface, but not inside the cells (Figure 15A). In contrast, the fluorescent signal of Pa in the UT-NP-treated group was found to be mainly concentrated in the cytoplasm of the cells. To test the retention and stability of the nanofibrillar network formed on the surface of tumor cells, unbound NPs were washed off after 6 h of incubation, fresh NP-free medium was added, and the cells were incubated for another 18 h. T-NP-treated cells still retained a strong red fluorescent signal on the cell surface even at 24 h (Figure 15B). In sharp contrast, only a very weak fluorescent signal was observed in cells treated with UT-NPs after 24 h. This is likely due to enzymatic degradation of the UT-NPs already internalized after 18 h of incubation, despite the absence of any new endocytic uptake during that period. TEM images confirmed the presence of nanofibrillar networks (T-NPs) on the surface and between 4T1 cells after 24 h of incubation with T-NPs, but the absence of such nanofibrillar structures on cells treated with UT-NPs (Figure 15C). The fibrillar structures far from the cell surface were likely caused by secreted tumor exosomes displaying the α3β1 integrin protein.We investigated the effect of esterase on the interaction between T-NPs and T cell surface α4β1 integrin after conversion of the parent ligand pro-LLP2A to LLP2A, which is presented on the surface of T-NPs. Live GFP-transfected Jurkat T lymphocytic leukemia cells, which have high expression levels of constitutively activated α4β1 integrin protein, were used to mimic T cells. As shown in Figure 15D, after 6 hours of incubation of Jurkat cells with T-NPs (pretreated with esterase), a highly abundant red fluorescent layer was observed around the Jurkat cells, indicating successful conversion of the parent ligand to an LLP2A ligand. Scanning electron microscopy (SEM) confirmed the presence of a fibrillar network on the surface of T-NP-treated 4T1 cells and Jurkat cells pretreated with esterase (Figure 15E).

[0190] To simulate the initial fibril transformation process of T-NPs on the 4T1 cell surface and subsequent T cell homing, 4T1 cells were first incubated with T-NPs for 6 h. Unbound T-NPs were then washed away, followed by the addition of fresh medium containing esterase but no T-NPs. After 1 h of incubation, Jurkat cells were added and incubated with the 4T1 cells for 2 or 4 h. Unbound Jurkat cells were then gently removed before CLSM imaging (Figure 15F). As expected, after 2 h of incubation, a fibril structure layer with red fluorescence was detected around the 4T1 cell surface, and Jurkat cells (GFP+) were found to interact with the red fluorescent fibril network and to be in close proximity to the 4T1 breast tumor cells. When the incubation time was extended to 4 h, more Jurkat cells were found to swarm around the 4T1 tumor cells, which was consistent with our concept that the fibril network would facilitate the homing of immune cells, such as T cells, to the tumor site. SEM imaging provided conclusive evidence that the nanofibril structure plays an important role in direct physical contact between 4T1 and Jurkat cells through the nanofibril network (Figure 15G).

[0191] Conversion of TAMs from an immunosuppressive M2-polarized phenotype to an anti-tumorigenic M1-polarized phenotype is one of the major immunotherapy strategies for restoring the immunosuppressive tumor microenvironment. Macrophage polarization demonstrates distinctive morphology, e.g., elongated processes of M2-like cells, in contrast to the rounded or plate-like morphology of their M1-like counterparts. IL-4 was used to induce bone marrow-derived macrophages (BMDMs) into M2-polarized macrophages, as reflected by increased expression levels of the metabolic checkpoint enzymes arginase-1 (Arg1) and mannose receptor 1 (Mrc1). R848 was reported to be a potent driver of the M1 phenotype in vitro, resulting in high levels of interleukin-12 (IL-12) and nitric oxide synthase (Nos2) produced by these cells. The potential use of T-NF for reeducating macrophages from the M2 phenotype to the M1 phenotype was investigated. In the nanoplatform, R848 was covalently linked to TMP2 via an ester bond. Therefore, not unexpectedly, incubation of 4T1 cells with T-NF preformed from T-NPs using soluble α3β1 integrin protein had no significant effect on IL-4-induced M2-polarized macrophages (Figure 15H). No significant changes in macrophage morphology or expression levels of Arg1 and Mrc1 were observed even after 12 h, which could be explained by the lack of R848 released from T-NF. In contrast, addition of esterase to the medium followed by 12 h of incubation resulted in morphological changes of M2-state macrophages toward the M1 state, a decrease in Arg1 and Mrc1, and increased expression of IL-12 and Nos2 as measured by qPCR. These changes were even more pronounced after 24 h, when macrophages completely transformed into round and plate-like morphologies (M1-like), further decrease in Arg1 and Mrc1, and increased expression of IL-12 and Nos2. The ability of T-NF to sequester the TME, providing sustained release of R848 from the fibril network, creates a durable anti-cancer immunoactive TME.

