Jonathan S. Rink

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.

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.

Lipoproteins and lipoprotein mimetics for imaging and drug delivery.

Lipoproteins are a set of natural nanoparticles whose main role is the transport of fats within the body. While much work has been done to develop synthetic nanocarriers to deliver drugs or contrast media, natural nanoparticles such as lipoproteins represent appealing alternatives. Lipoproteins are biocompatible, biodegradable, non-immunogenic and are naturally targeted to some disease sites. Lipoproteins can be modified to act as contrast agents in many ways, such as by insertion of gold cores to provide contrast for computed tomography. They can be loaded with drugs, nucleic acids, photosensitizers or boron to act as therapeutics. Attachment of ligands can re-route lipoproteins to new targets. These attributes render lipoproteins attractive and versatile delivery vehicles. In this review we will provide background on lipoproteins, then survey their roles as contrast agents, in drug and nucleic acid delivery, as well as in photodynamic therapy and boron neutron capture therapy.

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.

Conditional and specific inhibition of NF-κB in mouse pancreatic β 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 β-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 β 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.

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.

Knockdown of intraislet IKKβ by spherical nucleic acid conjugates prevents cytokine-induced injury and enhances graft survival.

Background The efficiency of islet graft survival after intraportal implantation is compromised by host innate immune responses and the production of proinflammatory cytokines that cause acute cellular injury. This reaction activates intraislet nuclear factor-&kgr;B (NF-&kgr;B), causing production of gene products that have detrimental effects on &bgr;-cell survival and function. We hypothesized that small interfering RNA targeting of IKK&bgr;, a crucial kinase in the NF-&kgr;B activation pathway, in islets before transplantation would ameliorate the detrimental effects of cytokines and improve islet survival after transplantation. Methods To test this hypothesis, we prepared small interfering RNA–based spherical nucleic acid nanoparticle conjugates targeting IKK&bgr; IKK&bgr; SNA-NCs). We treated isolated islets with IKK&bgr; SNA-NCs and assessed the functional consequences of IKK&bgr; knockdown in vitro and after intraportal transplantation in mice. Results Treatment of freshly isolated mouse islets with IKK&bgr; SNA-NCs reduced constitutive IKK&bgr; expression and protected against proinflammatory cytokine-induced NF-&kgr;B activation, resulting in improved cell viability and decreased expression of gene products associated with &bgr;-cell dysfunction. Intraportal transplantation of a marginal mass (50 islets) of syngeneic islets treated with nanoparticle conjugates targeting IKK&bgr; resulted in reversion to normoglycemia in 50% of streptozotocin-induced diabetic recipients (n=12) compared with 0% of controls (n=12). Histologic analyses showed reduced CD11b+ cellular infiltration and decreased islet apoptosis. Conclusions These results are consistent with the hypothesis that inhibition of intraislet NF-&kgr;B activation ameliorates the detrimental effects of host cytokines and demonstrates that preconditioning freshly isolated islets in culture with IKK&bgr; SNA-NCs may be a promising therapy to enhance islet graft function and survival after transplantation.

Systemically administered collagen‐targeted gold nanoparticles bind to arterial injury following vascular interventions

Surgical and endovascular therapies for severe atherosclerosis often fail due to the development of neointimal hyperplasia and arterial restenosis. Our objective was to synthesize, characterize, and evaluate the targeting specificity and biocompatibility of a novel systemically injected nanoparticle. We hypothesize that surface‐functionalization of gold nanoparticles (AuNPs) with a collagen‐targeting peptide will be biocompatible and target specifically to vascular injury. 13 nm AuNPs were surface functionalized with a peptide‐molecular fluorophore and targeted to collagen (T‐AuNP) or a scrambled peptide sequence (S‐AuNP). After rat carotid artery balloon injury and systemic injection of T‐AuNP or S‐AuNP, arteries and organs were harvested and assessed for binding specificity and biocompatibility. The T‐AuNP bound with specificity to vascular injury for a minimum of 24 h. No significant inflammation was evident locally at arterial injury or systemically in major organs. The T‐AuNP did not impact endothelial cell viability or induce apoptosis at the site of injury in vivo. No major changes were evident in hepatic or renal blood chemistry profiles. Herein, we synthesized a biocompatible nanoparticle that targets to vascular injury following systemic administration. These studies demonstrate proof‐of‐principle and serve as the foundation for further T‐AuNP optimization to realize systemic, targeted delivery of therapeutics to the sites of vascular injury.

Mosaic Interdigitated Structure in Nanoparticle-Templated Phospholipid Bilayer Supports Partial Lipidation of Apolipoprotein A-I

Using gold nanoparticle-templated high-density lipoprotein-like particles as a model, the nanoparticle-templated phospholipid bilayer is studied from the bottom-up. Data support the phospholipids have a mosaic interdigitated structure. The discontinuous lipid milieu supports partial lipidation of apolipoprotein A-I, different from an ordinary phospholipid bilayer, suggesting that synergy between nanoparticle templates and bound phospholipid layers can modulate amphiphilic proteins for desired functions.

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.