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Myeloid-derived suppressor cells (MDSC) are innate immune cells that potently inhibit T cells. In cancer, novel therapies aimed to activate T cells can be rendered ineffective due to the activity of MDSCs. Thus, targeted inhibition of MDSCs may greatly enhance T-cell–mediated antitumor immunity, but mechanisms remain obscure. Here we show, for the first time, that scavenger receptor type B-1 (SCARB1), a high-affinity receptor for spherical high-density lipoprotein (HDL), is expressed by MDSCs. Furthermore, we demonstrate that SCARB1 is specifically targeted by synthetic high-density lipoprotein-like nanoparticles (HDL NP), which reduce MDSC activity. Using in vitro T-cell proliferation assays, data show that HDL NPs specifically bind SCARB1 to inhibit MDSC activity. In murine cancer models, HDL NP treatment significantly reduces tumor growth, metastatic tumor burden, and increases survival due to enhanced adaptive immunity. Flow cytometry and IHC demonstrate that HDL NP–mediated suppression of MDSCs increased CD8+ T cells and reduced Treg cells in the metastatic tumor microenvironment. Using transgenic mice lacking SCARB1, in vivo data clearly show that the HDL NPs specifically target this receptor for suppressing MDSCs. Ultimately, our data provide a new mechanism and targeted therapy, HDL NPs, to modulate a critical innate immune cell checkpoint to enhance the immune response to cancer. Mol Cancer Ther; 17(3); 686–97. ©2017 AACR.

Medicine has been a great beneficiary of the nanotechnology revolution. Nanotechnology involves the synthesis of functional materials with at least one size dimension between 1 and 100 nm. Advances in the field have enabled the synthesis of bio-nanoparticles that can interface with physiological systems to modulate fundamental cellular processes. One example of a diverse acting nanoparticle-based therapeutic is synthetic high-density lipoprotein (HDL) nanoparticles (NP), which have great potential for treating diseases of the ocular surface. Our group has developed a spherical HDL NP using a gold nanoparticle core. HDL NPs: (i) closely mimic the physical and chemical features of natural HDLs; (ii) contain apoA-I; (iii) bind with high-affinity to SR-B1, which is the major receptor through which HDL modulates cell cholesterol metabolism and controls the selective uptake of HDL cargo into cells; (iv) are non-toxic to cells and tissues; and (v) can be chemically engineered to display nearly any surface or core composition desired. With respect to the ocular surface, topical application of HDL NPs accelerates re-epithelization of the cornea following wounding, attenuates inflammation resulting from chemical burns and/or other stresses, and effectively delivers microRNAs with biological activity to corneal cells and tissues. HDL NPs will be the foundation of a new class of topical eye drops with great translational potential and exemplify the impact that nanoparticles can have in medicine.

High-density lipoproteins (HDL) are constitutionally dynamic nanoparticles that circulate in the blood. The biological functions of HDLs are impacted by interchangeable surface chemical components, like cholesterol and HDL-associated proteins. Current methods to quantify the chemical constituents of HDL are largely restricted to clinical or academic laboratories and require expensive instrumentation, and there is no commonality to the techniques required to detect and quantify different analytes (e.g., cholesterol versus HDL-associated protein). To potentially facilitate and streamline the analysis of HDL composition, we hypothesized that mixing native HDLs with similarly sized gold nanoparticles whose surfaces are endowed with phospholipids, called complementary nanoparticle scaffolds (CNS), would enable interparticle exchange of surface components. Then, easy isolation of the newly formed particles could be accomplished using benchtop centrifugation for subsequent measurement of HDL components exchanged to the surface of the CNS. As proof-of-concept, data demonstrate that CNS incubated with only a few microliters of human serum rapidly (1 h) sequester cholesterol and HDL-associated proteins with direct correlation to native HDLs. As such, data show that the CNS assay is a single platform for rapid isolation and subsequent detection of the surface components of native HDLs.

Background ApoAI (apolipoproteins AI) and apoAII (apolipoprotein AII) are structural and functional proteins of high‐density lipoproteins (HDL) which undergo post‐translational modifications at specific residues, creating distinct proteoforms. While specific post‐translational modifications have been reported to alter apolipoprotein function, the full spectrum of apoAI and apoAII proteoforms and their associations with cardiometabolic phenotype remains unknown. Herein, we comprehensively characterize apoAI and apoAII proteoforms detectable in serum and their post‐translational modifications and quantify their associations with cardiometabolic health indices. Methods and Results Using top‐down proteomics (mass‐spectrometric analysis of intact proteins), we analyzed paired serum samples from 150 CARDIA (Coronary Artery Risk Development in Young Adults) study participants from year 20 and 25 exams. Measuring 15 apoAI and 9 apoAII proteoforms, 6 of which carried novel post‐translational modifications, we quantified associations between percent proteoform abundance and key cardiometabolic indices. Canonical (unmodified) apoAI had inverse associations with HDL cholesterol and HDL‐cholesterol efflux, and positive associations with obesity indices (body mass index, waist circumference), and triglycerides, whereas glycated apoAI showed positive associations with serum glucose and diabetes mellitus. Fatty‐acid‒modified ApoAI proteoforms had positive associations with HDL cholesterol and efflux, and inverse associations with obesity indices and triglycerides. Truncated and dimerized proteoforms of apoAII were associated with HDL cholesterol (positively) and obesity indices (inversely). Several proteoforms had no significant associations with phenotype. Conclusions Associations between apoAI and AII and cardiometabolic indices are proteoform‐specific. These results provide “proof‐of‐concept” that precise chemical characterization of human apolipoproteins will yield improved insights into the complex pathways through which proteins signify and mediate health and disease.