[0192] In vivo evaluation of nanoparticle fibril transformation and tumor homing of T effector cells. T-NP was found to be nontoxic: blood counts, platelets, creatinine, and liver function tests from normal Balb / c mice treated with eight consecutive qd intravenous (iv) doses of T-NP were within normal ranges (Figures 25-26). In vivo blood pharmacokinetic (PK) studies showed that T-NP had a long circulation time (T-half-life (α): 2.866 h and T-half-life (β): 23.186 h), indicating its stability in the circulation (Figure 27). For biodistribution studies, T-NP was injected via the tail vein into Balb / c mice bearing syngeneic orthotopic 4T1 breast cancer; at 10, 24, 48, 72, 120, and 168 h, tumors and major organs were removed for ex vivo fluorescence imaging (Figures 16A-16B). Significant fluorescent signals of Pa were found to persist in tumor tissue for over 168 hours, whereas those in normal organs began to decline after 10 hours and were barely detectable in major organs by 72 hours. In stark contrast, the fluorescent signals of Pa in tumor tissue treated with UT-NPs were found to gradually decline over time after reaching a peak at 24 hours (Figures 16C-16D). By 168 hours, less than 2.88% of the peak fluorescent signal of UT-NPs was maintained in the tumor, whereas for T-NPs, more than 59.89% of the signal was maintained in the tumor (Figure 16D). The prolonged retention of fluorescent signals in T-NP-treated mice may be a result of in situ receptor-mediated transformation of T-NPs into the T-NF network in the TME. TEM examination of excised tumor sections 72 hours after iv administration showed abundant nanofibrils embedded within the extracellular matrix, whereas no such nanofibrils were observed in negative control UT-NP-treated and saline-treated mice (Figure 16E). Fluorescence micrographs of the tumor and overlying skin revealed a strong fluorescent signal in the tumor area but negligible signal in normal skin.This is consistent with our concept that (1) T-NPs leak into the TME through leaky tumor vasculature (EPR effect) and subsequently produce T-NF by interacting with α3β1 integrin on tumor cells and tumor-associated exosomes, and (2) blood vessels in normal skin are not leaky (Figure 16F). The tissue distribution of R848 over time was also measured by high-pressure liquid chromatography-mass spectrometry (HPLC-MS). Using T-NPs, we found that tumor uptake of R848 was significantly higher than that of other normal organs at 24 hours, and retention of R848 at the tumor site was very high at 1.18 μg per gram of tissue even 7 days after injection (Figure 16G). Although UT-NPs could also deliver significant amounts of R848 to the tumor site (80% of that delivered by T-NPs), retention of R848 at the tumor site was much lower than that of T-NPs. The prolonged residence of R848 at the tumor site indicates that a sustained immune-type active TME can be achieved using T-NPs.

