High-density lipoprotein-like nanoparticles as inducers of ferroptosis in cancer

Abstract

To provide high-density lipoprotein-like nanoparticles as inducers of ferroptosis in cancer.SOLUTION: Disclosed herein are compositions and methods for treating a subject having cancer and other ferroptosis disorders with high-density lipoprotein-like nanoparticles that induce ferroptosis. Accordingly, one aspect of the present disclosure provides a method of treating a subject having cancer by administering to the subject a synthetic nanostructure comprising a nanostructure core and a shell, the shell comprising a lipid surrounding and attached to the nanostructure core, where the shell comprises a phospholipid; where the subject has cancer cells and the synthetic nanostructure is administered in an effective amount to induce ferroptosis in the cancer cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of the filing date of U.S. patent application Ser. No. 62 / 902,342, filed September 18, 2019, the contents of which are hereby incorporated by reference in their entirety. [Background technology]

[0002] Background of the Invention Cancer is the second leading cause of death in the United States and worldwide. Discovering targeted therapeutics with efficacy across multiple malignancies offers tremendous potential value, both in improving patient outcomes and from an economic standpoint. Despite long-term remissions observed in some patients with lymphoma, more than one-third of patients with diffuse large B-cell lymphoma (DLBCL), the most common subtype, will relapse or have disease that is refractory to first-line treatment (1-3). This is particularly true for patients in high-risk groups identified by molecular and clinical prognostic factors (4, 5). Experimental treatments for these patients, including immunotherapy and cell-based therapies, have limited success rates, high costs, and toxicity. Summary of the Invention [Means for solving the problem]

[0003] Summary of the Invention The present disclosure is based, at least in part, on compositions, kits, and methods for treating a subject with cancer by administering high-density lipoprotein nanoparticles (HDL-NPs) that target malignant cells and induce ferroptosis.

[0004] Accordingly, one aspect of the present disclosure provides a method of treating a subject having cancer by administering to the subject a synthetic nanostructure comprising a nanostructure core, a shell surrounding the nanostructure core and comprising a lipid bound thereto, wherein the shell comprises a phospholipid, the subject has cancer cells, and the synthetic nanostructure is administered in an amount effective to induce ferroptosis in the cancer cells.

[0005] Another aspect of the present disclosure provides a method of reducing the number of cancer cells in a cell population, comprising contacting the cancer cells with a synthetic nanostructure comprising a nanostructure core, a shell surrounding the nanostructure core and comprising a lipid bound thereto, wherein the shell comprises a phospholipid, wherein the synthetic nanostructure is present in an amount effective to induce ferroptosis in the cancer cells.

[0006] In some embodiments of the present disclosure, the nanostructure core is Ag, Au, Pt, Fe, Cr, Co, Ni, Cu, Zn, and other transition metals, semiconductors (e.g., silicon, silicides and alloys, cadmium selenide, cadmium sulfide, indium arsenide, and indium phosphide), or insulators (e.g., ceramics such as silicon oxide).

[0007] In some embodiments, the synthetic nanostructure further comprises an apolipoprotein. In some embodiments, the apolipoprotein is apolipoprotein AI, apolipoprotein A-II, or apolipoprotein E.

[0008] In some embodiments, the synthetic nanostructure further comprises cholesterol.

[0009] In some embodiments, the phospholipid shell comprises a lipid monolayer.

[0010] In some embodiments, the phospholipid shell comprises a lipid bilayer, hi some embodiments, at least a portion of the lipid bilayer is covalently attached to the nanostructure core.

[0011] In some embodiments, the nanostructure core has a maximum cross-sectional dimension less than or equal to about 500 nanometers (nm). In some embodiments, the nanostructure core has a maximum cross-sectional dimension less than or equal to about 250 nanometers (nm). In some embodiments, the nanostructure core has a maximum cross-sectional dimension less than or equal to about 100 nanometers (nm). In some embodiments, the nanostructure core has a maximum cross-sectional dimension less than or equal to about 75 nanometers (nm). In some embodiments, the nanostructure core has a maximum cross-sectional dimension less than or equal to about 50 nanometers (nm). In some embodiments, the nanostructure core has a maximum cross-sectional dimension less than or equal to about 30 nanometers (nm). In some embodiments, the nanostructure core has a maximum cross-sectional dimension less than or equal to about 15 nanometers (nm). In some embodiments, the nanostructure core has a maximum cross-sectional dimension less than or equal to about 10 nanometers (nm). In some embodiments, the nanostructure core has a maximum cross-sectional dimension less than or equal to about 5 nanometers (nm). In some embodiments, the nanostructure core has a maximum cross-sectional dimension less than or equal to about 3 nanometers (nm).

[0012] In some embodiments, the nanostructure core has an aspect ratio greater than about 1:1. In some embodiments, the nanostructure core has an aspect ratio greater than 3:1. In some embodiments, the nanostructure core has an aspect ratio greater than 5:1.

[0013] In some embodiments, the phospholipid comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (16:0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (18:0 PE), sphingomyelin, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), or a combination thereof.

[0014] In some embodiments, the subject has been diagnosed with cancer. In some embodiments, the subject has been diagnosed with a ferroptosis-sensitive malignancy or a cholesterol-auxotrophic malignancy. In some embodiments, the cancer is selected from B-cell lymphoma, renal cell carcinoma, T-cell lymphoma, gastric cancer, ovarian cancer, endometrial adenocarcinoma sarcoma, anaplastic large cell lymphoma, clear cell renal cell carcinoma (ccRCC), platinum-resistant ovarian cancer, and clear cell ovarian cancer.

[0015] In some embodiments, the synthetic nanostructure is administered to a subject or contacted with cells more than once. In some embodiments, the synthetic nanostructure is administered to a subject or contacted with cells at least once per month. In some embodiments, the synthetic nanostructure is administered to a subject or contacted with cells at least once per week. In some embodiments, the synthetic nanostructure is administered to a subject or contacted with cells at least once per day. In some embodiments, the synthetic nanostructure is administered to a subject or contacted with cells twice per day.

[0016] In some embodiments, any of the methods of the present disclosure further comprise administering to the subject a ferroptosis inducer compound.

[0017] In some embodiments, any of the methods of the present disclosure further include determining whether the cancer is susceptible to ferroptosis.

[0018] In some aspects, the present disclosure relates to a method of treating a subject having a ferroptosis-susceptible disorder, the method comprising: identifying a subject having a ferroptosis-susceptible disorder; and administering to the subject a synthetic nanostructure comprising a nanostructure core, a shell surrounding the nanostructure core and comprising a lipid bound thereto, in an amount effective to induce ferroptosis in affected cells of the subject, wherein the shell comprises a phospholipid.

[0019] In some aspects, the present disclosure relates to a composition comprising a synthetic nanostructure comprising a nanostructure core, a shell surrounding the nanostructure core and comprising a lipid bound thereto, wherein the shell comprises a phospholipid and a ferroptosis inducer compound.

[0020] In some aspects, the present disclosure relates to a method for inducing ferroptosis in a cell, the method comprising identifying the cell as a ferroptosis-susceptible cell and contacting the cell with a nanostructure core and a shell comprising a lipid surrounding the nanostructure core and bound to it, wherein the shell comprises a phospholipid in an amount effective to induce ferroptosis in the cell.

[0021] In some embodiments, the subject of any of the disclosed methods is a mammal, hi some embodiments, the subject of any of the disclosed methods is a human.

[0022] The details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will be apparent from the following drawings and detailed description of several embodiments, as well as from the appended claims.