The rapid spread of COVID-19 including recent emergence of new variants with its extreme range of pathologies create an urgent need to develop a versatile sensor for a rapid, precise, and highly sensitive detection of SARS-CoV-2. Herein, we report a microcantilever-based optical detection of SARS-CoV-2 antigenic proteins in just few minutes with high specificity by employing fluidic-atomic force microscopy (f-AFM) mediated nanomechanical deflection method. The corresponding antibodies against the target antigens were first grafted on the gold-coated microcantilever surface pre-functionalized with EDC-NHS chemistry for a suitable antibody-antigen interaction. Rapid detection of SARS-CoV-2 nucleocapsid (N) and spike (S1) receptor binding domain (RBD) proteins was first demonstrated at a clinically relevant concentration down to 1 ng/mL (33 pM) by real-time monitoring of nanomechanical signal induced by antibody-antigen interaction. More importantly, we further show high specific detection of antigens with nasopharyngeal swab specimens from patients pre-determined with qRT-PCR. The results take less than 5 min (swab to signal ≤5 min) and exhibit high selectivity and analytical sensitivity (LoD: 100 copies/ ml; 0.71 ng/ml of N protein). These findings demonstrate potential for nanomechanical signal transduction towards rapid antigen detection for early screening of SARS-CoV-2 and its related mutants.

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.

Primary tumours can establish long‐range communication with distant organs to transform them into fertile soil for circulating tumour cells to implant and proliferate, a process called pre‐metastatic niche (PMN) formation. Tumour‐derived extracellular vesicles (EV) are potent mediators of PMN formation due to their diverse complement of pro‐malignant molecular cargo and their propensity to target specific cell types (Costa‐Silva et al., 2015; Hoshino et al., 2015; Peinado et al., 2012; Peinado et al., 2017). While significant progress has been made to understand the mechanisms by which pro‐metastatic EVs create tumour‐favouring microenvironments at pre‐metastatic organ sites, comparatively little attention has been paid to the factors intrinsic to recipient cells that may modify the extent to which pro‐metastatic EV signalling is received and transduced. Here, we investigated the role of recipient cell cholesterol homeostasis in prostate cancer (PCa) EV‐mediated signalling and metastasis. Using a bone metastatic model of enzalutamide‐resistant PCa, we first characterized an axis of EV‐mediated communication between PCa cells and bone marrow that is marked by in vitro and in vivo PCa EV uptake by bone marrow myeloid cells, activation of NF‐κB signalling, enhanced osteoclast differentiation, and reduced myeloid thrombospondin‐1 expression. We then employed a targeted, biomimetic approach to reduce myeloid cell cholesterol in vitro and in vivo prior to conditioning with PCa EVs. Reducing myeloid cell cholesterol prevented the uptake of PCa EVs by recipient myeloid cells, abolished NF‐κB activity and osteoclast differentiation, stabilized thrombospondin‐1 expression, and reduced metastatic burden by 77%. These results demonstrate that cholesterol homeostasis in bone marrow myeloid cells regulates pro‐metastatic EV signalling and metastasis by acting as a gatekeeper for EV signal transduction.

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.

We report the identification of a molecular signature using the Scano-miR profiling platform based on the differential expression of circulating microRNAs (miRNA, miR) in serum samples specific to patients with very high-risk (VHR) prostate cancer (PCa). Five miRNA PCa biomarkers (miR-200c, miR-605, miR-135a*, miR-433, and miR-106a) were identified as useful for differentiating indolent and aggressive forms of PCa. All patients with VHR PCa in the study had elevated serum levels of miR-200c. Circulating miR-433, which was differentially expressed in patients with VHR versus low-risk (LR) forms of PCa, was not detectable by quantitative real-time PCR in samples from healthy volunteers. In blind studies, the five miRNA PCa biomarkers were able to differentiate patients with VHR PCas from those with LR forms as well as healthy individuals with at least 89% accuracy. Biological pathway analysis showed the predictive capability of these miRNA biomarkers for the diagnosis and prognosis of VHR aggressive PCa.

We report the development of a previously undescribed gold nanoparticle bio-barcode assay probe for the detection of prostate specific antigen (PSA) at 330 fg/mL, automation of the assay, and the results of a clinical pilot study designed to assess the ability of the assay to detect PSA in the serum of 18 men who have undergone radical prostatectomy for prostate cancer. Due to a lack of sensitivity, available PSA immunoassays are often not capable of detecting PSA in the serum of men after radical prostatectomy. This new bio-barcode PSA assay is ≈300 times more sensitive than commercial immunoassays. Significantly, with the barcode assay, every patient in this cohort had a measurable serum PSA level after radical prostatectomy. Patients were separated into categories based on PSA levels as a function of time. One group of patients showed low levels of PSA with no significant increase with time and did not recur. Others showed, at some point postprostatectomy, rising PSA levels. The majority recurred. Therefore, this new ultrasensitive assay points to significant possible outcomes: ( i ) The ability to tell patients, who have undetectable PSA levels with conventional assays, but detectable and nonrising levels with the barcode assay, that their cancer will not recur. ( ii ) The ability to assign recurrence earlier because of the ability to measure increasing levels of PSA before conventional tools can make such assignments. ( iii ) The ability to use PSA levels that are not detectable with conventional assays to follow the response of patients to adjuvant or salvage therapies.

