Pharmacodynamic Studies of Fluorescent Diamond Carriers of Doxorubicin in Liver Cancer Cells and Colorectal Cancer Organoids.

BACKGROUND: We recently reported on preferential deposition of bare fluorescent diamond particles FDP-NV-700/800nm (FDP-NV) in the liver following intravenous administration to rats. The pharmacokinetics of FDP-NV in that species indicated short residency in the circulation by rapid clearance by the liver. Retention of FDP-NV in the liver was not associated with any pathology. These observations suggested that cancer therapeutics, such as doxorubicin, linked to FDP-NV, could potentially serve for anti-cancer treatment while sparing toxicities of peripheral organs. PURPOSE: To generate proof-of-concept (POC) and detail mechanisms of action of doxorubicin-coated FDP-NV-700/800nm (FDP-DOX) as a prospective chemotherapeutic for metastatic liver cancer. METHODS: FDP-DOX was generated by adsorption chemistry. Experimental design included concentration and time-dependent efficacy studies as compared with naïve (baren) FDP-NV in in vitro liver cancer cells models. Uptake of FDP-NV and FDP-DOX by HepG-2, Hep-3B and hCRC organoids were demonstrated by flow-cytometry and fluorescent microscopy. FDP-DOX pharmacodynamic effects included metabolic as well as cell death biomarkers Annexin V, TUNEL and LDH leakage. DOX desorpted from FDP-DOX was assessed by confocal microscopy and chemical assay of cells fractions. RESULTS: FDP-DOX efficacy was dose- and time-dependent and manifested in both liver cancer cell lines and human CRC organoids. FDP-DOX was rapidly internalized into cancer cells/organoids leading to cancer growth inhibition and apoptosis. FDP-DOX disrupted cell membrane integrity as evident by LDH release and suppressing mitochondrial metabolic pathways (AlamarBlue assay). Access of free DOX to the nuclei was confirmed by direct UV-Visible fluorescent assay and confocal microscopy of DOX fluorescence. CONCLUSION: The rapid uptake and profound cancer inhibition observed using FDP-DOX in clinically relevant cancer models, highlight FDP-DOX promise for cancer chemotherapeutics. We also conclude that the in vitro data justify further investment in in vivo POC studies.

Effects of Fluorescent Diamond Particles FDP-NV-800nm on Essential Biochemical Functions of Primary Human Umbilical Vein Cells and Human Hepatic Cell Line, HepG-2 in vitro (Part VI): Acute Biocompatibility Studies.

BACKGROUND: Recently, we reported the safety and biocompatibility of fluorescent diamond particles, FDP-NV-Z-800nm (FDP-NV) injected intravenously into rats, where no morbidity and mortality were noted over a period of 3 months. The acute effects of FDP-NV-800nm particles on cultured human endothelial and hepatic cells remain unexplored. PURPOSE: In this study, we aimed to explore select cellular and biochemical functions in cultured human umbilical endothelial cells (HUVEC) and a human hepatic cancer cell line (HepG-2) exposed to FDP-NV-800 in vitro at exposure levels within the pharmacokinetics (Cmax and the nadir) previously reported in vivo. METHODS: Diverse cellular and biochemical functions were monitored, which cumulatively can provide insights into some vital cellular functions. Cell proliferation and migration were assessed by quantitative microscopy. Mitochondrial metabolic functions were tested by the MTT assay, and cytosolic esterase activity was studied by the calcein AM assay. Chaperons (CHOP), BiP and apoptosis (caspase-3 activation) were monitored by using Western blot (WB). MAPK Erk1/2 signaling was assessed by the detection of the phosphorylated form of the protein (P-Erk 1/2) and its translocation into the cell nucleus. RESULTS: At all concentrations tested (0.001-0.1mg/mL), FDP-NV did not affect any of the biomarkers of cell integrity of HepG2 cells. In contrast, the proliferation of HUVEC was affected at the highest concentration tested (0.1mg/mL, Cmax). Exposure of HUVEC to (0.01 mg/mL) FDP-NV had a mild-moderate effect on cell proliferation as evident in the MTT assay and was absent when proliferation was assessed by direct cell counting or by using the calcein AM assays. In both cell types, exposure to the highest concentration (0.1 mg/mL) of FDP-NV did neither affect FBS-stimulated cell signaling (MAPK Erk1/2 phosphorylation) nor did it activate of Caspase 3. CONCLUSION: Our data suggest that FDP-NV-800nm are largely biocompatible with HepG-2 cells proliferation within the pharmacokinetic data reported previously. In contrast, HUVEC proliferation at the highest exposure dose (0.1 mg/mL) responded adversely with respect to several biomarkers of cell integrity. However, since the Cmax levels are very short-living, the risk for endothelial injury is likely minimal for slow rate cell proliferation such as endothelial cells.