[0193] To assess whether nanofibrillar networks displaying LLP2A and R848 in the TME could promote in vivo T cell homing to tumor sites, tumors from T-NP-treated mice were excised 15 days after a single intravenous injection of T-NP, and immune cell populations within the tumor were analyzed by flow cytometry, immunohistochemistry (IHC), and qPCR. Experiments using UT-NP as a non-transferable / endocytosis-negative control were also performed simultaneously. It was found that tail vein injection of T-NP resulted in a sustained immune-active TME. First, it was found that T-NP significantly stimulated the production of the chemokine CXCL10 at the tumor site (Figure 16H), which is known to facilitate T effector cell recruitment. CD45 expression in T-NP-treated tumor tissue was significantly enhanced. + CD3 + and CD45 + CD3 + CD8 + We observed that the percentage of T cells was substantially higher than that from mice treated with endocytosed UT-NP or saline alone (Figures 16I-16J). More specifically, intratumoral CD3+ CD8 + The percentage of T effector cells was found to be increased 18-fold and 4-fold compared to that of mice treated with saline and UT-NP, respectively (Figure 16J). Second, CD4 + Foxp3 + The relative amount of Tregs was found to be substantially lower in mice receiving T-NP treatment than in mice treated with UT-NP (4.97% vs. 13.0%) or saline (4.97% vs. 14.6%) (Fig. 16K). Tumor-penetrating CD8, an indicator of antitumor immune balance, + Killer T cells vs. immunosuppressive Treg (CD3 + CD4 + Foxp3 + The ratio of CD8 / CD4 to TAMs was found to be highest in the T-NP-treated group (Figures 16J-16K). IHC staining of tumor tissue sections also confirmed an increase in CD8 / CD4 and a decrease in Foxp3 (Figure 16L). Third, IHC staining of tumor sections demonstrated an increase in the M1-polarized macrophage marker CD68 and a decrease in the M2-polarized macrophage marker CD163 in the T-NP-treated group compared with tumor tissue treated with UT-NP. This could be explained by the sustained release of R848 at the tumor site, which led to phenotypic re-education of TAMs. Fourth, the gene expression levels of cellular immune-related markers (IFN-γ, TGF-β) and macrophage markers (IL-12, IL-10, Nos2, and Arg-1) were also assessed by qPCR. As shown in Figure 16M, the high expression level of IFN-γ and the low expression level of TGF-β in tumor tissue confirmed the induction of a strong tumor-specific immune response. Furthermore, secretion of IL-12 and Nos2 was found to be significantly upregulated, whereas secretion of IL-10 and Arg-1 was significantly downregulated, indicating a significant phenotypic conversion of TAMs from an M2 state to an M1 state with T-NP treatment, but not with UT-NP treatment or saline control.

[0194] Treatment efficacy studies were conducted in mice bearing syngeneic orthotopic 4T1 breast cancer. Mice were randomly assigned to six groups and received different treatment regimens: (1) saline; (2) (EK)3-KLVFFK(Pa) / (EK)3-KLVFFK(R848); (3) proLLP2A-KLVFFK(R848) (single monomer); (4) LXY30-KAAGGK(Pa) / proLLP2A-KAAGGK(R848) (non-mutable UT-NP); (5) LXY30-KLVFFK(Pa) / proLLP2A-KLVFFK(Pa) (fibril transformation, but absence of R848); and (6) LXY30-KLVFFK(Pa) / proLLP2A-KLVFFK(R848). Regimen 6 is a complete T-NP containing all four key components: LXY30, proLLP2A, R848, and KLVFF, whereas regimens 2, 3, 4, or 5 all lack some component of T-NP. 3 When the tumor size reached 100 μg / mL, all treatment regimens were injected via the tail vein for eight consecutive qods, and the mice were continuously observed for 21 days (Figure 17A). As shown in Figure 17B, regimens 2, 3, and 4 were inactive. Regimen 5 (fibril-transformed but without R848) demonstrated significant tumor suppression compared with groups 2, 3, and 4. Regimen 6 (T-NPs, both fibril-transformed and R848) was found to be the most effective, with significant tumor growth suppression (Figure 17B) and long-term survival (Figure 17D), demonstrating the importance of combining a T cell homing strategy with sustained release of a TLR7 / 8 agonist. None of the mice in this treatment study showed symptoms of dehydration or significant weight loss throughout the entire treatment period (Figure 17C). The survival curve correlated well with tumor growth results. Mice treated with regimen 6 (or T-NP) achieved a longer median survival time (62 days) compared to the other treatment groups (29, 32.5, 33.5, 33.5, and 39 days for regimens 1, 2, 3, 4, and 5, respectively).