An ultrasensitive method for detecting protein analytes has been developed. The polymerase chain reaction (PCR) and other forms of target amplification have enabled rapid advances in the development of powerful tools for detecting and quantifying DNA targets of interest for research, forensic, and clinical applications. Although gel electrophoresis was routinely used to analyze the results of the assay, in general, the scanometric method provided higher sensitivity and was easier to implement than the gel-based method. Importantly, one can eliminate the PCR step and still obtain a high sensitivity assay by using larger nanoparticles, which can support larger absolute amounts of barcode DNA. The bio-barcode method offers several advantages over current protein detection methods. Homogeneous mixing makes this assay faster than heterogeneous immuno-PCR systems and also can increase sensitivity because the capturing step is more efficient (the equilibrium can be pushed toward the captured protein state by increasing the concentration of magnetic microparticle probe, which cannot be done in the heterogeneous assay).

Top-down proteomics (TDP) allows precise determination/characterization of the different proteoforms derived from the expression of a single gene. In this study, we targeted apolipoprotein A-I (ApoA-I), a mediator of high-density-lipoprotein cholesterol efflux (HDL-E), which is inversely associated with coronary heart disease risk. Absolute ApoA-I concentration and allelic variation only partially explain interindividual HDL-E variation. Therefore, we hypothesize that differences in HDL-E are associated with the abundances of different ApoA-I proteoforms. Here, we present a targeted TDP methodology to characterize ApoA-I proteoforms in serum samples and compare their abundances between individuals. We characterized 18 ApoA-I proteoforms using selected-ion monitoring coupled to electron-transfer dissociation mass spectrometry. We then compared the abundances of these proteoforms between two groups of four participants, representing the individuals with highest and lowest HDL-E values within the Chicago Healthy Aging Study ( n = 420). Six proteoforms showed significantly ( p0.0005) higher intensity in high HDL-E individuals: canonical ApoA-I [fold difference (fd) = 1.17], carboxymethylated ApoA-I (fd = 1.24) and, with highest difference, four fatty acylated forms: palmitoylated (fd = 2.16), oleoylated (fd = 2.08), arachidonoylated (fd = 2.31) and one bearing two modifications: palmitoylation and truncation (fd = 2.13). These results demonstrate translational potential for targeted TDP in revealing, with high sensitivity, associations between interindividual proteoform variation and physiological differences underlying disease risk.

Myeloid-derived suppressor cells (MDSC) are innate immune cells that potently inhibit T cells. In cancer, novel therapies aimed to activate T cells can be rendered ineffective due to the activity of MDSCs. Thus, targeted inhibition of MDSCs may greatly enhance T-cell–mediated antitumor immunity, but mechanisms remain obscure. Here we show, for the first time, that scavenger receptor type B-1 (SCARB1), a high-affinity receptor for spherical high-density lipoprotein (HDL), is expressed by MDSCs. Furthermore, we demonstrate that SCARB1 is specifically targeted by synthetic high-density lipoprotein-like nanoparticles (HDL NP), which reduce MDSC activity. Using in vitro T-cell proliferation assays, data show that HDL NPs specifically bind SCARB1 to inhibit MDSC activity. In murine cancer models, HDL NP treatment significantly reduces tumor growth, metastatic tumor burden, and increases survival due to enhanced adaptive immunity. Flow cytometry and IHC demonstrate that HDL NP–mediated suppression of MDSCs increased CD8+ T cells and reduced Treg cells in the metastatic tumor microenvironment. Using transgenic mice lacking SCARB1, in vivo data clearly show that the HDL NPs specifically target this receptor for suppressing MDSCs. Ultimately, our data provide a new mechanism and targeted therapy, HDL NPs, to modulate a critical innate immune cell checkpoint to enhance the immune response to cancer. Mol Cancer Ther; 17(3); 686–97. ©2017 AACR.

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.

We report the development of potential theranostic agents for cardiovascular disease that are based on high-density lipoprotein-like magnetic nanostructures (HDL-MNS). The HDL-MNS offer prospects for diagnosis via noninvasive magnetic resonance imaging for anatomic detection and also serve as effective cholesterol efflux agents to address atherosclerotic vascular lesions. The HDL-MNS are synthesized by adding phospholipids and the HDL-defining apolipoprotein A1 to the surface of magnetic nanostructures (MNS) to mimic some aspects of natural HDL particles. From a diagnostic perspective, HDL-MNS show a 5 times higher contrast (r2 relaxivity up to 383 mM–1 s–1) in magnetic resonance imaging (MRI) than commercially available T2 MRI contrast agents (e.g., Ferumoxytol). Internalization of HDL-MNS by macrophage cells was confirmed by transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX), inductive-coupled plasma mass spectrometry (ICP-MS), and successfully imaged via MRI. Also, the H...

HDL efflux capacity and HDL particle size are associated with atherosclerotic CVD (ASCVD) events in middle-aged individuals; however, it is unclear whether these associations are present in older adults. We sampled 402 Chicago Healthy Aging Study participants who underwent a dedicated carotid MRI assessment for lipid-rich necrotic core (LRNC) plaque. We measured HDL particle size, HDL particle number, and LDL particle number with NMR spectroscopy, as well as HDL efflux capacity. We quantified the associations between HDL particle size and HDL efflux using adjusted linear regression models. We quantified associations between the presence of LRNC and HDL and LDL particle number, HDL particle size, and HDL efflux capacity using adjusted logistic regression models. HDL efflux capacity was directly associated with large (β = 0.037, P < 0.001) and medium (β = 0.0065, P = 0.002) HDL particle concentration and inversely associated with small (β = −0.0049, P = 0.018) HDL particle concentration in multivariable adjusted models. HDL efflux capacity and HDL particle number were inversely associated with prevalent LRNC plaque in unadjusted models (odds ratio: 0.5; 95% confidence interval: 0.26, 0.96), but not after multivariable adjustment. HDL particle size was not associated with prevalent LRNC. HDL particle size was significantly associated with HDL efflux capacity, suggesting that differences in HDL efflux capacity may be due to structural differences in HDL particles. Future research is needed to determine whether HDL efflux is a marker of ASCVD risk in older populations.

