Targeted reduction of cholesterol uptake in cholesterol-addicted lymphoma cells blocks turnover of oxidized lipids to cause ferroptosis.

Normal human cells can either synthesize cholesterol or take it up from lipoproteins to meet their metabolic requirements. In some malignant cells, de novo cholesterol synthesis genes are transcriptionally silent or mutated, meaning that cholesterol uptake from lipoproteins is required for survival. Recent data suggest that lymphoma cells dependent upon lipoprotein-mediated cholesterol uptake are also subject to ferroptosis, an oxygen- and iron-dependent cell death mechanism triggered by accumulation of oxidized lipids in cell membranes unless the lipid hydroperoxidase, glutathione peroxidase 4 (GPX4), reduces these toxic lipid species. To study mechanisms linking cholesterol uptake with ferroptosis and determine the potential role of the high-density lipoprotein (HDL) receptor as a target for cholesterol depleting therapy, we treated lymphoma cell lines known to be sensitive to the reduction of cholesterol uptake with HDL-like nanoparticles (HDL NPs). HDL NPs are a cholesterol-poor ligand that binds to the receptor for cholesterol-rich HDLs, scavenger receptor type B1 (SCARB1). Our data reveal that HDL NP treatment activates a compensatory metabolic response in treated cells toward increased de novo cholesterol synthesis, which is accompanied by nearly complete reduction in expression of GPX4. As a result, oxidized membrane lipids accumulate, leading to cell death through a mechanism consistent with ferroptosis. We obtained similar results in vivo after systemic administration of HDL NPs in mouse lymphoma xenografts and in primary samples obtained from patients with lymphoma. In summary, targeting SCARB1 with HDL NPs in cholesterol uptake-addicted lymphoma cells abolishes GPX4, resulting in cancer cell death by a mechanism consistent with ferroptosis.

Biomimetic Magnetic Nanostructures: A Theranostic Platform Targeting Lipid Metabolism and Immune Response in Lymphoma.

B-cell lymphoma cells depend upon cholesterol to maintain pro-proliferation and pro-survival signaling via the B-cell receptor. Targeted cholesterol depletion of lymphoma cells is an attractive therapeutic strategy. We report here high-density lipoprotein mimicking magnetic nanostructures (HDL-MNSs) that can bind to the high-affinity HDL receptor, scavenger receptor type B1 (SR-B1), and interfere with cholesterol flux mechanisms in SR-B1 receptor positive lymphoma cells, causing cellular cholesterol depletion. In addition, the MNS core can be utilized for its ability to generate heat under an external radio frequency field. The thermal activation of MNS can lead to both innate and adaptive antitumor immune responses by inducing the expression of heat shock proteins that lead to activation of antigen presenting cells and finally lymphocyte trafficking. In the present study, we demonstrate SR-B1 receptor mediated binding and cellular uptake of HDL-MNS and prevention of phagolysosome formation by transmission electron microscopy, fluorescence microscopy, and ICP-MS analysis. The combinational therapeutics of cholesterol depletion and thermal activation significantly improves therapeutic efficacy in SR-B1 expressing lymphoma cells. HDL-MNS reduces the T2 relaxation time under magnetic resonance imaging (MRI) more effectively compared with a commercially available contrast agent, and the specificity of HDL-MNS toward the SR-B1 receptor leads to differential contrast between SR-B1 positive and negative cells suggesting its utility in diagnostic imaging. Overall, we have demonstrated that HDL-MNSs have cell specific targeting efficiency, can modulate cholesterol efflux, can induce thermal activation mediated antitumor immune response, and possess high contrast under MRI, making it a promising theranostic platform in lymphoma.

Supramolecular Assembly of High-Density Lipoprotein Mimetic Nanoparticles Using Lipid-Conjugated Core Scaffolds.

Synthetic high-density lipoprotein (HDL) mimics have emerged as promising therapeutic agents. However, approaches to date have been unable to reproduce key features of spherical HDLs, which are the most abundant human HDL species. Here, we report the synthesis and characterization of spherical HDL mimics using lipid-conjugated organic core scaffolds. The core design motif constrains and orients phospholipid geometry to facilitate the assembly of soft-core nanoparticles that are approximately 10 nm in diameter and resemble human HDLs in their size, shape, surface chemistry, composition, and protein secondary structure. These particles execute salient HDL functions, including efflux of cholesterol from macrophages, cholesterol delivery to hepatocytes, support lecithin:cholesterol acyltransferase activity, and suppress inflammation. These results represent a significant step toward a genuine functional mimic of human HDLs.