[0195] To elucidate the mechanism of the immune treatment effect induced by tunable nanoparticles, tumor tissues were harvested and tumor-penetrating CD3 + (CD45+ CD3 + ) and CD8 + (CD45 + CD3 + CD8 + Flow cytometry was used to quantify T cells (Figure 17E). Only treatment regimens that allowed in situ fibril transformation and pro-LLP2A presentation (regimen 5 and 6) significantly increased intratumoral CD3 T cells, especially in combination with the T-NP immunoadjuvant R848 (regimen 6). + and CD8 + The frequency of T cells was significantly enhanced, which was consistent with the strongest antitumor effect observed with T-NPs. Tumor sections (H&E) from mice treated with T-NPs showed a significant decrease in Ki-67 expression, CD8 expression, and IL-1 expression compared to other control groups. + T cell expansion and Foxp3(T reg A decrease in CD8 cells was evident (Figure 17F). There was an increase in CD68 and a decrease in CD163, indicating that the macrophage phenotype was restored after eight doses of T-NP. + T cells are known to secrete the cytokines IFN-γ and TNF-α to kill tumor cells. The expression levels of IFN-γ and TNF-α in tumor tissues were further evaluated by qPCR. As shown in Figure 17G, treatment regimen 6 (T-NP) was the most effective in restoring the immune activation state of the tumor microenvironment, with the highest expression levels of IFN-γ and TNF-α. In addition, T-NP also significantly induced the expression of IL-12, IL-6, and Nos2, and suppressed the expression of TGF-β, IL-10, and Arg-1, and significantly increased the expression of T reg This led to the suppression of cell recruitment and reeducation of M2-like macrophages to an M1 phenotype.

[0196] Although promising, T-NP alone has not completely eliminated tumors. This may be caused by insufficient activation and homing of T effector cells in the tumor microenvironment. It is well known that tumor cells hijack the PD-1 receptor on T cells by overexpressing PD-L1, which activates PD-1 and can lead to inhibition of T cell proliferation, activation, cytokine production, altered metabolism, and cytotoxic T lymphocyte killer function, as well as the eventual death of activated T cells. Clinically, antibodies targeting PD-1 or PD-L1 have demonstrated the ability to reactivate "exhausted" T cells within the tumor microenvironment. However, with the exception of melanoma and non-small cell lung cancer, the clinical response rate of ICB anti-PD-1 or anti-PD-L1 therapy is limited, and most patients remain refractory. One important reason is the lack of sufficient Teff cells in the tumor microenvironment. Our receptor-mediated fibril-tunable nanoplatform (which promotes T cell homing and improves the tumor microenvironment) may be able to correct such defects, thereby providing significant synergy to PD-1 and PD-L1 checkpoint blockade immunotherapy. Syngeneic orthotopic 4T1 breast cancer-bearing mice were randomized into four groups for anti-PD-1 antibody (anti-PD-1) therapy with or without the additional nanoplatform: (1) anti-PD-1 alone; (2) regimen 4 (UT-NP) + anti-PD-1; (3) regimen 5 + anti-PD-1; (4) regimen 6 (T-NP) + anti-PD-1. Tumors with tumor volumes of approximately 100 mm 3When tumor growth reached 100%, NP was given intravenously on day 1, and anti-PD-1 was given intravenously on day 2. The same cycle was repeated on days 3, 5, 7, and 9 for a total of five cycles, and mice were continuously observed for 21 days (Figure 18A). Not unexpectedly, anti-PD-1 alone and regimen 4 plus anti-PD-1 treatment were ineffective (Figure 18B). In contrast, regimen 5 plus anti-PD-1 treatment significantly suppressed tumor growth and resulted in a longer median survival (49.5 days vs. 39 days) compared with eight treatments of regimen 5 without anti-PD-1, as shown in Figures 18B and 18D. However, neither of these treatments completely eliminated tumors. Most unexpectedly, mice treated with regimen 6 (T-NP) + anti-PD-1 experienced progressive regression and ultimately complete elimination of tumors within 21 days without any signs of recurrence throughout the 90-day observation period, confirming the validity of the synergistic effect of our tunable nanoimmunoplatform T-NP with checkpoint blockade immunotherapy (Figure 18C).