[0023] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present disclosure, which may be more fully understood by reference to one or more of these drawings in conjunction with the detailed description of specific embodiments presented herein. For clarity, not every component may be labeled in every drawing. It should be understood that the data illustrated in the drawings in no way limit the scope of the present disclosure. [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1 contains a bar graph showing that HDL NPs downregulate GPX4 in both Ramos and SUDHL4 cells (*p<0.05 at dosages of 20 nM and 50 nM versus 0 nM). [Figure 2] FIG. 2 contains a bar graph showing that HDL NPs induce ferroptosis in SUDHL4 (diffuse large B-cell lymphoma) cells (*p<0.05 vs. control (PBS)). [Figure 3] FIG. 3 contains a bar graph showing that HDL NPs induce ferroptosis in Ramos (Burkitt's lymphoma) cells (*p<0.05 vs. control (PBS)). [Figure 4] 4A-4B contain plots showing that HDL NPs induce lipid peroxide accumulation in SUDHL4 cells (*p<0.05 vs. 0 hr). [Figure 5A] 5A-5B contain plots showing that HDL NPs induce lipid peroxide accumulation in Ramos cells (*p<0.05 vs. 0 hr). [Figure 5B] 5A-5B contain plots showing that HDL NPs induce lipid peroxide accumulation in Ramos cells (*p<0.05 vs. 0 hr). [Figure 6A]Figures 6A-6B contain plots showing SR-B1 expression and HDL NP efficacy (*p<0.05 for PBS) in cholesterol auxotrophic cell lines. SNU-1: Stomach cancer. SUDHL1:ALK+ anaplastic large T-cell lymphoma. SR:ALK+ anaplastic large T-cell lymphoma. U937: Histiocytic lymphoma. HEC-1B: Endometrial adenocarcinoma. U266B1: Myeloma. Ramos: Burkitt lymphoma (positive control). Jurkat: T-cell lymphoma (negative control). [Figure 6B] Figures 6A-6B contain plots showing SR-B1 expression and HDL NP efficacy (*p<0.05 for PBS) in cholesterol auxotrophic cell lines. SNU-1: Stomach cancer. SUDHL1:ALK+ anaplastic large T-cell lymphoma. SR:ALK+ anaplastic large T-cell lymphoma. U937: Histiocytic lymphoma. HEC-1B: Endometrial adenocarcinoma. U266B1: Myeloma. Ramos: Burkitt lymphoma (positive control). Jurkat: T-cell lymphoma (negative control). [Figure 7A] 7A-7B contain plots showing tumor volume, weight and in vivo GPX4 expression (*p=0.0339 and **p=0.0238) in a SUDHL1 tumor xenograft model. [Figure 7B] 7A-7B contain plots showing tumor volume, weight and in vivo GPX4 expression (*p=0.0339 and **p=0.0238) in a SUDHL1 tumor xenograft model. [Figure 8] FIG. 8 contains plots showing SUDHL1 in vivo ferroptosis assay (n=9 for PBS, 10 for HDL NP, *p=0.0006). [Figure 9] FIG. 9 contains a bar graph showing that HDL NPs induce ferroptosis in 786-O (renal cell carcinoma-clear cell) cells (*p<0.05 vs. HDL NPs+ferrostatin-1 and HDL NPs+DFO). [Figure 10]FIG. 10 contains a bar graph showing that HDL NPs induce ferroptosis in Caki-2 (renal cell carcinoma-papillary) cells (*p<0.05 vs. HDL NPs+ferrostatin-1 and HDL NPs+DFO). [Figure 11] 11A-11B contain plots showing that HDL NPs induce the accumulation of lipid peroxides in 786-O and Caki-2 cells (*p=0.0096 and **p=0.0011). [Figure 12A-B]Figures 12A-12G show results obtained in the 786-O renal cell carcinoma cell line. Figure 12A shows that siRNA knockdown of SR-B1 down-regulates GPX4 expression. Data are shown for 96 and 120 hours (hours). 25 μg of protein, SR-B1 antibody Abcam (ab52629, 1:2000), GPX4 Abcam (ab41787, 1:20,000), beta-actin Cell Signaling (13E5, 1:2,000). Figure 12B shows that down-regulation of SR-B1 by siRNA knockdown induces cell death. Figure 12C shows Western blots of GPX4 and SR-B1 over various time courses at various concentrations of HDL NP. As can be seen from the figure, HDL NPs do not directly regulate SR-B1 receptor expression, but they significantly down-regulate GPX4 expression in a time- and dose-dependent manner (e.g., HDL NP concentration) dependent manner. Figure 12D shows a Western blot demonstrating that HDL NPs significantly down-regulate GPX4 expression in the presence of Sutent. 8 μg of protein, GPX4 Abcam (ab41787, 1:5,000), beta-actin Cell Signaling (13E5, 1:2,000). Figure 12E shows that HDL NPs increase the expression of oxidized lipids. Figure 12F shows an MTS Rescue Assay demonstrating that cell death induced by HDL NPs is rescued by ferrostatin-1 and deferoxamine. Figure 12G shows in vivo data that after five treatments of HDL NPs, HDL NPs reduce 786-O tumor burden (top left panel), HDL NPs increase survival (top right panel), and HDL NPs increase oxidized lipids in the tumor (bottom middle panel). [Figure 12C-D]Figures 12A-12G show results obtained in the 786-O renal cell carcinoma cell line. Figure 12A shows that siRNA knockdown of SR-B1 down-regulates GPX4 expression. Data are shown for 96 and 120 hours (hours). 25 μg of protein, SR-B1 antibody Abcam (ab52629, 1:2000), GPX4 Abcam (ab41787, 1:20,000), beta-actin Cell Signaling (13E5, 1:2,000). Figure 12B shows that down-regulation of SR-B1 by siRNA knockdown induces cell death. Figure 12C shows Western blots of GPX4 and SR-B1 over various time courses at various concentrations of HDL NP. As can be seen from the figure, HDL NPs do not directly regulate SR-B1 receptor expression, but they significantly down-regulate GPX4 expression in a time- and dose-dependent manner (e.g., HDL NP concentration) dependent manner. Figure 12D shows a Western blot demonstrating that HDL NPs significantly down-regulate GPX4 expression in the presence of Sutent. 8 μg of protein, GPX4 Abcam (ab41787, 1:5,000), beta-actin Cell Signaling (13E5, 1:2,000). Figure 12E shows that HDL NPs increase the expression of oxidized lipids. Figure 12F shows an MTS Rescue Assay demonstrating that cell death induced by HDL NPs is rescued by ferrostatin-1 and deferoxamine. Figure 12G shows in vivo data that after five treatments of HDL NPs, HDL NPs reduce 786-O tumor burden (top left panel), HDL NPs increase survival (top right panel), and HDL NPs increase oxidized lipids in the tumor (bottom middle panel). [Figure 12E-F]Figures 12A-12G show results obtained in the 786-O renal cell carcinoma cell line. Figure 12A shows that siRNA knockdown of SR-B1 down-regulates GPX4 expression. Data are shown for 96 and 120 hours (hours). 25 μg of protein, SR-B1 antibody Abcam (ab52629, 1:2000), GPX4 Abcam (ab41787, 1:20,000), beta-actin Cell Signaling (13E5, 1:2,000). Figure 12B shows that down-regulation of SR-B1 by siRNA knockdown induces cell death. Figure 12C shows Western blots of GPX4 and SR-B1 over various time courses at various concentrations of HDL NP. As can be seen from the figure, HDL NPs do not directly regulate SR-B1 receptor expression, but they significantly down-regulate GPX4 expression in a time- and dose-dependent manner (e.g., HDL NP concentration) dependent manner. Figure 12D shows a Western blot demonstrating that HDL NPs significantly down-regulate GPX4 expression in the presence of Sutent. 8 μg of protein, GPX4 Abcam (ab41787, 1:5,000), beta-actin Cell Signaling (13E5, 1:2,000). Figure 12E shows that HDL NPs increase the expression of oxidized lipids. Figure 12F shows an MTS Rescue Assay demonstrating that cell death induced by HDL NPs is rescued by ferrostatin-1 and deferoxamine. Figure 12G shows in vivo data that after five treatments of HDL NPs, HDL NPs reduce 786-O tumor burden (top left panel), HDL NPs increase survival (top right panel), and HDL NPs increase oxidized lipids in the tumor (bottom middle panel). [Figure 12G]Figures 12A-12G show results obtained in the 786-O renal cell carcinoma cell line. Figure 12A shows that siRNA knockdown of SR-B1 down-regulates GPX4 expression. Data are shown for 96 and 120 hours (hours). 25 μg of protein, SR-B1 antibody Abcam (ab52629, 1:2000), GPX4 Abcam (ab41787, 1:20,000), beta-actin Cell Signaling (13E5, 1:2,000). Figure 12B shows that down-regulation of SR-B1 by siRNA knockdown induces cell death. Figure 12C shows Western blots of GPX4 and SR-B1 over various time courses at various concentrations of HDL NP. As can be seen from the figure, HDL NPs do not directly regulate SR-B1 receptor expression, but they significantly down-regulate GPX4 expression in a time- and dose-dependent manner (e.g., HDL NP concentration) dependent manner. Figure 12D shows a Western blot demonstrating that HDL NPs significantly down-regulate GPX4 expression in the presence of Sutent. 8 μg of protein, GPX4 Abcam (ab41787, 1:5,000), beta-actin Cell Signaling (13E5, 1:2,000). Figure 12E shows that HDL NPs increase the expression of oxidized lipids. Figure 12F shows an MTS Rescue Assay demonstrating that cell death induced by HDL NPs is rescued by ferrostatin-1 and deferoxamine. Figure 12G shows in vivo data that after five treatments of HDL NPs, HDL NPs reduce 786-O tumor burden (top left panel), HDL NPs increase survival (top right panel), and HDL NPs increase oxidized lipids in the tumor (bottom middle panel). [Figure 13A] Figures 13A-13B show results obtained with HDL NPs in the renal clear cell carcinoma cell line 769-P. Figure 13A shows Western blots of GPX4 and its downregulation by HDL NPs. Figure 13B shows MTS data showing that cell death induced by HDL NPs is rescued by ferrostatin-1 and deferoxamine. [Figure 13B] Figures 13A-13B show results obtained with HDL NPs in the renal clear cell carcinoma cell line 769-P. Figure 13A shows Western blots of GPX4 and its downregulation by HDL NPs. Figure 13B shows MTS data showing that cell death induced by HDL NPs is rescued by ferrostatin-1 and deferoxamine. [Figure 14A] Figures 14A-14D show results obtained with HDL NPs in the OVCAR5 cell line, a platinum-sensitive ovarian cancer cell line. Figure 14A shows a Western blot of GPX4 demonstrating that HDL NPs downregulate GPX4 expression. Figure 14B shows C11-BODIPY Flow Data demonstrating that HDL NPs increase the expression of oxidized lipids. Figure 14C shows an MTS assay demonstrating that cell death induced by HDL NPs is rescued by ferrostatin-1 and deferoxamine. Figure 14D shows a Western blot of SR-B1 and GPX4, and shows that siRNA knockdown of SR-B1 downregulates GPX4 expression. [Figure 14B-C] Figures 14A-14D show results obtained with HDL NPs in the OVCAR5 cell line, a platinum-sensitive ovarian cancer cell line. Figure 14A shows a Western blot of GPX4 demonstrating that HDL NPs downregulate GPX4 expression. Figure 14B shows C11-BODIPY Flow Data demonstrating that HDL NPs increase the expression of oxidized lipids. Figure 14C shows an MTS assay demonstrating that cell death induced by HDL NPs is rescued by ferrostatin-1 and deferoxamine. Figure 14D shows a Western blot of SR-B1 and GPX4, and shows that siRNA knockdown of SR-B1 downregulates GPX4 expression. [Figure 14D]Figures 14A-14D show results obtained with HDL NPs in the OVCAR5 cell line, a platinum-sensitive ovarian cancer cell line. Figure 14A shows a Western blot of GPX4 demonstrating that HDL NPs downregulate GPX4 expression. Figure 14B shows C11-BODIPY Flow Data demonstrating that HDL NPs increase the expression of oxidized lipids. Figure 14C shows an MTS assay demonstrating that cell death induced by HDL NPs is rescued by ferrostatin-1 and deferoxamine. Figure 14D shows a Western blot of SR-B1 and GPX4, and shows that siRNA knockdown of SR-B1 downregulates GPX4 expression. [Figure 15A] Figures 15A-15C show results obtained with HDL NPs in the platinum-resistant ovarian cancer cell line, OVCAR5 CP Resistant Cell Line. Figure 15A shows a Western blot of GPX4 and shows that HDL NPs downregulate GPX4 expression. Figure 15B shows an MTS assay demonstrating that cell death induced by HDL NPs is rescued by ferrostatin-1 and deferoxamine. Figure 15C shows that HDL NPs increase the expression of oxidized lipids. [Figure 15B-C] Figures 15A-15C show results obtained with HDL NPs in the platinum-resistant ovarian cancer cell line, OVCAR5 CP Resistant Cell Line. Figure 15A shows a Western blot of GPX4 and shows that HDL NPs downregulate GPX4 expression. Figure 15B shows an MTS assay demonstrating that cell death induced by HDL NPs is rescued by ferrostatin-1 and deferoxamine. Figure 15C shows that HDL NPs increase the expression of oxidized lipids. [Figure 16] Figure 16 shows the results obtained from HDL NPs in the ES2 cell line, a clear cell ovarian cancer cell line. Western blots of GPX4 and HDL NPs down-regulate GPX4 expression are shown. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description of the Invention The present invention relates to drugs containing high-density lipoprotein-like nanoparticles (HDL NPs) that are useful for treating subjects with cancer and other disorders by targeting cancerous malignant cells and causing targeted cell death.

[0026] Altered metabolism is a hallmark of cancer, and malignant cells require increased amounts of various nutrients, including cholesterol and cholesteryl esters. While this altered metabolic state promotes cell proliferation, it can also sensitize cells to an iron- and oxygen-dependent form of programmed cell death called ferroptosis. The present disclosure provides compositions and methods using biomimetic high-density lipoprotein-like nanoparticles (HDL NPs; also referred to as synthetic nanostructures or cholesterol-reduced high-density lipoprotein (HDL)-like nanoparticles) that mimic the size, surface composition, and shape of natural HDL.

[0027] The HDL NPs of the present invention bind to the receptor for mature HDL, i.e., scavenger receptor type B1 (SR-B1), also referred to as SCARB1 (these terms are used interchangeably herein) (a high-affinity receptor for cholesterol-rich high-density lipoprotein (HDL)), preventing the internalization of cholesteryl esters from native HDL and effluxing free cholesterol from cells, thereby depriving malignant cells of cholesterol. It has been discovered that HDL NPs can effectively induce ferroptosis in sensitive cells; for example, HDL NP therapy targeting SCARB1 induced lymphoma cell death through a mechanism involving GPX4 and ferroptosis. Initially, data presented in this application revealed that HDL NPs enforce cellular expression of de novo cholesterol biosynthetic genes, accompanied by a decrease in GPX4 expression. We further showed that reduced GPX4 expression led to increased membrane oxidized lipids and cell death in cell lines, in vivo xenograft models, and in primary samples obtained from patients with B-cell lymphoma by a mechanism consistent with ferroptosis.

