Brian M. Barth

Near-Infrared Emitting Fluorophore-Doped Calcium Phosphate Nanoparticles for In Vivo Imaging of Human Breast Cancer

Early detection is a crucial element for the timely diagnosis and successful treatment of all human cancers but is limited by the sensitivity of current imaging methodologies. We have synthesized and studied bioresorbable calcium phosphate nanoparticles (CPNPs) in which molecules of the near-infrared (NIR) emitting fluorophore, indocyanine green (ICG), are embedded. The ICG-CPNPs demonstrate exceptional colloidal and optical characteristics. Suspensions consisting of 16 nm average diameter particles are colloidally stable in physiological solutions (phosphate buffered 0.15 M saline (PBS), pH 7.4) with carboxylate or polyethylene glycol (PEG) surface functionality. ICG-doped CPNPs exhibit significantly greater intensity at the maximum emission wavelength relative to the free constituent fluorophore, consistent with the multiple molecules encapsulated per particle. The quantum efficiency per molecule of the ICG-CPNPs is 200% greater at 0.049 +/- 0.003 over the free fluorophore in PBS. Photostability based on fluorescence half-life of encapsulated ICG in PBS is 500% longer under typical clinical imaging conditions relative to the free dye. PEGylated ICG-CPNPs accumulate in solid, 5 mm diameter xenograft breast adenocarcinoma tumors via enhanced retention and permeability (EPR) within 24 h after systemic tail vein injection in a nude mouse model. Ex situ tissue imaging further verifies the facility of the ICG-CPNPs for deep-tissue imaging with NIR signals detectable from depths up to 3 cm in porcine muscle tissue. Our ex vivo and in vivo experiments verify the promise of the NIR CPNPs for diagnostic imaging in the early detection of solid tumors.

Targeted Indocyanine-Green-Loaded Calcium Phosphosilicate Nanoparticles for In Vivo Photodynamic Therapy of Leukemia

Leukemia is one of the most common and aggressive adult cancers, as well as the most prevalent childhood cancer. Leukemia is a cancer of the hematological system and can be divided into a diversity of unique malignancies based on the onset of the disease as well as the specific cell lineages involved. Cancer stem cells, including recently identified leukemia stem cells (LSCs), are hypothesized to be responsible for cancer development, relapse, and resistance to treatment, and new therapeutics targeting these cellular populations are urgently needed. Nontoxic and nonaggregating calcium phosphosilicate nanoparticles (CPSNPs) encapsulating the near-infrared fluoroprobe indocyanine green (ICG) were recently developed for diagnostic imaging and drug delivery as well as for photodynamic therapy (PDT) of solid tumors. Prior studies revealed that specific targeting of CPSNPs allowed for enhanced accumulation within breast cancer tumors, via CD71 targeting, or pancreatic cancer tumors, via gastrin receptor targeting. In the present study, ICG-loaded CPSNPs were evaluated as photosensitizers for PDT of leukemia. Using a novel bioconjugation approach to specifically target CD117 or CD96, surface features enhanced on leukemia stem cells, in vitro ICG-CPSNP PDT of a murine leukemia cell line and human leukemia samples were dramatically improved. Furthermore, the in vivo efficacy of PDT was dramatically enhanced in a murine leukemia model by utilizing CD117-targeted ICG-CPSNPs, resulting in 29% disease-free survival. Altogether, this study demonstrates that leukemia-targeted ICG-loaded CPSNPs offer the promise to effectively treat relapsing and multidrug-resistant leukemia and to improve the life of leukemia patients.

Bioconjugation of calcium phosphosilicate composite nanoparticles for selective targeting of human breast and pancreatic cancers in vivo.