[0197] Unlike traditional chemotherapy or targeted therapy in clinical oncology, immunotherapy can potentially induce responses tailored to memory capacity. Memory is crucial for achieving durable tumor responses and preventing recurrence, which often leads to mortality. To evaluate whether synergistic treatment with T-NPs (T-NPs and anti-PD-1 Abs) along with immune checkpoint anti-PD-1 therapy can induce memory responses, mice recovered from the previous experiment were rechallenged with 4T1 cells on the contralateral mammary fat pad on day 90 (Figures 18A-18C); age-matched naive mice served as negative controls (Figure 18D). In this experiment, mice were administered anti-PD-1 three times via i.p. on days 91, 93, and 95. The tumor volumes of all naive mice rapidly increased within 30 days, even with anti-PD-1 injection (Figure 18E). However, either no tumor growth or a significant delay in tumor growth was observed in mice successfully pretreated with T-NP + anti-PD-1 (Figure 18F), confirming the presence of an excellent immune memory response exerted by these pretreated mice. The survival curve of this experimental group correlated well with the tumor growth results (Figure 18G). All mice remained alive throughout the 60-day observation period (days 90–150). In addition, serum levels of cytokines such as TNF-α and IFN-γ in this experimental group were significantly higher than those in age-matched naive control mice after rechallenge with 4T1 tumor cells for 6 days (Figures 18H–18I). These results suggest that durable and robust T cell memory responses were generated by the pretreatment regimen 6 (or T-NP) + anti-PD-1.

[0198] In addition to the 4T1 syngeneic orthotopic breast cancer model, we performed a similar treatment study in the Lewis lung syngeneic subcutaneous mouse tumor model with excellent results (Figures 18J-18L). Treatment with T-NP and anti-PD-1 resulted in complete tumor regression and long-term survival. Systemic toxicity and weight loss were not detected.

[0199] Despite the clinical success of checkpoint blockade immunotherapy, only a small proportion of cancer patients benefit from this therapy. Defective Teff cell homing to tumor sites is likely one of the main reasons why many patients remain refractory to such treatments. The development of approaches to immunologically convert "cool" tumors into "hot" tumors is under intensive investigation worldwide. The receptor-mediated tunable nanoparticles (T-NPs) described herein offer a relatively simple solution to this challenge. By incorporating the parent ligands LLP2A and R848 into the nanoparticles, we demonstrated in syngeneic 4T1 breast cancer and Lewis lung cancer models that this non-toxic treatment (1) facilitates T cell homing to tumor sites, (2) promotes T cell retention in proximity to tumor cells, and (3) provides sustained release of R848 in the tumor microenvironment, potentially resulting in the reeducation of TAMs to an M1 phenotype. Because the nanoplatform is modular, options exist for incorporating various ligands, parent ligands, or immunomodulatory drugs into the nanoplatform via conjugation. One unique feature of the immuno-nanoplatform is that the nanofibrillar network formed in the tumor microenvironment is persistent, and it can manifest its remarkable in vivo antitumor immune response and memory effect without any signs of systemic immunotoxicity, even when given in conjunction with an anti-PD-1 antibody. The parent ligand concept of using LLP2A to capture T cells at tumor sites is innovative and could be applied to capture other beneficial immune cells, including natural killer cells. Other potent immunomodulators against other pathways, such as the stimulator of IFN gene pathway (STING), may also be tested. The nanoplatform appears complex because it is highly modular; however, in reality, it is very robust. Each variable peptide monomer is chemically well-defined, and the final immuno-nanoparticles can be assembled by simple mixing in DMSO followed by dilution in water. Scaling up manufacturing for clinical development should not be an issue.