[0028] Ferroptosis is an oxygen- and iron-dependent form of necroptosis characterized by the accumulation of plasma membrane lipid and cholesterol peroxides resulting from targeted inhibition of the lipid hydroperoxidase glutathione peroxidase 4 (GPX4). Cells become more susceptible to ferroptosis after GPX4 inhibition because this enzyme reduces and detoxifies lipid peroxides (L-OOH) by converting them to the corresponding lipid alcohols (L-OH). Malignant cells under oxidative stress are significantly more susceptible to ferroptosis due to higher levels of reactive oxygen species and their dependence on GPX4 activity to mitigate the accumulation of toxic L-OOH. Small molecule inhibitors of GPX4 have been developed and tested, but they are toxic and lack specificity, limiting their in vivo use and clinical relevance.

[0029] Cholesterol-poor HDL NPs target SCARB1 in lymphoma cells and other susceptible cells that depend on cholesterol uptake. Binding of HDL NPs to SCARB1 results in a switch from a baseline dependency on cholesterol uptake and high expression of GPX4 to one that favors de novo cholesterol biosynthesis, accompanied by a decrease in GPX4 expression. This metabolic switch renders cancer cells particularly vulnerable because GPX4 is absolutely required by cancer cells to reduce the burden of membrane lipid peroxidation. Therefore, increased accumulation of oxidized membrane lipids leads to cell death by the ferroptotic mechanism.

[0030] In some embodiments, the methods disclosed herein are useful for inducing ferroptosis in ferroptosis-susceptible cells. Recent studies have shown that ferroptosis is closely related to the pathophysiological processes of many diseases, such as tumors, nervous system diseases, ischemia-reperfusion injury, kidney injury, and blood diseases. Ferroptosis-susceptible cells are cells that are iron-dependent and may undergo programmed cell death, resulting in the accumulation of lipid peroxides and cell death. In some embodiments, the ferroptosis cells are tumor cells, such as pancreatic cancer, hepatocellular carcinoma (HCC), gastric cancer, colorectal cancer, breast cancer, lung cancer, renal clear cell carcinoma (ccRCC), adrenocortical carcinoma, ovarian cancer, head and neck cancer, and melanoma.

[0031] In addition to cancer, HDL NPs are useful for inhibiting ferroptosis in neurological diseases such as traumatic brain injury, stroke, neurodegenerative disorders such as Huntington's disease, Parkinson's disease, ALS, and Friedreich's ataxia, and acute kidney disease or injury.

[0032] Methods for determining susceptibility to ferroptosis are known. For example, knowledge of the role of NAD(P)H in various pathways can be used to predict susceptibility to ferroptosis. Furthermore, the expression level of FSP1 positively correlates with ferroptosis resistance in cells and can be used to detect susceptibility, particularly in cancer cells. FSP1 expression has been used to predict the efficacy of drugs to induce ferroptosis in cancer and to identify potential ferroptosis inducers.

[0033] We have found that HDL NPs potently induce ferroptosis in a wide range of malignancies, including ferroptosis-sensitive malignancies (B-cell lymphoma, renal cell carcinoma) and cholesterol-auxotrophic malignancies (T-cell lymphoma, gastric cancer, and endometrial adenocarcinoma). DLBCL (diffuse large B-cell lymphoma) is a cancer type particularly susceptible to cell death by ferroptosis. In some embodiments of the present invention, HDL NPs are synthesized by surface functionalization of a gold nanoparticle core (optionally 5 nm) with apolipoprotein A1 (ApoA1), which defines HDL, and a phospholipid bilayer. Once assembled, these nanoparticles mimic the surface composition, size, and shape of mature, cholesteryl ester-rich HDL; however, given the presence of the gold nanoparticle core occupying space (e.g., position and volume in HDL NPs) typically reserved for cholesteryl esters, they are a poor source of cholesterol. By binding to receptors for mature HDL and preventing cholesteryl ester uptake, HDL NPs induce a state of cholesterol depletion, leading to the induction of ferroptosis in B-cell lymphoma (diffuse large B-cell lymphoma, Burkitt's lymphoma), T-cell lymphoma (anaplastic large cell lymphoma), renal cell carcinoma (clear cell and papillary), gastric cancer, and endometrial adenocarcinoma.

[0034] The HDL NPs of the present invention take advantage of the metabolic state of malignant cells. They exhibit preferential targeting and high efficacy against malignant cells compared to normal, healthy cells. Their efficacy against malignant cells is determined by the metabolic profile of the cells rather than their origin. HDL NPs effectively reduce the viability of cancer cells, reduce malignant disease, and activate host immune cells against malignant cells. This allows for targeting of cells of various origins and treatment of a wide range of cancers. Furthermore, the compositions of the present invention exhibit better biodistribution and pharmacokinetics than other ferroptosis inducers (e.g., small molecule inhibitors).

[0035] cancer In some embodiments, the compositions of the present invention can be used to treat or prevent cancer, in some embodiments, the cancer is characterized by cells that express scavenger receptor class B type 1 (SR-B1).

[0036] Non-limiting examples of cancer include bladder cancer, breast cancer, colon and rectal cancer, endometrial cancer, kidney or renal cell carcinoma, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, gastric cancer, wasting disease, and thyroid cancer. Further non-limiting examples of cancer include cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma; lung: bronchogenic lung carcinoma (squamous cell, small undifferentiated cell, large undifferentiated cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, and thyroid cancer. hanlartoma), mesothelioma; gastrointestinal system: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); genitourinary tract: kidney (adenocarcinoma, Wilms' tumor [nephroblastoma]), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenocarcinoma, lipoma); liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, soft tissue Ewing's sarcoma, soft tissue sarcoma, synovial sarcoma, malignant lymphoma (reticulum cell sarcoma) cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, desmoid-type fibromatosis, fibroblastic sarcoma, gastrointestinal stromal tumor, retroperitoneal sarcoma, osteochondroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor;Nervous system: skull (osteoma, hemangioma, granuloma, xanthomas, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor [pinealoma], glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord (neurofibroma, meningioma, glioma, sarcoma); gynecological sarcoma, Kaposi's sarcoma, peripheral nerve sheath tumor; gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, preneoplastic cervical dysplasia), ovary (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumor tumor), Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma [embryonal rhabdomyosarcoma], fallopian tube [carcinoma]); hematological: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, nevi, dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and adrenal gland: neuroblastoma. Thus, the term "cancerous cell," as used herein, includes a cell afflicted with any one of the above-identified conditions.

[0037] As used herein, the terms "disease" and "disorder" refer to any condition that would benefit from treatment with a composition of the invention (e.g., any of the compositions or methods described herein). This includes chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder in question.

[0038] Synthetic Nanostructures In some embodiments of the present disclosure, a subject with cancer is treated by administering a synthetic nanostructure described herein. The synthetic nanostructure comprises a nanostructure core, a shell, and the shell comprises a lipid layer surrounding and attached to the nanostructure core. In some embodiments, the synthetic nanostructure further comprises a protein associated with the shell. Examples of synthetic nanostructures useful for purposes of the present invention are described below.

[0039] Examples of synthetic nanostructures that can be used in the present method are described herein. The structures (e.g., synthetic nanostructures, HDL NPs) have a core and a shell surrounding the core. In embodiments in which the core is a nanostructure, the core includes a surface to which one or more components can be optionally bound. For example, in some cases, the core is a nanostructure surrounded by a shell, which includes an interior and exterior surface. The shell may be at least partially formed from one or more components, such as multiple lipids, which can optionally associate with each other and / or with the surface of the core. For example, the components may be covalently bound to the core, physically adsorbed (physisorbed), chemically adsorbed (chemisorbed), or associated with the core by ionic interactions, hydrophobic and / or hydrophilic interactions, electrostatic interactions, van der Waals interactions, or a combination thereof. In one particular embodiment, the core includes a gold nanostructure, and the shell is attached to the core by a gold-thiol bond.

[0040] Optionally, the components may be cross-linked to one another. Cross-linking of shell components may, for example, allow for controlled transport of species into the shell or between the outer and inner regions of the shell. For example, a relatively high amount of cross-linking may allow certain small molecules to enter or pass through the shell but not large molecules, while relatively little or no cross-linking may allow larger molecules to enter or pass through the shell. Furthermore, the components forming the shell may be in the form of a single layer or multiple layers, which may facilitate or hinder the transport or sequestration of molecules. In an exemplary embodiment, the shell comprises a lipid bilayer configured to sequester cholesterol and / or control cholesterol efflux from cells, as described herein.

[0041] It should be understood that the shell surrounding the core need not completely surround the core, although such an embodiment may be possible. For example, the shell may surround at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the surface area of the core. In some cases, the shell substantially surrounds the core. In other cases, the shell completely surrounds the core. The components of the shell may be uniformly distributed across the surface of the core in some cases, or may be non-uniform in other cases. For example, the shell may include portions (e.g., pores) that are free of any substance in some cases. If desired, the shell may be designed to allow penetration and / or transport of certain molecules and components into or out of the shell, while preventing penetration and / or transport of other molecules and components into or out of the shell. The ability of certain molecules to penetrate and / or be transported into and / or across the shell may depend, for example, on the packing density of the components forming the shell and the chemical and physical properties of the components forming the shell. As described herein, the shell may comprise one layer of material, or in some embodiments, multiple layers of material.

[0042] In certain embodiments, the synthetic nanostructure may further comprise one or more agents, such as a therapeutic or diagnostic agent. The agent may be a diagnostic agent (sometimes known as an imaging agent), a therapeutic agent, or both a diagnostic and a therapeutic agent. In certain embodiments, the diagnostic agent is a tracer lipid. The tracer lipid may comprise a chromophore, a biotin subunit, or both a chromophore and a biotin subunit. The synthetic nanostructure (e.g., HDL NP) may be functionalized with other types of cargo, such as nucleic acids. In certain embodiments, the therapeutic agent may be a nucleic acid, an antiviral agent, an antineurological agent, or an antirheumatologic agent.

[0043] One or more agents may be associated with the core, the shell, or both, e.g., they may be associated with the surface of the core, the interior surface of the shell, the exterior surface of the shell, and / or embedded in the shell. For example, the one or more agents may be associated with the core, the shell, or both by covalent bonding, physisorption, chemisorption, or may be bound by ionic interactions, hydrophobic and / or hydrophilic interactions, electrostatic interactions, van der Waals interactions, or a combination thereof.

[0044] In some cases, the synthetic nanostructure is a synthetic cholesterol-binding nanostructure with a binding constant Kd for cholesterol. In some embodiments, Kd is less than or equal to about 100 μM, less than or equal to about 10 μM, less than or equal to about 1 μM, less than or equal to about 0.1 μM, less than or equal to about 10 nM, less than or equal to about 7 nM, less than or equal to about 5 nM, less than or equal to about 2 nM, less than or equal to about 1 nM, less than or equal to about 0.1 nM, less than or equal to about 10 pM, less than or equal to about 1 pM, less than or equal to about 0.1 pM, less than or equal to about 10 fM, or less than or equal to about 1 fM. Methods for determining the amount of sequestered cholesterol and binding constants are known in the art.