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.

Introduction: Significant clinical correlations have been observed between serum high-density lipoprotein (HDL) cholesterol and cancer risk, outcomes, and patient response to specific treatments. While the biological processes underlying these correlations remain unclear, evidence suggests that HDLs actively inhibit tumor progression through a variety of mechanisms. As a result, synthetic HDLs have emerged as attractive agents for targeted cancer therapy. Areas covered: We present a focused review of recent developments in the use of synthetic HDLs for cancer therapy, including roles in drug delivery, RNAi, monotherapy, and immunotherapy. In addition to historic references relevant to the field, we searched the following databases for recent articles published from January 1st, 2015 - May 1st, 2019: MEDLINE, Web of Science Core Collection, and Google Scholar. Expert opinion: Synthetic HDLs have already been used in human patients for cardiovascular disease, and have proven to be effective anticancer agents in pre-clinical testing, which should pave the way for future clinical trials in the setting of cancer. Given the growing notoriety of dysregulated cholesterol homeostasis as a key mechanism of cancer progression, and the immense success of synthetic HDLs in animal models, synthetic HDLs are well-poised to make significant strides toward the clinic as cancer therapy.

// Kaylin M. McMahon 1, * , Cristina Scielzo 2, 3, * , Nicholas L. Angeloni 1 , Elad Deiss-Yehiely 1 , Lydia Scarfo 2, 3 , Pamela Ranghetti 2, 3 , Shuo Ma 4 , Jason Kaplan 4, 5 , Federica Barbaglio 3 , Leo I. Gordon 4 , Francis J. Giles 4, 5 , C. Shad Thaxton 1, 4, 6, 7 , Paolo Ghia 2, 3 1 Department of Urology, Feinberg School of Medicine, Northwestern University, Tarry, Chicago, IL, USA 2 Universita Vita-Salute San Raffaele, Milan, Italy 3 Strategic Research Program On CLL and Unit of B cell Neoplasia, Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan, Italy 4 Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL, USA 5 Developmental Therapeutics Program of The Division of Hematology Oncology, Feinberg School of Medicine, Chicago, IL, USA 6 Simpson Querrey Institute (SQI) for BioNanotechnology, Chicago, IL, USA 7 International Institute for Nanotechnology, Evanston, IL, USA * These authors equally contributed to this work Correspondence to: C. Shad Thaxton, email: cthaxton003@md.northwestern.edu Paolo Ghia, email: ghia.paolo@hsr.it Keywords: lipoprotein, leukemia, scavenger receptor type B-I, nanoparticle, biomaterials Received: June 29, 2016 Accepted: December 26, 2016 Published: January 04, 2017 ABSTRACT Chronic lymphocytic leukemia (CLL) remains incurable despite the introduction of new drugs. Therapies targeting receptors and pathways active specifically in malignant B cells might provide better treatment options. For instance, in B cell lymphoma, our group has previously shown that scavenger receptor type B-1 (SR-B1), the high-affinity receptor for cholesterol-rich high-density lipoproteins (HDL), is a therapeutic target. As evidence suggests that targeting cholesterol metabolism in CLL cells may have therapeutic benefit, we examined SR-B1 expression in primary CLL cells from patients. Unlike normal B cells that do not express SR-B1, CLL cells express the receptor. As a result, we evaluated cholesterol-poor synthetic HDL nanoparticles (HDL NP), known for targeting SR-B1, as a therapy for CLL. HDL NPs potently and selectively induce apoptotic cell death in primary CLL cells. HDL NPs had no effect on normal peripheral blood mononuclear cells from healthy individuals or patients with CLL. These data implicate SR-B1 as a target in CLL and HDL NPs as targeted monotherapy for CLL.

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.

Toll-like receptor 4 (TLR4) plays a critical role in the innate immune system. Stimulation of TLR4 occurs upon binding lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls. Due to the potency of the induced inflammatory response, there is a growing interest in agents that can most proximally modulate this LPS/TLR4 interaction to prevent downstream cell signaling events and the production of inflammatory mediators. Building on the natural ability of human high-density lipoprotein (HDL) to bind LPS, we synthesized a suite of HDL-like nanoparticles (HDL-like NP). We identified one HDL-like NP that was particularly effective at decreasing TLR4 signaling caused by addition of purified LPS or Gram-negative bacteria to model human cell lines or primary human peripheral blood cells. The HDL-like NP functioned to inhibit TLR4-dependent inflammatory response to LPS derived from multiple bacterial species. Mechanistically, data show that the NP mainly functions by scavenging and neutralizing the LPS toxin. Taken together, HDL-like NPs constitute a powerful endotoxin scavenger with the potential to significantly reduce LPS-mediated inflammation.

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.