HDL nanoparticles targeting sonic hedgehog subtype medulloblastoma.

Medulloblastoma is the most common paediatric malignant brain cancer and there is a need for new targeted therapeutic approaches to more effectively treat these malignant tumours, which can be divided into four molecular subtypes. Here, we focus on targeting sonic hedgehog (SHH) subtype medulloblastoma, which accounts for approximately 25% of all cases. The SHH subtype relies upon cholesterol signalling for tumour growth and maintenance of tumour-initiating cancer stem cells (CSCs). To target cholesterol signalling, we employed biomimetic high-density lipoprotein nanoparticles (HDL NPs) which bind to the HDL receptor, scavenger receptor type B-1 (SCARB1), depriving cells of natural HDL and their cholesterol cargo. We demonstrate uptake of HDL NPs in SCARB1 expressing medulloblastoma cells and depletion of cholesterol levels in cancer cells. HDL NPs potently blocked proliferation of medulloblastoma cells, as well as hedgehog-driven Ewing sarcoma cells. Furthermore, HDL NPs disrupted colony formation in medulloblastoma and depleted CSC populations in medulloblastoma and Ewing sarcoma. Altogether, our findings provide proof of principle for the development of a novel targeted approach for the treatment of medulloblastoma using HDL NPs. These findings present HDL-mimetic nanoparticles as a promising therapy for sonic hedgehog (SHH) subtype medulloblastoma and possibly other hedgehog-driven cancers.

Rational Targeting of Cellular Cholesterol in Diffuse Large B-Cell Lymphoma (DLBCL) Enabled by Functional Lipoprotein Nanoparticles: A Therapeutic Strategy Dependent on Cell of Origin.

Cancer cells have altered metabolism and, in some cases, an increased demand for cholesterol. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative approach. Toward this end, we focused on diffuse large B-cell lymphoma (DLBCL) as a model because there is differential cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol uptake through the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). As we previously reported, GC DLBCL are exquisitely sensitive to HDL NP as monotherapy, while ABC DLBCL are less sensitive. Herein, we report that enhanced BCR signaling and resultant de novo cholesterol synthesis in ABC DLBCL drastically reduces the ability of HDL NPs to reduce cellular cholesterol and induce cell death. Therefore, we combined HDL NP with the BCR signaling inhibitor ibrutinib and the SYK inhibitor R406. By targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis, we achieved cellular cholesterol reduction and induced apoptosis in otherwise resistant ABC DLBCL cell lines. These results in lymphoma demonstrate that reduction of cellular cholesterol is a powerful mechanism to induce apoptosis. Cells rich in cholesterol require HDL NP therapy to reduce uptake and molecularly targeted agents that inhibit upstream pathways that stimulate de novo cholesterol synthesis, thus, providing a new paradigm for rationally targeting cholesterol metabolism as therapy for cancer.

Update on current and potential nanoparticle cancer therapies.

PURPOSE OF REVIEW: To summarize the most recent preclinical and clinical advancements in therapeutic nano-oncology. RECENT FINDINGS: First-generation nanotherapies are well tolerated in humans and evidence shows that they are efficacious, while at the same time reducing the burden of side-effects. Most of these therapies are not specifically targeted, but take advantage of enhanced passive accumulation within tumors to preferentially deliver chemotherapies that demonstrate off-target toxicities when administered as free drugs. Also, actively targeted nanotherapies are entering the clinical arena and preliminary data are encouraging. Finally, a number of exciting preclinical developments in nanotechnology provide clear evidence that nanotherapies will continue to enter the clinic and will have a significant impact in oncology. SUMMARY: A number of intriguing nanoparticle therapies are being tested in preclinical and clinical trials. Nanoparticles with increasing molecular sophistication, specific targeting properties, and unique mechanisms of action will find their way to the clinic. Certainly, nanoparticle-based therapies will be increasingly represented in drug development pipelines, and will continue to provide efficacious and well tolerated drug options for patients with cancer.

Biomimetic, synthetic HDL nanostructures for lymphoma.

New therapies that challenge existing paradigms are needed for the treatment of cancer. We report a nanoparticle-enabled therapeutic approach to B-cell lymphoma using synthetic high density lipoprotein nanoparticles (HDL-NPs). HDL-NPs are synthesized using a gold nanoparticle template to control conjugate size and ensure a spherical shape. Like natural HDLs, biomimetic HDL-NPs target scavenger receptor type B-1, a high-affinity HDL receptor expressed by lymphoma cells. Functionally, compared with natural HDL, the gold NP template enables differential manipulation of cellular cholesterol flux in lymphoma cells, promoting cellular cholesterol efflux and limiting cholesterol delivery. This combination of scavenger receptor type B-1 binding and relative cholesterol starvation selectively induces apoptosis. HDL-NP treatment of mice bearing B-cell lymphoma xenografts selectively inhibits B-cell lymphoma growth. As such, HDL-NPs are biofunctional therapeutic agents, whose mechanism of action is enabled by the presence of a synthetic nanotemplate. HDL-NPs are active in B-cell lymphomas and potentially, other malignancies or diseases of pathologic cholesterol accumulation.

