PIK-75 (F7) is a potent multikinase inhibitor that targets p110α, DNA-PK, and p38γ. PIK-75 has shown potential as a therapy in preclinical cancer models, but it has not been used in the clinic, at least in part, due to limited solubility. We therefore developed a nanoparticle to encapsulate PIK-75 and enable targeted cellular delivery. Scavenger receptor class B type 1 (SR-B1) is often overexpressed in cancer compared with normal cells, which enables targeting by synthetic lipid nanoparticles with some features of native high-density lipoprotein (HDL), the natural ligand of SR-B1. We investigated the use of organic core (oc) molecular platforms to synthesize HDL-like nanoparticles (oc-HDL NP). Employing an oc, we successfully formulated PIK-75 into oc-HDL NPs. The PIK-75 loaded oc-HDL NP (PIK-75 oc-HDL NP), comprising ∼20 PIK-75 molecules/NP, has similar size, surface charge, and surface composition as oc-HDL NP and natural human HDL. Using prostate cancer (PCa) and cutaneous T-cell lymphoma (CTCL) models known to be sensitive to inhibitors of p110α and p38γ, respectively, we found that PIK-75 oc-HDL NPs specifically targeted SR-B1 to deliver PIK-75 and potently induced cell death in vitro in PCa and CTCL and in vivo in a murine PCa model. Additionally, we found that PIK-75 oc-HDL NP, but not free PIK-75 or oc-HDL NP alone, reduced the IC50 in the NCI-60 cell line panel and additional pancreatic cancer cell lines. These data demonstrate the first example of drug-loaded oc-HDL NP that actively target SR-B1 and kill cancer cells in vitro and in vivo, encouraging further development and translation to human patients.

Introduction: Patients with relapse/refractory cutaneous T cell lymphomas (CTCL) have limited therapeutic options. p38γ, part of the mitogen-activated protein kinases (MAPK) pathway, has been identified as a critical driver of CTCL. PIK-75, a known p110α inhibitor, was found to inhibit p38γ and induced cell death in CTCL cells in vitro and in vivo tumor xenograft models. However, PIK-75 is very hydrophobic and difficult to systemically deliver and achieve clinical efficacy. Therefore, designing a carrier to formulate and enable targeted cellular delivery of PIK-75 is a logical next step to facilitate translation. We, and others, have shown that scavenger receptor type B1 (SR-B1) is often overexpressed in hematologic malignancies and can be targeted by synthetic lipid nanoparticles for efficient drug delivery. Our group pioneered the use of inorganic and organic core (oc) "templates” to synthesize lipid nanoparticles (LNP) that target SR-B1. The ocLNPs are an ideal construct for the delivery of hydrophobic drugs to malignant cells. Therefore, we hypothesized that ocLNPs could be adapted to formulate PIK-75 and deliver this drug via SR-B1 to CTCL cells to induce cell death. Methods: A suite of organic core molecules were synthesized and tested for their ability to support the synthesis of PIK-75 loaded ocLNPs. The PIK-75 ocLNPs were synthesized by combining an organic core with PIK-75, phospholipids, and apolipoprotein A-I. The drug loaded ocLNPs were purified by dialysis and filtration. PIK-75 ocLNPs were characterized for size (size exclusion chromatography, dynamic light scattering and electron microscopy), surface charge (zeta potential) and drug loading. SR-B1 expression in primary CTCL samples was assessed by RNA sequencing. SR-B1 positive CTCL cell lines (HH and HuT78), and SR-B1 positive normal cells, HepG2 (hepatocytes) and THP-1 (macrophages), as well as SR-B1 negative PIK-75-sensitive U266B1 (myeloma) cells were used for these studies. Expression of SR-B1 in all cell lines was confirmed by western blot and flow cytometry. Cell viability was measured using the MTS assay. An SR-B1 blocking antibody was used to demonstrate the necessity of SR-B1 for PIK-75 ocLNP targeting and drug delivery. Results: Nineteen out of 49 primary patient lesional CTCL skin samples (38.8%) demonstrated SR-B1 over-expression, defined as an expression level greater than two standard deviations above the mean expression measured in normal, CD3+ peripheral T cells using RNAseq data. Fourteen of the 19 samples (74%) were from patients with advanced disease (stage III and IV). PIK-75 was successfully incorporated into ocLNPs. Data showed that ~16 PIK-75 molecules were loaded per ocLNP. PIK-75 ocLNPs induced significant cell death in SR-B1 positive CTCL cell lines HH (IC50 = 3.95 nM) and HuT 78 (IC50 = 6.33 nM), while treatment of an PIK-75 sensitive, SR-B1 negative myeloma cell line (U266B1) with PIK-75 ocLNPs resulted in minimal cell death. Treatment of HH cells with PIK-75 ocLNPs for a 2 hour "pulse” followed by 72 hours of additional culture was completed to test active SR-B1 targeting. Data reveal significant cell death, which was significantly reduced by the addition of an SR-B1 blocking antibody (10 nM PIK-75 ocLNPs, viability 31.6% ± 4.3% w/o blocking antibody vs viability 83.6% ± 8.4% w/ blocking antibody). Treatment of hepatocytes and macrophages, both known to express SR-B1, with PIK-75 ocLNPs revealed no cell death even after 72 hours of culture. Conclusions: ocLNPs can be successfully loaded with drug cargo, in this case hydrophobic PIK-75, enabling active delivery via SR-B1 in CTCL. PIK-75 ocLNPs potently induced cell death in CTCL cells while having minimal negative effects on hepatocytes or macrophages. These results provide proof-of-principle that ocLNPs can be successfully leveraged to deliver hydrophobic small molecule drugs by actively targeting SR-B1.