Introduction: Exosomes produced by neoplastic cells can promote the development of a pro-tumorigenic microenvironment via intercellular communication, and in some cases promote metastasis. The role of exosomes in the spread of prostate cancer, however, remains poorly understood. Here, we show that exosomes derived from enzalutamide resistant prostate cancer cells (EnzR exos) mediate intercellular communication with bone marrow-resident cells, alter the expression of extracellular matrix (ECM) proteins, and enhance metastasis in a clinically relevant mouse model of metastatic prostate cancer. Methods: Exosomes were isolated via ultracentrifugation of conditioned media from enzalutamide resistant CWR-R1 cells. Mice were primed with exosomes via three systemic injections of 10 ug exosomal protein. Exosome-mediated changes in bone marrow ECM composition were assessed via RNA sequencing of CD11b+ bone marrow cells and immunohistochemistry of formalin-fixed, decalcified, paraffin-embedded mouse femurs and tibias. For metastasis studies, intracardiac injection of luciferase-expressing enzalutamide resistant CWR-R1 cells into C.B.-17 SCID mice was used to establish metastatic tumor burden, and animals were monitored via bioluminescence imaging. Exosomal RNA sequencing was performed on EnzR exos and clinical specimens, with exosomes from normal prostate epithelial cells (PNT2) used as a control. Results: Mice primed with EnzR exos developed significantly enhanced metastatic tumor burden compared to unprimed controls. EnzR exo priming also led to decreased expression of thrombospondin-1 in the bone marrow compartment, and increased expression of versican, as determined by RNA sequencing and immunohistochemistry. Exosomal RNA sequencing revealed that five miRNAs were significantly enriched in EnzR exos compared to PNT2 exosome controls. One of these, miR-4443, was found to target thrombospondin-1. Prostate cancer patient serum samples were also enriched in miR-4443 over healthy controls, and exosomal miR-4443 correlated with disease progression. Conclusions: These data indicate that exosomes derived from enzalutamide resistant prostate cancer cells can alter ECM composition in the bone marrow compartment in a pro-tumorigenic manner. Evidence for the clinical relevance of these data was observed in both serum samples and bone biopsies from prostate cancer patients, demonstrating abundant serum exosomal miR-4443 and similarly altered bone marrow ECM composition in advanced prostate cancer patients. Exosomal miR-4443 may be a promising diagnostic and prognostic marker, as well as a potential therapeutic target. Citation Format: Stephen E. Henrich, Kaylin McMahon, Michael Plebanek, Fabio Tavora, Andre De Souza, Anthony Mega, Benedito Carneiro, C. Shad Thaxton. Prostate cancer exosomes alter extracellular matrix composition in bone marrow and enhance metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6204.

Translating new discoveries into viable therapies is dependent upon communication between scientists and medical professionals, especially in the emerging field of nanomedicine. Understanding of mechanisms on the cellular and molecular scale is commonly facilitated using 3D animation. However, this project sought to validate the knowledge transfer of complex biomedical information in nanomedicine using an alternative medium, the comic book. This medium has been effective for science communication but remains largely untested in medical education. In order to explore the differences in knowledge gain, engagement, and preference between comics and 3D animations, a comic book about a synthetic high-density lipoprotein gold nanoparticle’s apoptotic effects on lymphoma cells was created and compared to a 3D animation with identical content. Thirty-five individuals consisting of medical students, physicians, graduate students, and research scientists in the biomedical sciences were randomly shown the comic or the animation following a pretest. A posttest and preferences survey was conducted afterward. Results indicated that both the comic and animation were similarly effective at increasing knowledge about the HDL AuNP mechanism of action and had a similar level of engagement.

Trinucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntington9s disease (HD). Pathology is caused by protein and RNA generated from the TNR regions including small siRNA‐sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR are toxic to cancer cells by targeting genes that contain long reverse complementary TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the six members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG TNR‐based siRNAs induce cell death in vitro in all tested cancer cell lines and slow down tumor growth in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR‐based siRNAs as a novel form of anticancer reagents.

Disorders of blood vessels cause a range of severe health problems. As a powerful vasodilator and cellular second messenger, nitric oxide (NO) is known to have beneficial vascular functions. However, NO typically has a short half-life and is not specifically targeted. On the other hand, high-density lipoproteins (HDLs) are targeted natural nanoparticles (NPs) that transport cholesterol in the systemic circulation and whose protective effects in vascular homeostasis overlap with those of NO. Evolving the AuNP-templated HDL-like nanoparticles (HDL NPs), a platform of bioinspired HDL, we set up a targeted biomimetic nanotherapy for vascular disease that combines the functions of NO and HDL. A synthetic S-nitrosylated (SNO) phospholipid (1,2-dipalmitoyl-sn-glycero-3-phosphonitrosothioethanol) was synthesized and assembled with S-containing phospholipids and the principal protein of HDL, apolipoprotein A-I, to construct NO-delivering HDL-like particles (SNO HDL NPs). SNO HDL NPs self-assemble under mild conditions similar to natural processes, avoiding the complex postassembly modification needed for most synthetic NO-release nanoparticles. In vitro data demonstrate that the SNO HDL NPs merge the functional properties of NO and HDL into a targeted nanocarrier. Also, SNO HDL NPs were demonstrated to reduce ischemia/reperfusion injury in vivo in a mouse kidney transplant model and atherosclerotic plaque burden in a mouse model of atherosclerosis. Thus, the synthesis of SNO HDL NPs provides not only a bioinspired nanotherapy for vascular disease but also a foundation to construct diversified multifunctional platforms based on HDL NPs in the future.

Trinucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntington’s disease (HD). Pathology is caused by protein and RNA generated from the TNR regions including small siRNA-sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR are toxic to cancer cells by targeting genes that contain long reverse complimentary TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the 6 members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG TNR-based siRNAs induce cell deathin vitroin all tested cancer cell lines and slow down tumor growth in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR-based siRNAs as a novel form of anti-cancer reagents.