[0045] The core of the nanostructure may have any suitable shape and / or size. For example, the core may be substantially spherical, non-spherical, ellipsoidal, rod-shaped, conical, cubic, disc-shaped, linear, or irregularly shaped. In a preferred embodiment of the present invention, the core is less than or equal to about 5 nm in diameter. The core (e.g., nanostructure core or hollow core) may have a maximum cross-sectional dimension (or sometimes, minimum cross-sectional dimension or diameter) of, for example, less than or equal to about 500 nm, less than or equal to about 250 nm, less than or equal to about 100 nm, less than or equal to about 75 nm, less than or equal to about 50 nm, less than or equal to about 40 nm, less than or equal to about 35 nm, less than or equal to about 30 nm, less than or equal to about 25 nm, less than or equal to about 20 nm, less than or equal to about 15 nm, less than or equal to about 10 nm, less than or equal to about 5 nm, less than or equal to about 4 nm, less than or equal to about 3 nm, less than or equal to about 2 nm, or less than or equal to about 1 nm. In some cases, the core has an aspect ratio of greater than about 1: 1, greater than 3: 1, or greater than 5: 1. As used herein, "aspect ratio" refers to the ratio of length to width, where length and width are measured perpendicular to one another and length refers to the largest dimension measured linearly.

[0046] In embodiments in which the core comprises a nanostructure core, the nanostructure core may be formed from any suitable material. In a preferred embodiment, the core is formed from gold (e.g., gold (Au)). In some embodiments, the core is formed from a synthetic material (e.g., a material that is not naturally occurring or that occurs naturally in the body). In one embodiment, the nanostructure core includes or is formed from an inorganic material. The inorganic material may include, for example, a metal (e.g., Ag, Au, Pt, Fe, Cr, Co, Ni, Cu, Zn, and other transition metals), a semiconductor (e.g., silicon, silicides and alloys, cadmium selenide, cadmium sulfide, indium arsenide, and indium phosphide), or an insulator (ceramics such as silicon oxide). The inorganic material may be present in the core in any suitable amount, for example, at least 1 wt%, 5 wt%, 10 wt%, 25 wt%, 50 wt%, 75 wt%, 90 wt%, or 99 wt%. In one embodiment, the core is formed from 100 wt% inorganic material. The nanostructure core may, in some cases, be in the form of a quantum dot, carbon nanotube, carbon nanowire, or carbon nanorod. In some cases, the nanostructure core includes or is formed from a non-biological material. In some embodiments, the nanostructure may include or be formed from one or more organic materials, such as synthetic and / or natural polymers. Examples of synthetic polymers include non-degradable polymers such as polymethacrylates and degradable polymers such as polybutyric acid, polyglycolic acid, and copolymers thereof. Examples of natural polymers include hyaluronic acid, chitosan, and collagen.

[0047] Furthermore, the shell of the structure may have any suitable thickness. For example, the shell thickness may be at least 10 angstroms, at least 0.1 nm, at least 1 nm, at least 2 nm, at least 5 nm, at least 7 nm, at least 10 nm, at least 15 nm, at least 20 nm, at least 30 nm, at least 50 nm, at least 100 nm, or at least 200 nm (e.g., from the inner surface to the outer surface of the shell). In some cases, the shell thickness is less than 200 nm, less than 100 nm, less than 50 nm, less than 30 nm, less than 20 nm, less than 15 nm, less than 10 nm, less than 7 nm, less than 5 nm, less than 3 nm, less than 2 nm, or less than 1 nm (e.g., from the inner surface to the outer surface of the shell). Such thicknesses may be determined before or after the isolation of molecules, as described herein.

[0048] Those skilled in the art are familiar with techniques for determining the size of structures and particles. Examples of suitable techniques include dynamic light scattering (DLS) (e.g., using a Malvern Zetasizer instrument), transmission electron microscopy, scanning electron microscopy, electroresistance counting, and laser diffraction. Other suitable techniques are known to those skilled in the art. Although many methods are known for determining the size of nanostructures, the sizes (e.g., maximum or minimum cross-sectional dimensions, thickness) described herein refer to those measured by dynamic light scattering.

[0049] The shell of the structures described herein may comprise any suitable material, such as a hydrophobic material, a hydrophilic material, and / or an amphiphilic material. While the shell may comprise one or more inorganic materials, such as those listed above for the nanostructure core, in many embodiments, the shell comprises an organic material, such as a lipid or certain polymers. The components of the shell may, in some embodiments, be selected to facilitate sequestration of cholesterol or other molecules. For example, cholesterol (or other sequestered molecules) may be bound to or otherwise associated with the surface of the shell, or the shell may include components that allow cholesterol to be internalized by the structure. Cholesterol (or other sequestered molecules) may be embedded within the shell, within a layer forming the shell, or between two layers forming the shell.

[0050] The components of the shell may be charged or uncharged, for example, to impart a charge to the surface of the structure. In some embodiments, the surface of the shell may have a zeta potential greater than or equal to about -75 mV, greater than or equal to about -60 mV, greater than or equal to about -50 mV, greater than or equal to about -40 mV, greater than or equal to about -30 mV, greater than or equal to about -20 mV, greater than or equal to about -10 mV, greater than or equal to about 0 mV, greater than or equal to about 10 mV, greater than or equal to about 20 mV, greater than or equal to about 30 mV, greater than or equal to about 40 mV, greater than or equal to about 50 mV, greater than or equal to about 60 mV, or greater than or equal to about 75 mV. The surface of the shell may have a zeta potential of less than or equal to about 75 mV, less than or equal to about 60 mV, less than or equal to about 50 mV, less than or equal to about 40 mV, less than or equal to about 30 mV, less than or equal to about 20 mV, less than or equal to about 10 mV, less than or equal to about 0 mV, less than or equal to about -10 mV, less than or equal to about -20 mV, less than or equal to about -30 mV, less than or equal to about -40 mV, less than or equal to about -50 mV, less than or equal to about -60 mV, or less than or equal to about -75 mV. Other ranges are possible. Combinations of the above ranges are also possible (e.g., greater than or equal to about -60 mV and less than or equal to about -20 mV). As described herein, the surface charge of the shell may be adjusted by altering the surface chemistry and constituents of the shell.

[0051] In one embodiment, the structures described herein or portions thereof (such as the shell of the structure) comprise one or more natural or synthetic lipids or lipid analogs (i.e., lipophilic molecules). The one or more lipids and / or lipid analogs can form a monolayer or multilayer (e.g., bilayer) of the structure. In some cases where multilayers are formed, the natural or synthetic lipids or lipid analogs interdigitate (e.g., between different layers). Non-limiting examples of natural or synthetic lipids or lipid analogs include fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, glycolipids (saccharolipids), and polyketides (derived from the condensation of ketoacyl subunits); and sterol lipids and prenol lipids (derived from the condensation of isoprene subunits).

[0052] In a particular embodiment, the structures described herein include one or more phospholipids, such as phosphatidylcholine, phosphatidylglycerol, lecithin, β,γ-dipalmitoyl-α-lecithin, sphingomyelin, phosphatidylserine, phosphatidic acid, N-(2,3-di(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammonium chloride, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylinositol, cephalin, cardiolipin, cerebroside, dicetyl phosphate, dioleoylphosphatidylcholine, dipalmitoyl phosphatidylcholine ...sphingomyelin, sphingomyelin, sphingomyelin, sphingomyelin, sphingomyelin, sphingomyelin, sphingomyelin, sphingomyelin, sphingomyelin, sphingomyelin, sphingomyelin, sphingomyelin, sphingomyelin, sphingomyelin, sphingomyelin, The lipids may include 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, ... For example, the shell may comprise, e.g., depending on the size of the structure, fewer than about 500, fewer than about 400, fewer than about 300, fewer than about 200, or fewer than about 100 natural or synthetic lipids or lipid analogs (e.g., phospholipids).

[0053] Non-phosphorus-containing lipids such as stearylamine, dodecylamine, acetyl palmitate, and fatty acid amides may also be used. In other embodiments, other lipids such as fats, oils, waxes, cholesterol, sterols, fat-soluble vitamins (e.g., vitamins A, D, E, and K), glycerides (e.g., monoglycerides, diglycerides, triglycerides), etc., can be used to form part of the structures described herein.

[0054] Portions of the structures described herein, such as the shell or surface of a nanostructure, may optionally include one or more alkyl groups, e.g., alkane-, alkene-, or alkyne-containing species, which optionally impart hydrophobic properties to the structure. An "alkyl" group refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. The alkyl groups may have a range of carbon numbers from C2 to C40, and in some embodiments, greater than C5, C10, C15, C20, C25, C30, or C35. In some embodiments, a straight-chain or branched-chain alkyl may have 30 or fewer carbon atoms in its backbone, and in some cases, 20 or fewer carbon atoms. In some embodiments, a straight-chain or branched-chain alkyl may have 12 or fewer carbon atoms (e.g., C1-C12 for straight chain, C3-C12 for branched chain), 6 or fewer, or 4 or fewer carbon atoms in its backbone. Likewise, cycloalkyls may have from 3-10 carbon atoms in their ring structure, or 5, 6 or 7 carbons in the ring structure. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, hexyl, cyclohexyl, and the like.

[0055] The alkyl group may include any suitable terminal group, such as a thiol group, an amino group (e.g., unsubstituted or substituted amine), an amide group, an imine group, a carboxyl group, or a sulfate group, which may enable, for example, direct or linker-mediated attachment of a ligand to the nanostructure core. For example, when an inert metal is used to form the nanostructure core, the alkyl species may include a thiol group to form a metal-thiol bond. In some cases, the alkyl species includes at least a second terminal group. For example, these species may be attached to a hydrophilic moiety such as polyethylene glycol. In other embodiments, the second terminal group may be a reactive group capable of covalently bonding to another functional group. In some cases, the second terminal group may participate in a ligand / receptor interaction (e.g., biotin / streptavidin).

[0056] In some embodiments, the shell comprises a polymer. For example, an amphiphilic polymer may be used. The polymer may be, for example, a diblock copolymer, a triblock copolymer, or the like, in which one block is a hydrophobic polymer and another block is a hydrophilic polymer. For example, the polymer may be a copolymer of an alpha-hydroxy acid (e.g., butyric acid) and polyethylene glycol. In some cases, the shell comprises a hydrophobic polymer, such as polymers that may include certain acrylics, amides and imides, carbonates, dienes, esters, ethers, fluorocarbons, olefins, styrenes, vinyl acetals, vinyl and vinylidene chlorides, vinyl esters, vinyl ethers and ketones, and vinylpyridines and vinylpyrrolidones. In other cases, the shell comprises a hydrophilic polymer, such as polymers that include certain acrylics, amines, ethers, styrenes, vinyl acids, and vinyl alcohols. The polymer may be charged or uncharged. As noted herein, the specific components of the shell can be selected to impart specific functionality to the structure.

[0057] When the shell comprises amphiphiles, the materials may be arranged in any suitable manner with respect to the nanostructure core and / or with respect to each other. For example, the amphiphiles may comprise hydrophilic groups extending toward the core and hydrophobic groups extending away from the core, or the amphiphiles may comprise hydrophobic groups extending toward the core and hydrophilic groups extending away from the core. Bilayers of each configuration may be formed.