Conditional and specific inhibition of NF-κB in mouse pancreatic ß cells prevents cytokine-induced deleterious effects and improves islet survival posttransplant.

BACKGROUND: Islets are susceptible to damage by proinflammatory cytokines via activation of transcription factor NF-κB. We hypothesized that inhibition of NF-κB activity will decrease cytokine-mediated ß-cell injury and improve islet transplant functional outcome. METHODS: We created a transgenic mouse expressing a degradation resistant N-terminally deleted IκBα (ΔNIκBα) under the control of a commercially available tetracycline-controlled transcriptional activation system using a rat insulin promoter. Isolated islets from transgenic and control mouse strains were exposed to cytokines in vitro and assayed or transplanted. RESULTS: Western blot analysis showed that ΔNIκBα was significantly increased with doxycycline treatment. Cytokine-induced NF-κB activation was significantly decreased in transgenic (0.065 ± 0.013 absorbance value/µg protein) vs control islets (0.128 ± 0.006; P < .05). Suppression of cytokine-mediated NF-κB activity decreased expression of inducible nitric oxide synthase, monocyte chemoattractant protein-1, and interferon-γ inducible protein-10 RNA transcripts, and significantly decreased nitric oxide production in transgenic islets (0.084 ± 0.043 µM/µg protein) vs. controls (0.594 ± 0.174; P < .01). The insulin stimulation index in islets exposed to cytokines was higher in transgenic vs controls (1.500 ± 0.106 vs 0.800 ± 0.098; P < .01). Syngeneic transplants of a marginal mass of intraportally infused transgenic islets resulted in a reversion to euglycemia in 69.2% of diabetic recipients at a mean of 7.8 ± 1.1 days vs. 35.7% of control islet recipients reverting at a mean of 15.8 ± 2.9 days (P < .05). CONCLUSION: Conditional and specific suppression of NF-κB activity in ß cells protected islets from cytokine-induced dysfunction in vitro and in vivo. These results provide a proof of principle that inhibition of NF-κB activity in donor islets enhances function and improves the outcome of islet transplantation.

Transfection of pancreatic islets using polyvalent DNA-functionalized gold nanoparticles.

BACKGROUND: Transplantation of pancreatic islets is an effective treatment for select patients with type 1 diabetes. Improved cellular therapy results may be realized by altering the gene expression profile of transplanted islets. Current viral and nonviral vectors used to introduce nucleic acids for gene regulation hold promise, but safety and efficacy shortcomings motivate the development of new transfection strategies. Polyvalent gold nanoparticles (AuNPs) densely functionalized with covalently immobilized DNA oligonucleotides (AuNP-DNA) are new single entity transfection and gene regulating agents (ie, not requiring lipids, polymers, or viral vectors for cell entry) able to enter cells with high efficiency and no evidence of toxicity. We hypothesize that AuNP-DNA conjugates can efficiently transfect pancreatic islets with no impact on viability or functionality, and can function to regulate targeted gene expression. METHODS: AuNPs were surface-functionalized with control and antisense DNA oligonucleotides. Purified murine and human islets were exposed to AuNP-DNA conjugates for 24 hours. Islet AuNP-DNA uptake, cell viability, and functionality were measured. Furthermore, the ability of antisense AuNP-DNA conjugates to regulate gene expression was measured using murine islets expressing eGFP. RESULTS: Collectively, fluorescent confocal microscopy, transmission electron microscopy, mass spectrometry, and flow cytometry revealed substantial penetration of the AuNP-DNA conjugates into the inner core of the islets and within islet cells. No change in cellular viability occurred and the insulin stimulation index was unchanged in treated versus untreated islets. Transplantation of AuNP-DNA treated islets cured diabetic nude mice. Functionally, antisense eGFP AuNP-DNA conjugates reduced eGFP expression in MIP-eGFP islets. CONCLUSION: Polyvalent AuNP-DNA conjugates may represent the next generation of nucleic acid-based therapeutic agents for improving pancreatic islet engraftment, survival, and long-term function.