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.

The ability of HDL to support macrophage cholesterol efflux is an integral part of its atheroprotective action. Augmenting this ability, especially when HDL cholesterol efflux capacity from macrophages is poor, represents a promising therapeutic strategy. One approach to enhancing macrophage cholesterol efflux is infusing blood with HDL mimics. Previously, we reported the synthesis of a functional mimic of HDL (fmHDL) that consists of a gold nanoparticle template, a phospholipid bilayer, and apo A-I. In this work, we characterize the ability of fmHDL to support the well-established pathways of cellular cholesterol efflux from model cell lines and primary macrophages. fmHDL received cell cholesterol by unmediated (aqueous) and ABCG1- and scavenger receptor class B type I (SR-BI)-mediated diffusion. Furthermore, the fmHDL holoparticle accepted cholesterol and phospholipid by the ABCA1 pathway. These results demonstrate that fmHDL supports all the cholesterol efflux pathways available to native HDL and thus, represents a promising infusible therapeutic for enhancing macrophage cholesterol efflux. fmHDL accepts cholesterol from cells by all known pathways of cholesterol efflux: unmediated, ABCG1- and SR-BI-mediated diffusion, and through ABCA1.

Metastatic cancers produce exosomes that condition pre-metastatic niches in remote microenvironments to favor metastasis. In contrast, here we show that exosomes from poorly metastatic melanoma cells can potently inhibit metastasis to the lung. These “non-metastatic” exosomes stimulate an innate immune response through the expansion of Ly6Clow patrolling monocytes (PMo) in the bone marrow, which then cause cancer cell clearance at the pre-metastatic niche, via the recruitment of NK cells and TRAIL-dependent killing of melanoma cells by macrophages. These events require the induction of the Nr4a1 transcription factor and are dependent on pigment epithelium-derived factor (PEDF) on the outer surface of exosomes. Importantly, exosomes isolated from patients with non-metastatic primary melanomas have a similar ability to suppress lung metastasis. This study thus demonstrates that pre-metastatic tumors produce exosomes, which elicit a broad range of PMo-reliant innate immune responses via trigger(s) of immune surveillance, causing cancer cell clearance at the pre-metastatic niche., Exosomes are extracellular vesicles that can favor tumor development and metastasis. Here, the authors show that cancer exosomes may also exert a suppressive function; in fact, exosomes from non-metastatic melanoma cells can lead to the recruitment of patrolling monocytes, which clear cancer cells at the pre-metastatic niche.

// Andrea E. Murmann 1 , Kaylin M. McMahon 3,6 , Ashley Haluck-Kangas 1 , Nandini Ravindran 1 , Monal Patel 1 , Calvin Y. Law 1 , Sonia Brockway 1 , Jian-Jun Wei 4 , C. Shad Thaxton 3,5,6,7 and Marcus E. Peter 1,2,5 1 Department of Medicine/Division of Hematology/Oncology, Feinberg School of Medicine, Chicago, IL, USA 2 Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Chicago, IL, USA 3 Department of Urology, Feinberg School of Medicine, Chicago, IL, USA 4 Department of Pathology and Obstetrics and Gynecology, Feinberg School of Medicine, Chicago, IL, USA 5 Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Northwestern University, Chicago, IL, USA 6 Simpson Querrey Institute (SQI) for BioNanotechnology, Chicago, IL, USA 7 International Institute for Nanotechnology, Evanston, IL, USA Correspondence to: Marcus E. Peter, email: // Keywords : RNAi, Fas, ovarian cancer, TLP nanoparticles, cell death Received : January 23, 2017 Accepted : September 13, 2017 Published : October 04, 2017 Abstract The death receptor CD95/Fas can be activated by immune cells to kill cancer cells. shRNAs and siRNAs derived from CD95 or CD95 ligand (CD95L) are highly toxic to most cancer cells. We recently found that these sh/siRNAs kill cancer cells in the absence of the target by targeting the 3’UTRs of critical survival genes through canonical RNAi. We have named this unique form of off-target effect DISE (for death induced by survival gene elimination). DISE preferentially kills transformed cells and cancer stem cells. We demonstrate that DISE induction occurs in cancer cells in vivo after introducing a lentiviral CD95L derived shRNA (shL3) into HeyA8 ovarian cancer cells grown as i.p. xenografts in mice, when compared to a scrambled shRNA. To demonstrate the possibility of therapeutically inducing DISE, we coupled siRNAs to templated lipoprotein nanoparticles (TLP). In vitro , TLPs loaded with a CD95L derived siRNA (siL3) selectively silenced a biosensor comprised of Venus and CD95L ORF and killed ovarian cancer cells. In vivo , two siRNA-TLPs (siL2-TLP and siL3-TLP) reduced tumor growth similarly as observed for cells expressing the shL3 vector. These data suggest that it is possible to kill ovarian cancer cells in vivo via DISE induction using siRNA-TLPs.