[0058] Examples of suitable proteins that may associate with the structures described herein are apolipoproteins such as apolipoprotein A (e.g., apo AI, apo A-II, apo A-IV, and apo AV), apolipoprotein B (e.g., apo B48 and apo B100), apolipoprotein C (e.g., apo CI, apo C-II, apo C-III, and apo C-IV), and apolipoproteins D, E, and H. Specifically, apo A1, apo A2, and apo E promote the import of cholesterol and cholesteryl esters into the liver for metabolism and may be useful for inclusion in the structures described herein. Additionally or alternatively, the structures described herein may include one or more peptide analogs of apolipoproteins, such as those described above. The structures may include any suitable number of apolipoproteins or analogs thereof, e.g., at least 1, 2, 3, 4, 5, 6, or 10. In certain embodiments, the structures include 1 to 6 apolipoproteins similar to naturally occurring HDL particles. Of course, other proteins (eg, non-apolipoproteins) may be included in the structures described herein.

[0059] It should be understood that the components described herein, such as lipids, phospholipids, alkyl groups, polymers, proteins, polypeptides, peptides, enzymes, bioactive agents, nucleic acids, and targeting species (if desired), can be associated with the structure in any suitable manner, and with any suitable portion of the structure, e.g., the core, the shell, or both. For example, one or more such components can be associated with the surface of the core, the interior of the core, the interior surface of the shell, the exterior surface of the shell, and / or embedded in the shell. Furthermore, in some embodiments, such components can be used to facilitate the sequestration, exchange, and / or transport of substances (e.g., proteins, peptides, polypeptides, nucleic acids, nutrients) from one or more components of a subject (e.g., cells, tissues, organs, particles, body fluids (e.g., blood), and portions thereof) to the structures described herein and / or from the structures to one or more components of the subject. In some cases, the components have chemical and / or physical properties that allow for favorable interaction (e.g., binding, adsorption, transport) with one or more substances from the subject.

[0060] combination In some embodiments, the HDL NPs disclosed herein are formulated or administered together with a ferroptosis inducer. As used herein, a ferroptosis inducer is a compound that plays a role in initiating, promoting, or supporting the process of ferroptosis. The HDL NPs are administered together with the compound in any manner that allows the compounds to be delivered to a subject together. For example, the HDL NPs and the compound can be co-administered at the same time or at different times. The two compounds can be administered to the same or different sites using the same or different administration routes. In some embodiments, the HDL NPs may be administered before the compound, such as about 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 1, 2, 3, 4, 5, 6, or 7 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or 1 month, 3 months, or 6 months before. In other embodiments, the HDL NPs may be administered after the compound, such as about 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 1, 2, 3, 4, 5, 6, or 7 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or 1 month, 3 months, or 6 months after. The HDL NPs and compounds may be administered multiple times in various administration cycles. Ferroptosis inducer compounds include, for example, compounds that inhibit iron chelation (i.e., iron chelators), compounds that reduce cellular antioxidant capacity and ROS accumulation, mitochondrial VDAC modulators, modulators of sulfur transfer pathways, and compounds related to polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA) or its derivatives, such as phosphatidylethanolamine (PE), containing adrenaline.

[0061] Specific inhibitors of ferroptosis include ferrostatin-1 (Fer-1), liproxystatin-1, vitamin E, and iron chelators. These substances typically inhibit ferroptosis by inhibiting the formation of lipid peroxides. Fer-1 has been shown to inhibit cell death in several in vitro models of diseases such as Huntington's disease (HD), periventricular white matter (PVL), and renal failure. RSL3, DPI7, and DPI10 are ferroptosis inducers that directly inhibit the activity of GPX4 and also directly act on GPX4 to induce ferroptosis.

[0062] Pharmaceutical Composition As described herein, synthetic nanostructures can be used in "pharmaceutical compositions" or "pharmaceutically acceptable" compositions (also referred to as drugs) comprising a therapeutically effective amount of one or more of the structures described herein formulated together with one or more pharmaceutically acceptable carriers, additives, and / or diluents. The pharmaceutical compositions described herein can be useful for treating cancer or other conditions. It should be understood that any suitable structure described herein can be used in such pharmaceutical compositions, including those described in conjunction with the drawings. In some cases, the structures in the pharmaceutical compositions have a nanostructure core comprising inorganic material and a shell substantially surrounding and attached to the nanostructure core.

[0063] Pharmaceutical compositions can be formulated for administration in solid or liquid form, including those adapted for oral administration, e.g., as drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal and sublingually targeted tablets, boluses, powders, granules, pastes for application to the tongue; as sterile solutions or suspensions, or sustained release formulations; as sprays to be applied to the oral cavity; e.g., as creams or foams.

[0064] The phrase "pharmaceutically acceptable" is used herein to refer to structures, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0065] The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting a subject compound from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0066] Wetting agents, emulsifying agents and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0067] Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; oil-soluble antioxidants such as ascorbic acid palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0068] The pharmaceutical compositions described herein include those suitable for oral administration. The formulations may be conveniently presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of compound that produces a therapeutic effect. Generally, this amount ranges from about 1% to about 99% active ingredient, from about 5% to about 70%, or from about 10% to about 30%.

[0069] Compositions of the present invention suitable for oral administration may be present in the form of capsules, sachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powder, granules, each containing a predetermined amount of the structures described herein as the active ingredient, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a troche (using an inert base, e.g., gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, etc. The structures of the present invention may also be administered as a bolus, electuary, or paste.

[0070] In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; humectants, such as glycerol; disintegrating agents, such as PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, PEG-9, PEG-10, PEG-11, PEG-12, PEG-13, PEG-14, PEG-15, PEG-16, PEG-17, PEG-18, PEG-19, PEG-20, PEG-21, PEG-22, PEG-23, PEG-24, PEG-25, PEG-26, PEG-27, PEG-28, PEG-29, PEG-30, PEG-31, PEG-32, PEG-33, PEG-44, PEG-45, PEG-46, PEG-47, PEG-48, PEG-49 ...50, PEG-51, PEG-52, PEG-53, PEG-54, PEG-55, PEG-55, PEG-55, Ingredients that may be used include, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; dissolution retarders, for example, paraffin; absorption accelerators, for example, quaternary ammonium compounds; wetting agents, for example, cetyl alcohol, glycerol monostearate, and nonionic surfactants; absorbents, for example, kaolin and bentonite clay; lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets, and pills, pharmaceutical compositions may also contain buffering agents. Similar types of solid compositions can also be used as fillers in soft and hard-shelled filled gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols.

[0071] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants or dispersants. Molded tablets can be made in a suitable machine (in which a mixture of powdered structures is moistened with an inert liquid diluent).

[0072] Tablets, as well as other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills, and granules, may be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the art of pharmaceutical formulation. They may be formulated to provide delayed or controlled release of the active ingredient therein, for example, using hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres in various proportions to provide the desired release profile. They may also be formulated for rapid release, for example, lyophilized. They may be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other injectable sterile medium before use. These compositions may optionally contain opacifying agents and may be composed to release the active ingredient only, or, if desired, in a delayed manner, only in a certain part of the gastrointestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient may optionally be in microencapsulated form with one or more of the above-mentioned excipients.

[0073] Oral liquid dosage forms of the structures described herein include pharmaceutically acceptable emulsions, microemulsions, solutions, dispersions, suspensions, syrups and elixirs.In addition to the structures of the present invention, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.

[0074] Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0075] In addition to the active compound, suspensions may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof.

[0076] Formulations of the pharmaceutical compositions described herein (e.g., for rectal or vaginal administration) may be present as suppositories, which can be prepared by mixing one or more compounds of the present invention with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, which are solid at room temperature but liquid at body temperature and will therefore melt and release their structure in the body.

[0077] The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[0078] Pastes, creams and gels may contain, in addition to the structures of the present invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0079] Powders and sprays may contain, in addition to the structures described herein, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, for example, butane and propane.

[0080] Suitable examples of aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0081] These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action on the structures of the present invention can be facilitated by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. Furthermore, prolonged absorption of injectable pharmaceutical forms may be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0082] Therapeutically effective dose The phrase "therapeutically effective amount," as used herein, refers to an amount of a substance or composition, including a structure of the present invention, that is effective to produce some desired therapeutic effect in a subject, at a reasonable benefit / risk ratio applicable to any medical treatment. Thus, a therapeutically effective amount can, for example, prevent, minimize, or reverse disease progression associated with a disease or physical condition. Disease progression can be monitored by clinical observation, laboratory investigations, and imaging studies that are apparent to those skilled in the art. A therapeutically effective amount can be an amount effective in a single dose, or an amount effective as part of a multi-dose treatment, e.g., an amount administered in two or more doses, or an amount administered chronically.

[0083] Effective amount can depend on the specific condition to be treated.Of course, effective amount will depend on factors such as the severity of the condition to be treated; the parameters of individual patients, including age, health condition, size and weight; concurrent treatment; treatment frequency; or administration method.These factors are well known to those skilled in the art and can be dealt with within the scope of no more than routine experimentation.In some cases, maximum dose, i.e., the highest safe dose, is used according to sound medical judgment.

[0084] Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0085] A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start the dosage of the structures described herein used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and then gradually increase the dosage until the desired effect is achieved.

[0086] Subject As used herein, "subject" or "patient" refers to any mammal (e.g., a human), for example, a mammal that may be susceptible to a disease or condition, such as a secondary disease or condition disclosed herein. Examples of subjects or patients include humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents such as mice, rats, hamsters, or guinea pigs. Generally, the present invention is intended for use in humans. A subject may be a subject that has been diagnosed with a particular disease or condition or is otherwise known to have a disease or condition. In some embodiments, a subject may be diagnosed with or known to be at risk for developing a disease or condition. In some embodiments, a subject may be diagnosed with or otherwise known to have a disease or condition associated with abnormal lipid levels, as described herein. In certain embodiments, a subject may be selected for treatment based on a known disease or condition in the subject. In some embodiments, a subject may be selected for treatment based on a suspected disease or condition in the subject. In some embodiments, the composition may be administered to prevent the onset of a disease or condition. However, in some embodiments, the existence of an existing disease or condition may be suspected but not yet identified, and the composition of the present invention may be administered to diagnose or prevent further onset of the disease or condition.

[0087] Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present invention to its fullest extent. The following specific embodiments, therefore, are to be construed as merely illustrative, and not limiting in any way to the remainder of the disclosure. All publications cited herein are incorporated by reference for the purposes or subject matter discussed herein.

[0088] method In some embodiments, the subject has cancer. In some embodiments, a composition of the present disclosure (e.g., synthetic nanostructures) can be delivered to (e.g., contacted with) cancer cells in vitro or ex vivo. The subject may have had cancer in the past and is currently in remission. The subject may currently have a diagnosis of active cancer (e.g., not in remission). The subject may have been diagnosed by any means known in the art to qualify for the status of having cancer.

[0089] In some embodiments, a subject is administered, or cells are contacted with, any of the compositions (e.g., synthetic nanostructures) described herein. The compositions disclosed herein may be administered by any route of administration known in the art. For example, in some embodiments, one skilled in the art can administer the composition by conventional routes, such as orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or by implanted reservoir.