The early diagnosis of cancer is the critical element in successful treatment and long-term favorable patient prognoses. The high rate of mortality is mainly attributed to the tendency for late diagnoses as symptoms may not occur until the disease has metastasized, as well as the lack of effective systemic therapies. Late diagnosis is often associated with the lack of timely sensitive imaging modalities. The promise of nanotechnology is presently limited by the inability to simultaneously seek, treat, and image cancerous lesions. This study describes the design and synthesis of fluorescent calcium phosphosilicate nanocomposite particles (CPNPs) that can be systemically targeted to breast and pancreatic cancer lesions. The CPNPs are a approximately 20 nm diameter composite composed of an amorphous calcium phosphate matrix doped with silicate in which a near-infrared imaging agent, indocyanine green (ICG), is embedded. In the present studies, we describe and validate CPNP bioconjugation of human holotransferrin, anti-CD71 antibody, and short gastrin peptides via an avidin-biotin or a novel PEG-maleimide coupling strategy. The conjugation of biotinylated human holotransferrin (diferric transferrin) and biotinylated anti-CD71 antibody (anti-transferrin receptor antibody) to avidin-conjugated CPNPs (Avidin-CPNPs) permits targeting of transferrin receptors, which are highly expressed on breast cancer cells. Similarly, the conjugation of biotinylated pentagastrin to Avidin-CPNPs and decagastrin (gastrin-10) to PEG-CPNPs via PEG-maleimide coupling permits targeting of gastrin receptors, which are overexpressed in pancreatic cancer lesions. These bioconjugated CPNPs have the potential to perform as a theranostic modality, simultaneously enhancing drug delivery, targeting, and imaging of breast and pancreatic cancer tumors.

Nanoliposomal ceramide prevents in vivo growth of hepatocellular carcinoma

Background and objectives Hepatocellular carcinoma (HCC) affects an increasing number of people worldwide. The poor survival rate of patients with HCC is manifested by an aggressive and metastatic phenotype, as well as a poor response to common therapeutic strategies. The purpose of this study was to evaluate the efficacy of nanoliposomal C6-ceramide as an antineoplastic agent in an in vivo model of human HCC. Methods The growth-arresting and pro-apoptotic properties of nanoliposomal C6-ceramide were first evaluated in vitro in human SK-HEP-1 cells by assessing cellular viability, caspase 3/7 activity, annexin-V expression, DNA fragmentation, cell cycle distribution and AKT phosphorylation. SK-HEP-1 cells were then engrafted subcutaneously into athymic nude mice and nanoliposomal C6-ceramide was administered by tail vein injection. Tumour size was monitored over time, followed by excision of tumours to evaluate tumour vascularisation, proliferation, apoptosis and cellular signalling. Results Nanoliposomal C6-ceramide, but not ghost (no ceramide) nanoliposomes, induced apoptotic cell death of SK-HEP-1 cells in vitro, concomitant with an accumulation of cells in the G2 phase of the cell cycle and decreased phosphorylation of AKT. Systemic administration of nanoliposomal C6-ceramide to mice engrafted with SK-HEP-1 tumours reduced tumour vascularisation and proliferation, induced tumour cell apoptosis, decreased phosphorylation of AKT and ultimately blocked tumour growth. Conclusions These studies show that nanoliposomal ceramide is an efficacious antineoplastic agent for the treatment of in vitro and in vivo models of human HCC.

Ceramide-Based Therapeutics for the Treatment of Cancer

The bioactive sphingolipid, ceramide, has garnered major interest as a principle regulator of cellular stress, proliferation, senescence, and death. Of particular interest to cancer biologists and clinical oncologist, dysregulated ceramide metabolism has been documented in both solid and non-solid malignancies. Moreover, most anticancer chemotherapeutics stimulate ceramide accumulation through increased ceramide synthesis or through the inhibition of ceramide catabolism. In fact, neutralization of ceramide via glycosylation or phosphorylation in malignant cells has been linked to multidrug chemoresistance. New therapeutic strategies to overcome chemoresistance focus on increasing endogenous ceramide levels by stimulating ceramide synthesis, by inhibiting ceramide neutralization, or by the direct delivery of exogenous ceramide. This review will discuss new therapeutic strategies designed specifically to modulate ceramide metabolism, as well as nanoscale delivery systems engineered to selectively deliver ceramide to cancerous cells and tissues.

Combinatorial therapies improve the therapeutic efficacy of nanoliposomal ceramide for pancreatic cancer