Introduction: Exosomes are 30-150 nm membrane-bound vesicles that mediate intercellular communication and are secreted in abundance by neoplastic cells. Tumor-derived exosomes are capable of transmitting pro-malignant signals to target cells at local or distant sites to enhance tumor invasiveness and promote metastasis. A sub-set of this pro-malignant intercellular communication is dependent upon target cell cholesterol homeostasis. Here, we show that prostate cancer exosome communication with myeloid cells can be inhibited by treatment with high density lipoprotein mimetic nanoparticles (HDL NPs), which reduce cholesterol in myeloid cells in a targeted fashion. Methods: Exosomes were isolated by ultracentrifugation of conditioned media from enzalutamide resistant CWR-R1 cells and used for all experiments. In vitro assays were conducted using murine bone marrow macrophages obtained from culture of total murine bone marrow in M-CSF for 7 days. Confocal microscopy and flow cytometry were used for in vitro and in vivo uptake assays. Osteoclastogenesis assays were performed using a commercially available TRAP staining kit (Sigma-Aldrich). NF-kB signaling experiments were performed using a reporter human monocyte cell line (THP1-Dual). HDL NPs were synthesized using 5 nm gold nanoparticle templates, apolipoprotein A-1, and phospholipids. Results: HDL NPs were found to inhibit the cellular uptake of prostate cancer exosomes in mouse bone marrow macrophages in vitro. Furthermore, HDL NPs inhibited exosome-induced osteoclastogenesis and exosome-induced monocyte NF-kB signaling. Finally, HDL NP-mediated inhibition of exosome communication was found to be dependent upon scavenger receptor type B-1 (SR-B1). SR-B1 was shown to be expressed ubiquitously in mouse bone marrow macrophages; and HDL NPs were unable to inhibit exosome communication in bone marrow macrophages derived from SR-B1-/- mouse bone marrow. Conclusion: These data demonstrate that HDL NPs inhibit prostate cancer exosome communication with murine myeloid cells, as evidenced by cellular uptake, osteoclastogenesis, and NF-kB signaling. Moreover, SR-B1 is required for HDL NP inhibition of PCa exosome communication. In sum, these results indicate that target cell cholesterol homeostasis may be important for exosome-mediated signaling in prostate cancer, particularly in immune cells and in the bone microenvironment, and that HDL NPs are potent inhibitors of PCa exosome-mediated signaling. Citation Format: Stephen E. Henrich, Kaylin M. McMahon, Michael P. Plebanek, C. Shad Thaxton. High density lipoprotein mimics inhibit prostate cancer exosome-mediated communication with myeloid cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3005.

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-κB (NF-κB), causing production of gene products that have detrimental effects on β-cell survival and function. We hypothesized that small interfering RNA targeting of IKKβ, a crucial kinase in the NF-κB 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 IKKβ IKKβ SNA-NCs). We treated isolated islets with IKKβ SNA-NCs and assessed the functional consequences of IKKβ knockdown in vitro and after intraportal transplantation in mice. RESULTS Treatment of freshly isolated mouse islets with IKKβ SNA-NCs reduced constitutive IKKβ expression and protected against proinflammatory cytokine-induced NF-κB activation, resulting in improved cell viability and decreased expression of gene products associated with β-cell dysfunction. Intraportal transplantation of a marginal mass (50 islets) of syngeneic islets treated with nanoparticle conjugates targeting IKKβ 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-κB activation ameliorates the detrimental effects of host cytokines and demonstrates that preconditioning freshly isolated islets in culture with IKKβ SNA-NCs may be a promising therapy to enhance islet graft function and survival after transplantation.

High density lipoproteins (HDLs) are dynamic natural nanoparticles best known for their role in cholesterol transport and the inverse correlation that exists between blood HDL levels and the risk of developing coronary heart disease. In addition, enhanced HDL-cholesterol uptake has been demonstrated in several human cancers. As such, the use of HDL as a therapeutic and as a vehicle for systemic delivery of drugs and as imaging agents is increasingly important. HDLs exist on a continuum from the secreted HDL-scaffolding protein, apolipoprotein A-1 (Apo A1), to complex, spherical "mature" HDLs. Aspects of HDL particles including their size, shape, and surface chemical composition are being recognized as critical to their diverse biological functions. Here we review HDL biology; strategies for synthesizing HDLs; data supporting the clinical use and benefit of directly administered HDL; a rationale for developing synthetic methods for spherical, mature HDLs; and, the potential to employ HDLs as therapies, imaging agents, and drug delivery vehicles. Importantly, methods that utilize nanoparticle templates to control synthetic HDL size, shape, and surface chemistry are highlighted.

The death receptor CD95/Fas can be activated by immune cells to kill cancer cells. shRNAs and siRNAs derived from CD95 or CD95 ligand (CD95L) are highly toxic to most cancer cells. We recently found that these sh/siRNAs kill cancer cells in the absence of the target by targeting the 3’UTRs of critical survival genes through canonical RNAi. We have named this unique form of off-target effect DISE (for death induced by survival gene elimination). DISE preferentially kills transformed cells and cancer stem cells. We demonstrate that DISE induction occurs in cancer cells in vivo after introducing a lentiviral CD95L derived shRNA (shL3) into HeyA8 ovarian cancer cells grown as i.p. xenografts in mice, when compared to a scrambled shRNA. To demonstrate the possibility of therapeutically inducing DISE, we coupled siRNAs to templated lipoprotein nanoparticles (TLP). In vitro, TLPs loaded with a CD95L derived siRNA (siL3) selectively silenced a biosensor comprised of Venus and CD95L ORF and killed ovarian cancer cells. In vivo, two siRNA-TLPs (siL2-TLP and siL3-TLP) reduced tumor growth similarly as observed for cells expressing the shL3 vector. These data suggest that it is possible to kill ovarian cancer cells in vivo via DISE induction using siRNA-TLPs.