[0090] In some embodiments, a subject is administered a composition (e.g., a synthetic nanostructure) or a cell is contacted with a composition at least once. In some embodiments, a subject receives multiple administrations or a cell is contacted multiple times. For example, without limitation, a subject may receive at least two administrations or a cell may be contacted at least two times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more times). In some embodiments, the administrations or contactings are irregularly spaced (e.g., not equal periods of time between administrations or contactings). In some embodiments, the administrations or contactings are equally spaced (e.g., equal periods of time between administrations or contactings). In some embodiments, the subject receives at least one administration per month, or the cells are contacted with at least one administration per month. In some embodiments, the subject receives at least one administration per week, or the cells are contacted with at least one administration per week. In some embodiments, the subject receives at least one administration per day, or the cells are contacted with at least one administration per day. In some embodiments, the subject receives at least two administrations per day, or the cells are contacted with at least two administrations per day. In some embodiments, when there is more than one administration or contacting, the administrations or contactings are by the same route. In some embodiments, when there is more than one administration or contacting, the administrations or contactings are by different routes. [Example]

[0091] The data presented in the following examples demonstrate that GPX4 gene and protein expression is downregulated after HDL NP exposure, likely mediated through SCARB1, a high-affinity receptor for cholesterol-rich high-density lipoproteins. RT-qPCR data support the finding that HDL NP mediates the reduction in GPX4 levels by reducing transcription. We have shown that cholesterol depletion of lymphoma cells increases the activation of SREBP-1a, thereby increasing de novo cholesterol biosynthesis. SREBP-1a has been reported as a negative regulator of GPX4 expression. Western blot data showed a significant reduction in GPX4, suggesting a post-translational mechanism for further reduction of GPX4. Inhibiting de novo cholesterol biosynthesis using statins did not reduce GPX4 expression or induce ferroptosis, suggesting that manipulation of de novo cholesterol biosynthesis cannot reproduce the effects of HDL NP treatment.

[0092] For ALK+ALCL (SR-786, SUDHL1) and U937 cell lines, pathways involving intermediates in the cholesterol biosynthesis pathway are of interest due to their common inability to synthesize cholesterol, resulting from enzymatic blockade induced by hypermethylation or mutation, respectively. The data presented herein show that HDL NP treatment increased the expression of de novo cholesterol synthesis genes and reduced the expression of GPX4. In theory, this could serve to further increase intermediates in the cholesterol biosynthesis pathway. This may function as an antioxidant, but is only effective in preventing ferroptosis in the presence of GPX4.

[0093] The data suggest that investigating HDL NPs in cholesterol-auxotrophic cell lines is warranted, despite the fact that these cells can take up cholesterol via LDL binding to the LDLR. SCARB1 expression was measured in all three auxotrophic cell lines examined, suggesting that both LDLR and SCARB1 play a role in providing cholesterol to cells. Possible explanations for the observed effective reduction of GPX4 and ferroptosis after treatment with HDL NPs include: a) reduced LDLR-mediated cholesterol uptake by HDL NPs; b) dependence on both LDLR and SCARB1 for sufficient cholesterol uptake; or c) different cellular mechanisms for cholesterol uptake by HDL (cell membrane binding) via SCARB1 versus LDL (particle internalization) via LDLR. In contrast to LDL / LDLR, HDL binding to SCARB1 is associated with intracellular signaling pathways, including the pro-survival PI3K / AKT pathway. Recent reports suggest that reduced GPX4 expression correlates with decreased AKT phosphorylation. The association of HDL NPs with SCARB1 not only prevents cholesterol influx but also disrupts membrane-anchored pro-survival signaling pathways that may ultimately affect GPX4 expression. Nevertheless, targeted inhibition of cholesterol uptake by synthetic nanoparticles constructed on an inert core appears to be an important target in certain cholesterol auxotrophic or cholesterol uptake-dependent cancers.

[0094] Interestingly, HDL NPs exhibit potent toxicity to ferroptosis-sensitive cancer cells, but no toxicity has been observed for normal cells in vitro or in vivo. Based on the data presented herein on cancer cells, it is believed that normal cells do not have the same oxidative burden as cancer cells and are able to maintain flexibility with regard to cholesterol metabolism.

[0095] method cell line Ramos (RRID: CVCL_0597), SUDHL4 (CVCL_0539), Raji (CVCL_0511), Daudi (CVCL_0008), SUDHL6 (CVCL_2206), Namalwa (CVCL_0067), Jurkat (CVCL_0367), SUDHL1 (CVCL_0538), SR-786 (CVCL_1711), and U937 (CVCL_0007) human cell lines were obtained from ATCC and used within 3 months of receipt and / or resuscitation. ATCC uses short tandem repeat (STR) profiling to authenticate their cell lines before shipping. For SUDHL4 cells, Charles River Laboratories was contracted to test for mycoplasma contamination before use in animal studies. All cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C in a humidified 5% CO incubator.

[0096] Synthesis of HDL NPs HDL NPs were synthesized and quantified as previously described (36). 5-nm-diameter citrate-stabilized gold nanoparticles (AuNPs) were surface-functionalized with apolipoprotein AI, followed by addition of phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (PDP PE) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). HDL NPs were purified using a KrosFlo TFF (Tangential Flow Filtration) system with a 50 kDa PES module. The concentration of HDL NPs was calculated using UV-Vis spectroscopy and Beer's law.

[0097] To synthesize fluorescently labeled HDL NPs, the intercalating dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) was added to a final concentration of 1 μM during the phospholipid addition step. Purification and quantification of fluorescently labeled HDL NPs were performed as described above.

[0098] HDL NP binding assay to SCARB1 Ramos, SUDHL4, and Jurkat cells were incubated with DiI HDL NPs (10 nM) in standard culture medium for 2 hours at 37°C in the presence or absence of a SCARB1 blocking antibody (Novus Biologicals; 1:100; RRID:AB_1291690) and / or a rabbit IgG isotype control antibody (Novus Biologicals; 1:100). Cells were washed once with 1 mL of ice-cold FACS buffer (PBS, 1% bovine serum albumin, 0.1% sodium azide) and resuspended in 500 μl of ice-cold FACS buffer prior to flow cytometry (BD LSR II Fortessa). Data were analyzed using FCS Express software.

[0099] Western blot analysis Western blots were performed as previously described. Blots were imaged using an Azure 3000 imager. SCARB1 antibody (Abcam, RRID:AB_882458; 1:1,000), GPX4 antibody (Abcam, AB_941790; 1:5,000), β-actin antibody (Cell Signaling Technologies, AB_2223172; 1:3,000), and secondary antibody (goat anti-rabbit HRP, Bio-Rad, AB_11125142; 1:2,000) were used in these experiments.

[0100] RT-qPCR analysis Ramos, SUDHL4, SUDHL1, SR-786, and U937 cells were treated with HDL NPs (20 nM, 50 nM), human HDL (hHDL; 50 nM), or PBS for up to 72 hours, and RNA was isolated using the RNeasy Mini Kit (Qiagen). In all cases, hHDL was added to HDL NPs at equimolar concentrations based on protein concentration. RNA samples (500 ng RNA / 30 μl reaction) were reverse transcribed using the TaqMan Reverse Transcription Kit, and qPCR was performed using TaqMan Gene Expression Assays (Life Technologies) on a BioRad CFX-Connect iCycler. Samples were normalized to β-actin, and relative expression was calculated using the ΔΔCt method. Each condition was run in biological triplicate.

[0101] C11-BODIPY assay for lipid peroxidation Ramos, SUDHL4, SUDHL1, SR-786, and U937 cells (2.5 × 10 per ml) 5 Cells (1000 x 1000 cells) were treated with HDL NPs (50 nM) or PBS for 24, 48, or 72 hours. After treatment, C11-BODIPY (1 μM final concentration; Thermo Fisher Scientific) was added to each well, and the cells were incubated at 37°C and 5% CO2 for 30 minutes. Cells were then washed twice with 1x PBS, resuspended in ice-cold FACS buffer, and C11-BODIPY fluorescence in the FITC channel was quantified using a BD LSR II Fortessa flow cytometer. Data were analyzed using FCS Express software.

[0102] Cell death (MTS) assay MTS assays (CellTiter; Promega) were performed as previously described. Ramos, SUDHL4, Raji, Daudi, Namalwa, SUDHL6, and Jurkat cells were cultured at 2 × 10 per mL. 5SUDHL1, SR-786, and U937 cells were plated at a density of 5 × 10 cells per mL and cultured for 72 hours before assay. 4 Cells were plated at a density of 1 / 300 cells per well and cultured for 5 days before the MTS assay. SCARB1 blocking antibody and isotype control antibody were added at dilutions of 1:1000 to 1:250. Ferrostatin-1 and deferoxamine (DFO) were obtained from Sigma-Aldrich and added to a final concentration of 1 µM. MTS values were normalized to the PBS control.

[0103] Tumor xenograft model SCID-beige mice (4-6 weeks old; Charles River) were used for SUDHL4 tumor xenograft studies. Flank tumors were grown at 1 × 10 per mouse. 7 SUDHL4 cells were used to initiate the treatment. Tumors were grown to approximately 100 mm before HDL NP treatment began. 3 Based on their initial tumor volume, mice were randomly divided into two groups: PBS (100 μL) and HDL NP (100 μL of 1 μM NP). Treatment (intravenous) was administered three times per week for one week. Tumors were then harvested, and single-cell suspensions were generated by mechanically dissociating the tumors and passing the cells through a 70-micron filter. C11-BODIPY (1 μM final concentration) was added to the resulting cell suspension (1 × 10 6 A fraction of the cells (1000 cells) was added to the sera and subjected to flow analysis as described above. RNA was isolated from the remaining portion of the cells, and GPX4 expression was quantified by RT-qPCR as described above.

[0104] Human tissue analysis Archival, formalin-fixed, paraffin-embedded tissue sections from patients with large B-cell lymphoma and follicular lymphoma were analyzed. All samples were anonymized for all information except the final diagnosis. A total of 104 archival samples from follicular lymphoma and 49 from diffuse large B-cell lymphoma were obtained and stained for SCARB1 expression. Immunohistochemical staining of sections was performed at the Pathology Core of the Robert H. Lurie Comprehensive Cancer Center at Northwestern University using a monoclonal SCARB1 antibody (Abcam, AB_882458; 1:100 dilution). Liver and thymus specimens served as positive and negative controls, respectively. Brightfield images were captured at 10x and 40x magnification.

[0105] result HDL NP downregulates GPX4 A study was conducted to determine whether HDL NPs increase the expression of de novo cholesterol synthesis genes and decrease the expression of GPX4. Data are shown in Figure 1. Ramos and SUDHL4 cells are well-studied models of Burkitt's lymphoma (BL) and germinal center DLBCL (GC-DLBCL), respectively. HDL NPs result in cellular cholesterol depletion and significant in vitro and in vivo cell death of SUDHL4 and Ramos cells. RT-qPCR analysis was performed to evaluate GPX4 expression in Ramos cells (left panel) and SUDHL4 cells (right panel) treated with 0 nM (control), 20 nM, or 50 nM HDL NPs for 24 or 48 hours. Western blot analysis and conventional RT-qPCR were used to confirm the decrease in GPX4 expression. We observed that HDL NP treatment significantly decreased GPX4 expression in both cell lines relative to the PBS control (0 nM) at both the protein (not shown) and mRNA levels (Figure 1). In contrast, treatment with equimolar concentrations of cholesterol-rich HDL did not alter GPX4 protein or gene expression (not shown).