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.

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.

Abstract 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.

The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in Wuhan, China in late 2019 and has now caused a global pandemic. The disease caused by SARS-CoV-2 is known as COVID-19. To date, few treatments for COVID-19 have proven effective, and the current standard of care is primarily supportive. As a result, novel therapeutic strategies are in high demand. Viral entry into target cells is frequently sensitive to cell membrane lipid composition and membrane organization. Evidence suggests that cell entry of SARS-CoV-2 is most efficient when the target cell plasma membrane is replete with cholesterol; and recent data implicate cholesterol flux through the high-affinity receptor for cholesterol-rich high-density lipoprotein (HDL), called scavenger receptor type B-1 (SR-B1), as critical for SARS-CoV-2 entry. Here, we demonstrate that a cholesterol-poor synthetic biologic high-density lipoprotein (HDL NP) targets SR-B1 and inhibits cell entry of a SARS-CoV-2 spike protein pseudovirus. Human cells expressing SR-B1 are susceptible to SARS-CoV-2 infection, and viral entry can be inhibited by 50-80% using HDL NPs in an SR-B1-dependent manner. These results indicate that HDL NP targeting of SR-B1 is a powerful potential therapy to combat COVID-19 and other viral diseases.

Hierarchical assemblies of proteins into fibrillar structures occur in both physiologic and pathologic extracellular spaces and often involve interactions between oppositely charged peptide domains. However, the interplay between tertiary structure dynamics and quaternary hierarchical structure formation remains unclear. In this work, we investigate supramolecular mimics of these systems by mixing one-dimensional assemblies of small alkylated peptides bearing opposite charge and varying in peptide sequence. We found that assemblies with weak cohesive interactions readily create fibrous superstructures of bundled filaments as molecules redistribute upon mixing. Low cohesion allows molecules to escape from the original assemblies and exchange dynamics help them reassemble into electrostatically stable bundles. However, we also found that kinetic barriers can be encountered in these systems and limit formation of the hierarchical structures at pH values where charge densities are high. Increasing intermolecular cohesion using longer peptide sequences that form stable β-sheets was found to suppress superstructure formation. Our findings suggest that low internal cohesion in protein systems could facilitate the conformational rearrangements required to create hierarchical structures.

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.

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.

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.

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 1(st), 2015 - May 1(st), 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.

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.

Schlemm's canal (SC) plays central roles in ocular physiology. These roles depend on the molecular phenotypes of SC endothelial cells (SECs). Both the specific phenotype of SECs and development of SC remain poorly defined. To allow a modern and extensive analysis of SC and its origins, we developed a new whole-mount procedure to visualize its development in the context of surrounding tissues. We then applied genetic lineage tracing, specific-fluorescent reporter genes, immunofluorescence, high-resolution confocal microscopy, and three-dimensional (3D) rendering to study SC. Using these techniques, we show that SECs have a unique phenotype that is a blend of both blood and lymphatic endothelial cell phenotypes. By analyzing whole mounts of postnatal mouse eyes progressively to adulthood, we show that SC develops from blood vessels through a newly discovered process that we name "canalogenesis." Functional inhibition of KDR (VEGFR2), a critical receptor in initiating angiogenesis, shows that this receptor is required during canalogenesis. Unlike angiogenesis and similar to stages of vasculogenesis, during canalogenesis tip cells divide and form branched chains prior to vessel formation. Differing from both angiogenesis and vasculogenesis, during canalogenesis SECs express Prox1, a master regulator of lymphangiogenesis and lymphatic phenotypes. Thus, SC development resembles a blend of vascular developmental programs. These advances define SC as a unique vessel with a combination of blood vascular and lymphatic phenotypes. They are important for dissecting its functions that are essential for ocular health and normal vision.