[0200] Statistical analysis. Data are presented as mean ± standard deviation (SD). Comparisons between groups were analyzed using Student's t-test (two-tailed). Significance levels were defined as *p<0.05, **p<0.01, and ***p<0.001. All statistical tests were two-tailed.

[0201] Although the foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, those skilled in the art will recognize that certain changes and modifications may be made within the scope of the appended claims. Additionally, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference were individually incorporated by reference. In the event of a conflict between this application and a reference provided herein, the present application shall control. Sequence Listing: SEQ ID NO: 1: KLVFF Sequence number 2: klvff SEQ ID NO: 3: FFVLK SEQ ID NO: 4: YCDGFYACYMDV

Claims

1. Compounds of formula (I): ABC (I) {During the ceremony, A is a hydrophobic moiety; B is a peptide, wherein the peptide forms a β-sheet; and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting ligand is an LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, an LHRH peptide, a HER2 ligand, an EGFR ligand, or a toll-like receptor agonist CpG oligonucleotide; and wherein the hydrophobic moiety is bis-pyrene and C is an LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, LHRH peptide, EGFR ligand, or toll-like receptor agonist CpG oligonucleotide}.

2. 2. The compound of claim 1, wherein the hydrophobic moiety is a dye or a drug.

3. 3. The compound of claim 1 or 2, wherein the hydrophobic moiety is a chemotherapeutic agent, a fluorescent dye, an immunomodulatory agent, a toll-like receptor agonist, a small molecule agonist of stimulator of interferon genes (STING), a porphyrin, cholesterol, vitamin D, or vitamin E.

4. 4. The compound of any one of claims 1 to 3, wherein the hydrophobic moiety is paclitaxel, bis-pyrene, cyanine dye, resiquimod, gardikimod, amidobenzimidazole, porphyrin, cholesterol, vitamin D, or vitamin E.

5. The compound of any one of claims 1 to 4, wherein the hydrophobic moiety is resiquimod or a porphyrin.

6. The compound according to any one of claims 3 to 5, wherein the porphyrin is pyropheophorbide-a, pheophorbide, chlorin e6, purpurin or purpurinimide.

7. The porphyrin has the following structure: 【Chemistry 1】 The compound according to any one of claims 3 to 6, having the formula:

8. The compound according to any one of claims 3 to 6, wherein the porphyrin is pheophorbide-a.

9. The compound according to any one of claims 1 to 8, wherein the peptide is a peptide sequence of 5 to 20 amino acids in length.

10. The compound according to any one of claims 1 to 9, wherein the peptide is a peptide sequence of 5 to 15 amino acids in length.

11. The compound according to any one of claims 1 to 10, wherein the peptide comprises a peptide sequence from the β-sheet peptide domain of β-amyloid peptide.

12. 12. The compound of claim 11, wherein the β-amyloid peptide is β-amyloid 40.

13. The compound of any one of claims 1 to 12, wherein the peptide comprises at least 50% sequence identity to SEQ ID NO:

1.

14. The compound of any one of claims 1 to 13, wherein the peptide comprises SEQ ID NO:

1.

15. The compound of any one of claims 1 to 12, wherein the peptide comprises at least 50% sequence identity to SEQ ID NO:

2.

16. The compound of any one of claims 1 to 13, wherein the peptide comprises SEQ ID NO:

2.

17. The compound of any one of claims 1 to 12, wherein the peptide comprises at least 50% sequence identity to SEQ ID NO:

3.

18. 18. The compound of claim 17, wherein the peptide comprises at least 80% sequence identity to SEQ ID NO:

3.

19. 19. The compound of claim 17 or 18, wherein the peptide comprises SEQ ID NO:

3.