[0106] HDL NPs induce ferroptosis in B-cell lymphoma cell lines At least two metrics have been proposed to distinguish ferroptosis from apoptosis and other forms of cell death: 1) cell death correlates with an increase in oxidized membrane lipids, quantified using C11-BODIPY, a lipophilic fluorescent dye used to measure lipid peroxidation, and flow cytometry; and 2) cell death can be reduced by the addition of lipophilic antioxidants (e.g., ferrostatin-1) or iron chelators, such as deferoxamine (DFO). Using these metrics, we evaluated whether HDL NPs induced ferroptosis in Ramos and SUDHL4 cells. In both cell lines, HDL NP treatment resulted in a dose-dependent increase in the C11-BODIPY signal over time. We evaluated whether HDL NPs induced the accumulation of lipid peroxides in SUDHL4 cells, and the data are shown in Figures 4A-4B. Similarly, we evaluated whether HDL NPs induce the accumulation of lipid peroxides in Ramos cells, and the data are shown in Figures 5A-5B. A dose-dependent increase in the accumulation of lipid peroxides was observed in both cell lines.

[0107] Ramos and SUDHL4 cells were then cultured with HDL NPs in the presence of either ferrostatin-1 or DFO and assayed for cell viability. The addition of ferrostatin-1 and DFO significantly inhibited HDL NP-induced cell death in SUDHL4 cells (diffuse large B-cell lymphoma, Figure 2) and Ramos cells (Burkitt's lymphoma, Figure 3). These data demonstrate that HDL NPs induce ferroptosis in Ramos and SUDHL4 cells.

[0108] HDL NPs induce ferroptosis in cholesterol-auxotrophic lymphoma cell lines Several cell lines, including SR-786 (ALK+ ALCL), SUDHL1 (ALK+ ALCL), and U937 (isolated from a histiocytic lymphoma but of myeloid lineage), are auxotrophic for cholesterol. ALK+ ALCL cells were identified based on their reduced viability when cultured in lipoprotein-deficient serum, and the cell death phenotype was rescued by the addition of cholesterol-rich low-density lipoprotein (LDL) or free cholesterol. HDL NPs target SCARB1 in SUDHL4 and Ramos cells, resulting in cellular cholesterol depletion and significant cell death in vitro and in vivo. The requirement for SCARB1 as a target for HDL NPs in these lymphoma cells was verified using an anti-SCARB1 blocking antibody and fluorescently labeled HDL NPs (data not shown). SCARB1 expression was investigated in ALK+ ALCL and U937 cells. The data reveal SCARB1 expression in SR-786, SUDHL1, and U937 cells (Figure 6A). Treatment of each cell line with HDL NPs strongly induced cell death (Figure 6B). The data in Figure 6 show the effect of SR-B1 expression and HDL NP efficacy in cholesterol-auxotrophic cell lines. Tested cells include: SNU-1: gastric cancer; SUDHL1: ALK+ anaplastic large T-cell lymphoma; SR: ALK+ anaplastic large T-cell lymphoma; U937: histiocytic lymphoma; HEC-1B: endometrial adenocarcinoma; and U266B1: melanoma. Ramos and Jurkat represent positive and negative controls for SCARB1 expression, respectively. β-actin was used as a loading control. Cells were treated with HDL NPs for 120 hours.

[0109] HDL NPs induce ferroptosis in vivo As shown in SUDHL4 and Ramos cells, HDL NPs specifically target and significantly reduce tumor burden in xenograft models. To determine whether systemic HDL NP treatment reduces GPX4 expression and increases lipid peroxide accumulation in tumor cells in vivo, SUDHL4 tumor xenografts (approximately 100 mm in volume) were cultured. 3 ) was established in SCID-beige mice. Mice were then treated with PBS or HDL NPs (100 μl of 1 μM HDL NPs, iv, three times per week for one week). After treatment, tumors were excised, and GPX4 expression and lipid peroxide accumulation were quantified by RT-qPCR and C11-BODIPY staining, respectively. HDL NP treatment resulted in downregulation of GPX4 as measured by RT-qPCR compared with PBS controls (Figure 7B), which correlated with increased membrane lipid peroxide accumulation. Changes in tumor volume are shown in Figure 7A. No adverse side effects were observed after systemic administration of HDL NPs. These data indicate that HDL NPs induce molecular changes consistent with ferroptosis in the SUDHL4 flank tumor xenograft model of lymphoma. Lipid accumulation in SUDHL1 cells in an in vivo ferroptosis assay is shown in the plots in Figure 8.

[0110] Studies extending these findings to renal cell carcinoma are shown in Figures 9-11. Figure 9 is a bar graph showing that HDL NPs induce ferroptosis in 786-O (renal cell carcinoma-clear cell) cells (HDL NPs + ferrostatin-1 and HDL NPs + DFO). Ferrostatin-1, especially at high concentrations, had a dramatic effect on HDL NP function. Data in Figure 10 show that HDL NPs also induce ferroptosis in Caki-2 (renal cell carcinoma-papillary) cells. Similarly, HDL NPs induce the accumulation of lipid peroxides in 786-O and Caki-2 cells (Figures 11A-11B).

[0111] Expression of GPX4 and SR-B1 in response to HDL NP in renal cell carcinoma and ovarian cancer cell lines The role of SR-B1 in GPX4 expression was analyzed using siRNA. Specific knockdown of SR-B1 using siRNA was shown to downregulate GPX4 expression. After treatment with SR-B1 siRNA, siRNA control (siCtrl), or PBS at 96 and 120 hours (h), proteins were isolated and examined by Western blot (25 μg protein, SR-B1 Abcam antibody (ab52629, 1:2,000), GPX4 Abcam antibody (ab41787, 1:20,000)). Beta-actin Cell Signaling (13E5, 1:2,000) served as a protein control. Data demonstrating a complete loss of protein expression upon SR-B1 knockdown are shown in Figure 12A. Data in Figure 12B show that siRNA knockdown of SR-B1 downregulation induces cell death.

[0112] Renal cell carcinoma cell lines were further treated with HDL NPs to evaluate the effects of HDL NPs on the expression of SR-B1 and GPX4. Western blot analysis of GPX4 and SR-B1 over various time courses with varying concentrations of HDL NPs was performed. The data are shown in Figure 12C. The data demonstrate that HDL NPs do not directly regulate SR-B1 receptor expression, but that HDL NPs significantly down-regulate GPX4 expression in a time- and dose- (e.g., HDL NP concentration-) dependent manner.

[0113] We also investigated the ability of HDL NPs to affect GPX4 expression in the presence of Sutent, a targeted receptor protein-tyrosine kinase inhibitor therapy. Results are shown in Figure 12D, demonstrating that HDL NPs significantly down-regulate GPX4 expression in the presence of Sutent. Eight micrograms of protein were used: GPX4 (Abcam) (ab41787, 1:5,000); beta-actin (Cell Signaling) (13E5, 1:2,000). HDL NPs were also shown to increase the expression of oxidized lipids (Figure 12E). Using an MTS Rescue Assay, we found that cell death induced by HDL NPs was rescued by ferrostatin-1 and deferoxamine (Figure 12F). Figure 12G shows in vivo data that after five treatments of HDL NPs, HDL NPs reduce 786-O tumor burden (top left panel); HDL NPs increase survival (top right panel); and HDL NPs increase oxidized lipids in tumors (bottom middle panel).

[0114] We further investigated the effects of HDL NPs in another renal cell carcinoma cell line, 769-P. The results, shown in Figures 13A-13B, indicate that HDL NPs also down-regulated GPX4 expression in these cells (Figure 13A, Western blot analysis) and induced cell death, which was rescued by ferrostatin-1 and deferoxamine (MTS data in Figure 13B).

[0115] Similar experiments were performed in ovarian cancer cells, yielding consistent data shown in Figures 14-16. Figures 14A-14D show results obtained with HDL NPs in the OVCAR5 cell line, a platinum-sensitive ovarian cancer cell line. Figure 14A shows a Western blot of GPX4, demonstrating that HDL NPs downregulate GPX4 expression. Figure 14B shows C11-BODIPY flow data demonstrating that HDL NPs increase the expression of oxidized lipids. Figure 14C shows the results of an MTS assay, demonstrating that cell death induced by HDL NPs is rescued by ferrostatin-1 and deferoxamine. Figure 14D shows a Western blot of SR-B1 and GPX4, and that siRNA knockdown of SR-B1 downregulates GPX4 expression.

[0116] Similar data were obtained using the platinum-resistant ovarian cancer cell line, OVCAR5 CP Resistant Cell Line, and are shown in Figures 15A-15C. Figure 15A shows a Western blot of GPX4 and shows that HDL NPs downregulate GPX4 expression. Figure 15B shows an MTS assay demonstrating that cell death induced by HDL NPs is rescued by ferrostatin-1 and deferoxamine. Figure 15C shows that HDL NPs increase the expression of oxidized lipids.

[0117] Similarly, similar data was obtained using the ES2 cell line, a clear cell ovarian cancer cell line, and the results are shown in Figure 16. Western blot for GPX4 and HDL NPs are shown to downregulate GPX4 expression.