Poor prognosis cancers, such as pancreatic cancer, represent inherent challenges for ceramide-based nanotherapeutics due to metabolic pathways, which neutralize ceramide to less toxic or pro-oncogenic metabolites. We have recently developed a novel 80 nanometer diameter liposomal formulation that incorporates 30 molar percent C6-ceramide, a bioactive lipid that is pro-apoptotic to many cancer cells, but not to normal cells. In this manuscript, we evaluated the efficacy of combining nanoliposomal C6-ceramide (Lip-C6) with either gemcitabine or an inhibitor of glucosylceramide synthase. We first assessed the biological effect of Lip-C6 in PANC-1 cells, a gemcitabine-resistant human pancreatic cancer cell line, and found that low doses alone did not induce cell toxicity. However, cytotoxicity was achieved by combining Lip-C6 with either non-toxic sub-therapeutic concentrations of gemcitabine or with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Furthermore, these combinations with Lip-C6 cooperatively inhibited PANC-1 tumor growth in vivo. Mechanistically, Lip-C6 inhibited pro-survival Akt and Erk signaling, whereas the nucleoside analog gemcitabine did not. Furthermore, by including PDMP within the nanoliposomes, which halted ceramide neutralization as evidenced by LC-MS3, the cytotoxic effects of Lip-C6 were enhanced. Collectively, we have demonstrated that nanoliposomal ceramide can be an effective anti-pancreatic cancer therapeutic in combination with gemcitabine or an inhibitor of ceramide neutralization.

Metabolism of short-chain ceramide by human cancer cells—Implications for therapeutic approaches

Due to recent use of short-chain ceramides in preclinical studies, we characterized C6-ceramide metabolism in cancer cell lines and assessed metabolic junctures for enhancing efficacy. MDA-MB-231 breast cancer cells decreased the amount of C6-ceramide metabolized to C6-sphingomyelin (C6-SM) and increased the amount metabolized to C6-glucosylceramide (C6-GC) in response to increasing concentrations. A similar trend was seen in DU-145 (prostate cancer), PANC-1 (pancreatic cancer), and LoVo (colorectal cancer) cells. KG-1 leukemia cells favored C6-SM synthesis at low (0.6muM) and high-dose (12muM) C6-ceramide. Partnering C6-ceramide with tamoxifen, a P-glycoprotein antagonist that impedes ceramide glycosylation, was an effective regimen for enhancing cytotoxicity in cells. Experiments to assess the mechanism of cell death using KG-1 cells showed that tamoxifen inhibited synthesis of C6-GC and C6-SM from C6-ceramide by 80% and 50%, respectively, which was accompanied by enhanced apoptosis. Radiolabeling of KG-1 cells with [(3)H]palmitic acid produced a 2-fold increase in (3)H-long-chain ceramides when unlabeled C6-ceramide was added and a 9-fold increase when C6-ceramide and tamoxifen were added. The increase in (3)H-palmitate radiolabeling of long-chain ceramides was blocked by inclusion of a ceramide synthase inhibitor; however, inhibiting synthesis of long-chain ceramide did not rescue cells. These studies show that tamoxifen enhances the apoptotic effects of C6-ceramide. The proposed mechanism involves blocking short-chain ceramide anabolism to favor hydrolysis and generation of sphingosine. We propose that use of tamoxifen and other P-glycoprotein antagonists can be an effective means for enhancing cytotoxic potential of short-chain ceramides in the treatment of cancer.

Neutral sphingomyelinase activation precedes NADPH oxidase-dependent damage in neurons exposed to the proinflammatory cytokine tumor necrosis factor-α

Inflammation accompanied by severe oxidative stress plays a vital role in the orchestration and progression of neurodegeneration prevalent in chronic and acute central nervous system pathologies as well as in aging. The proinflammatory cytokine tumor necrosis factor‐α (TNFα) elicits the formation of the bioactive ceramide by stimulating the hydrolysis of the membrane lipid sphingomyelin by sphingomyelinase activities. Ceramide stimulates the formation of reactive oxygen species (ROS) and apoptotic mechanisms in both neurons and nonneuronal cells, establishing a link between sphingolipid metabolism and oxidative stress. We demonstrated in SH‐SY5Y human neuroblastoma cells and primary cortical neurons that TNFα is a potent stimulator of Mg2+‐dependent neutral sphingomyelinase (Mg2+‐nSMase) activity, and sphingomyelin hydrolysis, rather than de novo synthesis, was the predominant source of ceramide increases. Mg2+‐nSMase activity preceded an accumulation of ROS by a neuronal NADPH oxidase (NOX). Notably, TNFα provoked an NOX‐dependent oxidative damage to sphingosine kinase‐1, which generates sphingosine‐1‐phosphate, a ceramide metabolite associated with neurite outgrowth. Indeed, ceramide and ROS inhibited neurite outgrowth of dorsal root ganglion neurons by disrupting growth cone motility. Blunting ceramide and ROS formation both rescued sphingosine kinase‐1 activity and neurite outgrowth. Our studies suggest that TNFα‐mediated activation of Mg2+‐nSMase and NOX in neuronal cells not only produced the neurotoxic intermediates ceramide and ROS but also directly antagonized neuronal survival mechanisms, thus accelerating neurodegeneration. Journal of Neuroscience Research (2011)