20. 20. The compound of any one of claims 1 to 19, wherein the hydrophilic targeting ligand is a HER2 ligand, wherein the HER2 ligand is an anti-HER2 antibody peptidomimetic derived from the primary sequence of the CDR-H3 loop of anti-HER2 rhumAb 4D5.

21. 21. The compound of claim 20, wherein the HER2 ligand has at least 50% sequence identity to SEQ ID NO:

4.

22. 22. The compound of claim 20 or 21, wherein the HER2 ligand has at least 80% sequence identity to SEQ ID NO:

4.

23. The compound of any one of claims 20 to 22, wherein the HER2 ligand is SEQ ID NO:

4.

24. 20. The compound of any one of claims 1 to 19, wherein the hydrophilic targeting ligand is an LLP2A prodrug, LLP2A, LXY30, DUPA, folate, an LHRH peptide, or an EGFR ligand.

25. 25. The compound of any one of claims 1 to 19, or 24, wherein the hydrophilic targeting ligand is an LLP2A prodrug, LLP2A, or LXY30.

26. The hydrophilic targeting ligand has the following structure: 【Chemistry 2】 26. The compound of any one of claims 1 to 19, or 24 to 25, which is an LLP2A prodrug having the formula:

27. The hydrophilic targeting ligand has the following structure: 【Transformation 3】 26. The compound of any one of claims 1 to 19, or 24 to 25, which is LLP2A having the formula:

28. The hydrophilic targeting ligand has the following structure: 【Chemistry 4】 26. The compound of any one of claims 1 to 19, or 24 to 25, wherein LXY30 has the formula:

29. The following structure: 【Transformation 5】 30. The compound of any one of claims 1-7, 9-14, 24-25, or 28, having the formula:

30. The following structure: 【Transformation 6】 27. The compound of any one of claims 1-5, 9-14, or 24-26, having the formula:

31. The compound has the following structure: 【Transformation 7】 31. The compound of claim 30, which is converted in situ to

32. The following structure: 【Transformation 8】 30. The compound of any one of claims 1-5, 9-14, 24-25 or 28, having the formula:

33. 33. A nanocarrier having an interior and an exterior, comprising a plurality of compounds according to any one of claims 1 to 32, wherein each compound self-assembles in an aqueous medium to form a nanocarrier such that a hydrophobic pocket is formed on the interior of the nanocarrier and hydrophilic groups self-assemble on the exterior of the nanocarrier.

34. 34. The nanocarrier of claim 33, wherein the nanocarrier further comprises a hydrophobic drug or imaging agent sequestered within the hydrophobic pocket of the nanocarrier.

35. A nanocarrier having an interior and an exterior, comprising a plurality of first conjugates and second conjugates, wherein the first conjugates are represented by Formula (I): ABC (I) and the second conjugate comprises formula (II): A'-B'-C' (II) a nanocarrier comprising {During the ceremony, A and A' are each independently a hydrophobic moiety; B and B' are each independently a peptide, wherein each peptide independently forms a β-sheet; and C and C' are each independently a hydrophilic targeting ligand, wherein each hydrophilic targeting ligand is independently an LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, an LHRH peptide, a HER2 ligand, an EGFR ligand, or a radiometal chelator; and where A and A' are different hydrophobic moieties and / or C and C' are different hydrophilic targeting ligands}.

36. 36. The nanocarrier of claim 35, wherein each hydrophobic moiety is independently a dye, a drug, or a radioactive metal chelator.

37. 37. The nanocarrier of claim 35 or 36, wherein each hydrophobic moiety is independently bis-pyrene, porphyrin, resiquimod, or gardikimod.

38. 38. The nanocarrier of any one of claims 35 to 37, wherein each hydrophobic moiety is independently a porphyrin or resiquimod.

39. The nanocarrier of claim 37 or 38, wherein the porphyrin is pyropheophorbide-a, pheophorbide, chlorin e6, purpurin, or purpurinimide.