Atherosclerosis is one of the leading causes of morbidity and mortality worldwide. Nanotechnology has provided the possibility of designing nanoparticles that can translocate through tissues and home in to atherosclerotic plaques to achieve desired diagnostic, therapeutic, theranostic or 'theralivery' outcomes. Although nanomedicine approaches have demonstrated exciting possibilities, clinical reality is still distant and challenges are aplenty, such as specificity of targeting and nanotoxicity. Nevertheless, developments in formulations, delivery strategies and experimental models over the coming years will generate new knowledge to define the true potential of this field. This review discusses the most recent developments, current challenges and future possibilities.

High-density lipoprotein (HDL) plays an important role in the transport and metabolism of cholesterol. Mimics of HDL are being explored as potentially powerful therapeutic agents for removing excess cholesterol from arterial plaques. Gold nanoparticles (AuNPs) functionalized with apolipoprotein A-I and with the lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] have been demonstrated to be robust acceptors of cellular cholesterol. However, detailed structural information about this functionalized HDL AuNP is still lacking. In this study, we have used X-ray photoelectron spectroscopy and lecithin/cholesterol acyltransferase activation experiments together with coarse-grained and all-atom molecular dynamics simulations to model the structure and cholesterol uptake properties of the HDL AuNP construct. By simulating different apolipoprotein-loaded AuNPs, we find that lipids are oriented differently in regions with and without apoA-I. We also show that in this functionalized HDL AuNP, the distribution of cholesteryl ester maintains a reverse concentration gradient that is similar to the gradient found in native HDL.

High-density lipoproteins (HDL) are a class of natural nanostructures found in the blood and are composed of lipids, proteins, and nucleic acids (e.g. microRNA). Their size, which appears to be well-suited for both tissue penetration/retention as well as payload delivery, long circulation half-life, avoidance of endosomal sequestration, and potential low toxicity are all excellent properties to model in a drug delivery vehicle. In this review, we consider high-density lipoproteins for therapeutic delivery systems. First we discuss the structure and function of natural HDL, describing in detail its biogenesis and transformation from immature, discoidal forms, to more mature, spherical forms. Next we consider features of HDL making them suitable vehicles for drug delivery. We then describe the use of natural HDL, discoidal HDL analogs, and spherical HDL analogs to deliver various classes of drugs, including small molecules, lipids, and oligonucleotides. We briefly consider the notion that the drug delivery vehicles themselves are therapeutic, constituting entities that exhibit “theralivery.” Finally, we discuss challenges and future directions in the field.

Exosomes are produced by cells to mediate intercellular communication and have been shown to perpetuate diseases, including cancer. New tools are needed to understand exosome biology, detect exosomes from specific cell types in complex biological media and to modify exosomes. Our data demonstrate a cellular pathway whereby membrane-bound scavenger receptor type B-1 (SR-B1) in parent cells becomes incorporated into exosomes. We tailored synthetic HDL-like nanoparticles (HDL NP), high-affinity ligands for SR-B1, to carry a fluorescently labeled phospholipid. Data show SR-B1-dependent transfer of the fluorescent phospholipid from HDL NPs to exosomes. Modified exosomes are stable in serum and can be directly detected using flow cytometry. As proof-of-concept, human serum exosomes were found to express SR-B1 and HDL NPs can be used to label and isolate them. Ultimately, we discovered a natural cellular pathway and nanoparticle-receptor pair that enables exosome modulation, detection and isolation.

We report the development of a novel Scanometric MicroRNA (Scano-miR) platform for the detection of relatively low abundance miRNAs with high specificity and reproducibility. The Scano-miR system was able to detect 1 fM concentrations of miRNA in serum with single nucleotide mismatch specificity. Indeed, it provides increased sensitivity for miRNA targets compared to molecular fluorophore-based detection systems, where 88% of the low abundance miRNA targets could not be detected under identical conditions. The application of the Scano-miR platform to high density array formats demonstrates its utility for high throughput and multiplexed miRNA profiling from various biological samples. To assess the accuracy of the Scano-miR system, we analyzed the miRNA profiles of samples from victims of prostate cancer (CaP), the most common noncutaneous malignancy and the second leading cause of cancer death among American men. The platform exhibits 98.8% accuracy when detecting deregulated miRNAs involved in CaP, which demonstrates its potential utility in profiling and identifying clinical and research biomarkers.

We report a gold nanoparticle-templated high density lipoprotein (HDL AuNP) platform for gene therapy which combines lipid-based nucleic acid transfection strategies with HDL biomimicry. For proof-of-concept, HDL AuNPs are shown to adsorb antisense cholesterylated DNA. The conjugates are internalized by human cells, can be tracked within cells using transmission electron microscopy (TEM), and regulate target gene expression. Overall, the ability to directly image the AuNP core within cells, the chemical tailorability of the HDL AuNP platform, and the potential for cell-specific targeting afforded by HDL biomimicry make this platform appealing for nucleic acid delivery.

Atherosclerosis is the disease mechanism responsible for coronary heart disease (CHD), the leading cause of death worldwide. One strategy to combat atherosclerosis is to increase the amount of circulating high density lipoproteins (HDL), which transport cholesterol from peripheral tissues to the liver for excretion. The process, known as reverse cholesterol transport, is thought to be one of the main reasons for the significant inverse correlation observed between HDL blood levels and the development of CHD. This article highlights the most common strategies for treating atherosclerosis using HDL. We further detail potential treatment opportunities that utilize nanotechnology to increase the amount of HDL in circulation. The synthesis of biomimetic HDL nanostructures that replicate the chemical and physical properties of natural HDL provides novel materials for investigating the structure-function relationships of HDL and for potential new therapeutics to combat CHD.

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.