Exogenous Ceramide-1-phosphate Reduces Lipopolysaccharide (LPS)-mediated Cytokine Expression

Background: Reports on the role of ceramide to regulate LPS signaling have been inconsistent; thus, we have investigated the role of ceramide metabolites to differentially regulate LPS signaling. Results: Ceramide-1-phosphate limits LPS-mediated NF-κB activation, MAPK activation, and cytokine secretion. Conclusion: Ceramide-1-phosphate, but not ceramide, limits LPS signaling. Significance: Ceramide-1-phosphate may function as an anti-inflammatory lipid. Toll-like receptor 4 (TLR4) is a component of the innate immune system that recognizes a diverse group of molecular structures, such as lipopolysaccharide (LPS) from Gram-negative bacteria. TLR4 signaling ultimately leads to activation of the transcription factor, nuclear factor κB (NF-κB), and the production of cytokines. Ceramide is a bioactive sphingolipid that has been suggested to regulate TLR4-induced NF-κB signaling, although reports on the role of ceramide in TLR4 activation conflict. We investigated the possibility that ceramide metabolites, such as ceramide-1-phosphate (C-1-P), may explain these discrepancies. We now report that exogenous C-1-P, but not ceramide, reduces NF-κB-mediated gene transcription in HEK 293 cells stably transfected with human TLR4, CD14, and MD-2. We demonstrate that inhibition of NF-κB by exogenous C-1-P is dose-dependent and specific to TLR4 in a reporter assay. We further demonstrate a requirement for both the phosphate moiety and the sphingoid backbone to inhibit LPS-activated NF-κB transcription. Specifically, C-1-P prevents the degradation of IκB, the phosphorylation of the p65 subunit of NF-κB, and LPS-stimulated MAPK activation. The functional consequence of C-1-P inhibition of NF-κB is a reduction in LPS-mediated cytokine release from HEK 293 TLR4-expressing cells and human peripheral blood mononuclear cells. Taken together, these data demonstrate that C-1-P may function as an anti-inflammatory lipid mediator of immune response.

Ceramide 1-Phosphate Mediates Endothelial Cell Invasion via the Annexin a2-p11 Heterotetrameric Protein Complex

Background: Extracellular ceramide 1-phosphate is presumed to interact with extracellular proteins to mediate cellular invasion. These proteins are unidentified. Results: C-1-P interacts with both annexin a2 and p11 proteins. C-1-P-mediated vascular endothelial cell invasion requires expression of these proteins. Conclusion: Extracellular C-1-P mediates invasion via an interaction with the annexin a2-p11 heterotetramer. Significance: Gradients of C-1-P may guide vascular endothelial cell invasion during wound healing. The bioactive sphingolipid, ceramide 1-phosphate (C-1-P), has been implicated as an extracellular chemotactic agent directing cellular migration in hematopoietic stem/progenitor cells and macrophages. However, interacting proteins that could mediate these actions of C-1-P have, thus far, eluded identification. We have now identified and characterized interactions between ceramide 1-phosphate and the annexin a2-p11 heterotetramer constituents. This C-1-P-receptor complex is capable of facilitating cellular invasion. Herein, we demonstrate in both coronary artery macrovascular endothelial cells and retinal microvascular endothelial cells that C-1-P induces invasion through an extracellular matrix barrier. By employing surface plasmon resonance, lipid-binding ELISA, and mass spectrometry technologies, we have demonstrated that the heterotetramer constituents bind to C-1-P. Although the annexin a2-p11 heterotetramer constituents do not bind the lipid C-1-P exclusively, other structurally similar lipids, such as phosphatidylserine, sphingosine 1-phosphate, and phosphatidic acid, could not elicit the potent chemotactic stimulation observed with C-1-P. Further, we show that siRNA-mediated knockdown of either annexin a2 or p11 protein significantly inhibits C-1-P-directed invasion, indicating that the heterotetrameric complex is required for C-1-P-mediated chemotaxis. These results imply that extracellular C-1-P, acting through the extracellular annexin a2-p11 heterotetrameric protein, can mediate vascular endothelial cell invasion.