40. The nanocarrier of any one of claims 37 to 39, wherein the porphyrin is pheophorbide-a.

41. The porphyrin has the following structure: 【Chemistry 9】 40. The nanocarrier of any one of claims 37 to 39, comprising:

42. The resiquimod has the following structure: 【Chemistry 10】 39. The nanocarrier of claim 37 or 38, comprising:

43. 43. The nanocarrier of any one of claims 35 to 42, wherein each peptide is independently a peptide sequence of 5 to 20 amino acids in length.

44. The nanocarrier of any one of claims 35 to 43, wherein each peptide independently comprises a peptide sequence from a β-sheet peptide domain of β-amyloid peptide.

45. The nanocarrier of claim 44, wherein the β-amyloid peptide is β-amyloid 40.

46. 46. ​​The nanocarrier of any one of claims 35 to 45, wherein each peptide independently comprises at least 50% sequence identity to SEQ ID NO:

1.

47. The nanocarrier of any one of claims 35 to 46, wherein each peptide independently comprises SEQ ID NO:

1.

48. 46. ​​The nanocarrier of any one of claims 35 to 45, wherein each peptide independently comprises at least 50% sequence identity to SEQ ID NO:

2.

49. The nanocarrier of any one of claims 35 to 46, wherein each peptide independently comprises SEQ ID NO:

2.

50. 50. The nanocarrier of any one of claims 35 to 49, wherein each hydrophilic targeting ligand is independently an LLP2A prodrug, LLP2A, LXY30, folate, an LHRH peptide, a HER2 ligand, an EGFR ligand, a Gd(III) chelator, a DOTA chelator, or a NOTA chelator.

51. The nanocarrier of any one of claims 35 to 50, wherein each hydrophilic targeting ligand is independently an LLP2A prodrug, LLP2A, or LXY30.

52. Each hydrophilic targeting ligand independently has the structure: 【Chemistry 11】 The nanocarrier of any one of claims 35 to 51, which is an LLP2A prodrug having the formula:

53. Each hydrophilic targeting ligand independently has the structure: 【Chemistry 12】 The nanocarrier of any one of claims 35 to 51, which is LLP2A having the following structure:

54. Each hydrophilic targeting ligand independently has the structure: 【Chemistry 13】 The nanocarrier of any one of claims 35 to 51, wherein the nanocarrier is LXY30 having the formula: 。

55. The first conjugate has the following structure: 【Chemistry 14】 The nanocarrier of any one of claims 35 to 51, comprising:

56. The second conjugate has the following structure: 【Chemistry 15】 56. The nanocarrier of any one of claims 35 to 55, comprising:

57. The second conjugate has the following structure: 【Chemistry 16】 57. The nanocarrier of claim 56, which is converted in situ into

58. 58. The nanocarrier of any one of claims 35 to 57, wherein the ratio of the first conjugate to the second conjugate is from about 10:1 to about 1:

10.

59. 59. The nanocarrier of any one of claims 35 to 58, wherein the ratio of the first conjugate to the second conjugate is about 1:

1.

60. 60. A method of forming nanofibrils, comprising contacting the nanocarrier of any one of claims 33 to 59 with a cell surface or acellular components in a tumor microenvironment, wherein the nanocarrier undergoes deformation in situ to form a fibril structure, thereby forming nanofibrils.

61. 60. A method of treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of the nanocarrier of any one of claims 33 to 59, wherein the nanocarrier forms nanofibrils in situ after binding to cell surfaces or acellular components in the tumor microenvironment, thereby treating the disease.

62. 62. The method of claim 61, wherein the disease is cancer.

63. 62. The method of claim 61, wherein the disease is selected from the group consisting of bladder cancer, brain cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, intestinal cancer, head and neck cancer, leukemia, liver cancer, lung cancer, melanoma, myeloma, ovarian cancer, pancreatic cancer, and uterine cancer.

64. 60. A method of imaging, comprising administering to a subject to be imaged an effective amount of the nanocarrier of any one of claims 33 to 59.

Images