[0118] Other embodiments Embodiment 1. A method of treating a subject having, suspected of having, or at risk of having cancer, comprising administering to the subject a synthetic nanostructure comprising a nanostructure core, a shell surrounding the nanostructure core and comprising a lipid bound thereto, wherein the shell comprises a phospholipid, wherein the subject has cancer cells, and the synthetic nanostructure is administered in an amount effective to induce ferroptosis in the cancer cells. Embodiment 2. A method of reducing the number of cancer cells in a cell population, comprising contacting the cancer cells with a synthetic nanostructure comprising a nanostructure core, a shell surrounding the nanostructure core and comprising a lipid bound thereto, wherein the shell comprises a phospholipid, wherein the synthetic nanostructure is present in an amount effective to induce ferroptosis in the cancer cells. Embodiment 3. The method of any one of embodiments 1 to 2, wherein the nanostructure core is gold. Embodiment 4. The method of any one of embodiments 1 to 3, wherein the synthetic nanostructure further comprises an apolipoprotein. Embodiment 5. The method of embodiments 1 to 4, wherein the apolipoprotein is apolipoprotein AI, apolipoprotein A-II, or apolipoprotein E. Embodiment 6 The method of any one of embodiments 1 to 5, wherein the synthetic nanostructure further comprises cholesterol. Embodiment 7 The method of any one of embodiments 1 to 6, wherein the phospholipid shell comprises a lipid monolayer. Embodiment 8 The method of any one of embodiments 1 to 6, wherein the phospholipid shell comprises a lipid bilayer. Embodiment 9. The method of embodiment 8, wherein at least a portion of the lipid bilayer is covalently attached to the nanostructure core. Embodiment 10. The method of any one of embodiments 1 to 9, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 500 nanometers (nm). Embodiment 11. The method of any one of embodiments 1 to 10, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 250 nanometers (nm). Embodiment 12. The method of any one of embodiments 1 to 11, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 100 nanometers (nm). Embodiment 13. The method of any one of embodiments 1 to 12, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 75 nanometers (nm). Embodiment 14. The method of any one of embodiments 1 to 13, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 50 nanometers (nm). Embodiment 15. The method of any one of embodiments 1 to 14, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 30 nanometers (nm). Embodiment 16. The method of any one of embodiments 1 to 15, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 15 nanometers (nm). Embodiment 17. The method of any one of embodiments 1 to 16, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 10 nanometers (nm). Embodiment 18. The method of any one of embodiments 1 to 17, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 5 nanometers (nm). Embodiment 19. The method of any one of embodiments 1 to 18, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 3 nanometers (nm). Embodiment 20. The method of any one of embodiments 1 to 19, wherein the nanostructure core has an aspect ratio greater than about 1:1. Embodiment 21. The method of any one of embodiments 1 to 20, wherein the nanostructure core has an aspect ratio greater than 3:1. Embodiment 22. The method of any one of embodiments 1 to 21, wherein the nanostructure core has an aspect ratio greater than 5:1. Embodiment 23. The method of any one of embodiments 1 to 22, wherein the phospholipid comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (16:0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (18:0 PE), sphingomyelin, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), or a combination thereof. Embodiment 24 The method of embodiments 1 to 23, wherein the subject has been diagnosed with cancer. Embodiment 25 The method of any one of embodiments 1 to 24, wherein the subject has been diagnosed with a ferroptosis-sensitive malignancy or a cholesterol auxotrophic malignancy. Embodiment 26 The method of any one of embodiments 1 to 25, wherein the cancer is selected from B-cell lymphoma, renal cell carcinoma, T-cell lymphoma, gastric cancer, ovarian cancer, and endometrial adenocarcinoma. Embodiment 27. The method of any one of embodiments 1 to 26, wherein the cancer is selected from sarcoma, lymphoma, gastric cancer, anaplastic large cell lymphoma, clear cell renal cell carcinoma (ccRCC), ovarian cancer, platinum-resistant ovarian cancer, and clear cell ovarian cancer. Embodiment 28 The method of any one of embodiments 1 to 27, wherein the synthetic nanostructure is administered to the subject or contacted with the cells more than once. Embodiment 29 The method of embodiment 28, wherein the synthetic nanostructures are administered to the subject or contacted with the cells at least once per month. Embodiment 30 The method of any one of embodiments 28 to 29, wherein the synthetic nanostructure is administered to the subject or contacted with the cells at least once per week. Embodiment 31 The method of any one of embodiments 28 to 30, wherein the synthetic nanostructure is administered to the subject or contacted with the cells at least once per day. Embodiment 32 The method of any one of embodiments 28 to 31, wherein the synthetic nanostructures are administered to the subject or contacted with the cells twice per day. Embodiment 33 The method of any one of 1 to 32, wherein the subject is a mammal. Embodiment 34 The method of any one of embodiments 1 to 33, wherein the subject is a human. Embodiment 35 The method of any one of embodiments 1 to 33, further comprising administering to the subject a ferroptosis inducer compound. Embodiment 36 The method of any one of embodiments 1 to 33, further comprising determining whether the cancer is susceptible to ferroptosis. Embodiment 37. A method of treating a subject having a ferroptosis-susceptible disorder, comprising: identifying a subject having a ferroptosis-susceptible disorder; and administering to the subject a synthetic nanostructure comprising a nanostructure core and a shell surrounding the nanostructure core and comprising a lipid bound thereto, in an amount effective to induce ferroptosis in affected cells of the subject, wherein the shell comprises a phospholipid. Embodiment 38. A composition comprising a synthetic nanostructure comprising a nanostructure core and a shell surrounding the nanostructure core and comprising a lipid bound thereto, wherein the shell comprises a phospholipid and a ferroptosis inducer compound. Embodiment 39. A method for inducing ferroptosis in a cell, comprising: identifying the cell as a ferroptosis-susceptible cell; and contacting the cell with a nanostructure core, a shell surrounding the nanostructure core and comprising a lipid bound thereto, in an amount effective to induce ferroptosis in the cell, wherein the shell comprises a phospholipid.

[0119] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced with an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0120] From the above description, those skilled in the art can easily ascertain the essential features of the present invention, and can make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are within the scope of the appended claims.

[0121] equivalent While several inventive embodiments have been described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures for performing the functions described herein and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily recognize that all parameters, dimensions, materials, and shapes described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or shapes will vary depending on the specific application(s) for which the teachings of this invention are used. Those skilled in the art will recognize and be able to ascertain, using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. Accordingly, the embodiments are presented by way of example only, and it should be understood that, within the scope of the appended claims and their equivalents, the inventive embodiments may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual requirement, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not inconsistent with one another.

[0122] All definitions defined and used herein should be understood to govern dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0123] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, and in some cases may include the entire document. The indefinite articles "a" and "an," as used herein, should be understood to mean "at least one," unless the specification and claims clearly indicate to the contrary.

[0124] The term "and / or," as used herein, should be understood in the specification and claims to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and non-conjunctively present in other cases. Multiple elements listed with "and / or," i.e., "one or more" of the elements so conjoined, should be construed in the same manner. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause (whether related to those specifically identified elements). Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," may refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements), etc.

[0125] As used herein, in the specification and claims, "or" should be understood to have the same meaning as "and / or," as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive (i.e., including at least one of, but also including more than one of, and optionally additional unlisted items). Terms without clear indication to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," only refer to the inclusion of exactly one element of, or a list of elements. In general, the term "or," as used herein, shall only be construed as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by exclusive terms such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0126] As used herein, in the specification and claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each element specifically listed within the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements, whether related or unrelated to the specifically identified elements, that may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer, in one embodiment, to at least one that optionally includes two or more As and no Bs (and optionally includes elements other than B); in another embodiment, to at least one that optionally includes two or more Bs and no As (and optionally includes elements other than A); in yet another embodiment, to at least one that optionally includes two or more As, and at least one that optionally includes two or more Bs (and optionally includes other elements), etc.

[0127] It should also be understood that, unless expressly indicated to the contrary, in any method claimed herein that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method of treating a subject having cancer, comprising: Identifying a subject with a ferroptosis-sensitive malignancy; The subject is administering a synthetic nanostructure comprising a nanostructure core and a shell surrounding and bound to the nanostructure core, the shell comprising a lipid, wherein the shell comprises a phospholipid; Including, The method, wherein the subject has cancer cells and the synthetic nanostructure is administered in an amount effective to induce ferroptosis in the cancer cells. (Item 2) 1. A method for reducing the number of cancer cells in a cell population, comprising: The cancer cells with a synthetic nanostructure comprising a nanostructure core and a shell surrounding and associated with said nanostructure core comprising a lipid, said shell comprising a phospholipid; The method, wherein the synthetic nanostructure is present in an amount effective to induce ferroptosis in the cancer cells. (Item 3) 3. The method of any one of items 1 to 2, wherein the nanostructure core is gold. (Item 4) 4. The method of any one of items 1 to 3, wherein the synthetic nanostructure further comprises an apolipoprotein. (Item 5) 5. The method of any one of items 1 to 4, wherein the apolipoprotein is apolipoprotein AI, apolipoprotein A-II, or apolipoprotein E. (Item 6) 6. The method of any one of items 1 to 5, wherein the synthetic nanostructure further comprises cholesterol. (Item 7) 7. The method of any one of items 1 to 6, wherein the phospholipid shell comprises a lipid monolayer. (Item 8) 7. The method of any one of items 1 to 6, wherein the phospholipid shell comprises a lipid bilayer. (Item 9) 9. The method of claim 8, wherein at least a portion of the lipid bilayer is covalently bound to the nanostructure core. (Item 10) 10. The method of any one of items 1 to 9, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 500 nanometers (nm). (Item 11) 11. The method of any one of items 1 to 10, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 250 nanometers (nm). (Item 12) 12. The method of any one of items 1 to 11, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 100 nanometers (nm). (Item 13) 13. The method of any one of items 1 to 12, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 75 nanometers (nm). (Item 14) 14. The method of any one of items 1 to 13, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 50 nanometers (nm). (Item 15) 15. The method of any one of items 1 to 14, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 30 nanometers (nm). (Item 16) 16. The method of any one of items 1 to 15, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 15 nanometers (nm). (Item 17) 17. The method of any one of items 1 to 16, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 10 nanometers (nm). (Item 18) 18. The method of any one of items 1 to 17, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 5 nanometers (nm). (Item 19) 19. The method of any one of items 1 to 18, wherein the nanostructure core has a maximum cross-sectional dimension less than or equal to about 3 nanometers (nm). (Item 20) 20. The method of any one of items 1 to 19, wherein the nanostructure core has an aspect ratio greater than about 1:1. (Item 21) 21. The method of any one of items 1 to 20, wherein the nanostructure core has an aspect ratio greater than 3:1. (Item 22) 22. The method of any one of the preceding claims, wherein the nanostructure core has an aspect ratio greater than 5:1. (Item 23) 23. The method of any one of items 1 to 22, wherein the phospholipid comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (16:0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (18:0 PE), sphingomyelin, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), or a combination thereof. (Item 24) 24. The method of any one of items 1 to 23, wherein the subject has been diagnosed with cancer. (Item 25) 25. The method of any one of items 1 to 24, wherein the subject has been diagnosed with a ferroptosis-sensitive malignancy or a cholesterol-auxotrophic malignancy. (Item 26) 26. The method of any one of items 1 to 25, wherein the cancer is selected from B-cell lymphoma, renal cell carcinoma, T-cell lymphoma, gastric cancer, ovarian cancer, and endometrial adenocarcinoma. (Item 27) 27. The method of any one of items 1 to 26, wherein the cancer is selected from sarcoma, lymphoma, gastric cancer, anaplastic large cell lymphoma, clear cell renal cell carcinoma (ccRCC), ovarian cancer, platinum-resistant ovarian cancer, and clear cell ovarian cancer, B-cell lymphoma, and T-cell lymphoma. (Item 28) 28. The method of any one of items 1 to 27, wherein the synthetic nanostructure is administered to the subject or contacted with the cells more than once. (Item 29) 29. The method of claim 28, wherein the synthetic nanostructure is administered to the subject or contacted with the cells at least once per month. (Item 30) 30. The method of any one of items 28 to 29, wherein the synthetic nanostructure is administered to the subject or contacted with the cells at least once per week. (Item 31) 31. The method of any one of items 28 to 30, wherein the synthetic nanostructure is administered to the subject or contacted with the cells at least once per day. (Item 32) 32. The method of any one of items 28 to 31, wherein the synthetic nanostructure is administered to the subject or contacted with the cells twice per day. (Item 33) 33. The method of any one of items 1 to 32, wherein the subject is a mammal. (Item 34) 34. The method of any one of items 1 to 33, wherein the subject is a human. (Item 35) 34. The method of any one of items 1 to 33, further comprising administering to the subject a ferroptosis inducer compound. (Item 36) 34. The method of any one of items 1 to 33, further comprising determining whether the cancer is susceptible to ferroptosis. (Item 37) 1. A method of treating a subject having a ferroptosis-susceptible disorder, comprising: Identifying a subject with a ferroptosis-susceptible disorder; The subject is administering to the subject a synthetic nanostructure comprising a nanostructure core and a shell surrounding the nanostructure core and comprising a lipid bound thereto, in an amount effective to induce ferroptosis in diseased cells of the subject, wherein the shell comprises a phospholipid; A method comprising: (Item 38) A composition comprising a synthetic nanostructure comprising a nanostructure core and a shell surrounding the nanostructure core and comprising a lipid bound thereto, wherein the shell comprises a phospholipid and a ferroptosis inducer compound. (Item 39) 1. A method for inducing ferroptosis in a cell, comprising: 1. A method comprising: identifying a cell as a ferroptosis-susceptible cell; and contacting the cell with a nanostructure core, a shell surrounding the nanostructure core and comprising a lipid bound thereto, in an amount effective to induce ferroptosis in the cell, wherein the shell comprises a phospholipid.

Claims

[Claim 1] The invention described in the specification.

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