The Use of Near-Infrared Light-Emitting Fluorescent Nanodiamond Particles to Detect Ebola Virus Glycoprotein: Technology Development and Proof of Principle.

BACKGROUND: There is a dire need for rapid diagnostic tests of high sensitivity, efficiency, and point-of-test reporting capability to mitigate lethal viral epidemic outbreaks. PURPOSE: To develop a new operating system within the lateral flow assay (LFA) format for Ebola virus (EBOV), based on fluorescent nanodiamond particles (FNDP) nitrogen vacancy (NV) emitting near-infrared (NIR) light. Specifically, we aimed to detail technical issues and the feasibility of mobilizing FNDP-NV on nitrocellulose membranes (NCM) and capturing them at test and control lines. METHODS: FNDP-NV-200nm, 400nm or 800nm were linked to anti-EBOV glycoprotein (GP) monoclonal antibodies (mAb) and tested for LFA performance by monitoring NIR emissions using an in vivo imaging system or optoelectronic device (OED). Anti-EBOV recombinant glycoprotein (GP) humanized mAb c13C6 was linked to FNDP-NV-200nm for the mobile phase; and a second anti-GP mouse mAb, 6D8, was printed on NCM at the test line. Goat anti-human IgG (GAH-IgG) served as a nonspecific antibody for conjugated FNDP-NV-200nm at the control line. RESULTS: FNDP-NV-200nm-c13C6 specifically and dose-dependently bound to recombinant EBOV GP in vitro and was effectively captured in a sandwich configuration at the test line by mAb 6D8. FNDP-NV-200nm-c13C6 was captured on the control line by GAH-IgG. The OED quantitative analysis of NIR (obtained in less than 1 minute) was further validated by an in vivo imaging system. CONCLUSION: FNDP-NV-200nm performance as a reporter for EBOV GP rapid diagnostic tests suggests an opportunity to replace contemporary visual tests for EBOV GP and other highly lethal viral pathogens. Mobile, battery-operated OED adds portability, quantitative data, rapid data collection, and point-of-test reporting capability. Further development of FNDP-NV-200nm within a LFA format is justified.

Biocompatibility studies of fluorescent diamond particles-(NV)~800nm (part V): in vitro kinetics and in vivo localization in rat liver following long-term exposure.

BACKGROUND: We recently reported on long-term comprehensive biocompatibility and biodistribution study of fluorescent nanodiamond particles (NV)-Z-average 800nm (FNDP-(NV)) in rats. FNDP-(NV) primary deposition was found in the liver, yet liver function tests remained normal. PURPOSE: The present study aimed to gain preliminary insights on discrete localization of FNDP-(NV) in liver cells of the hepatic lobule unit and venous micro-vasculature. Kinetics of FDNP-(NV) uptake into liver cells surrogates in culture was conducted along with cell cytokinesis as markers of cells' viability. METHODS: Preserved liver specimens from a pilot consisting of two animals which were stained for cytoskeletal elements (fluorescein-isothiocyanate-phalloidin) were examined for distribution of FNDP-(NV) by fluorescent microscopy (FM) and Confocal-FM (CFM) using near infra-red fluorescence (NIR). Hepatocellular carcinoma cells (HepG-2) and human umbilical vein endothelial cells (HUVEC) were cultured with FNDP-(NV) and assayed for particle uptake and location using spectrophotometric technology and microscopy. RESULTS: HepG-2 and HUVEC displayed rapid (<30 mins) onset and concentration-dependent FNDP-(NV) internalization and formation of peri-nuclear corona. FM/CFM of liver sections revealed FNDP-(NV) presence throughout the hepatic lobules structures marked by spatial distribution, venous microvascular spaces and parenchyma and non-parenchyma cells. CONCLUSION: The robust presence of FNDP-(NV) throughout the hepatic lobules including those internalized within parenchyma cells and agglomerates in the liver venous micro-circulation were not associated with macro or micro histopathological signs nor vascular lesions. Cells cultures indicated normal cytokinesis in cells containing FNDP-(NV) agglomerates. Liver parenchyma cells and the liver microcirculation remain agnostic to presence of FNDP-(NV) in the sinusoids or internalized in the hepatic cells.

Long-term biocompatibility of fluorescent diamonds-(NV)-Z~800 nm in rats: survival, morbidity, histopathology, particle distribution and excretion studies (part IV).

BACKGROUND: Thromboembolic events are a major cause of heart attacks and strokes. However, diagnosis of the location of high risk vascular clots is hampered by lack of proper technologies for their detection. We recently reported on bio-engineered fluorescent diamond-(NV)-Z~800nm (FNDP-(NV)) conjugated with bitistatin (Bit) and proven its ability to identify iatrogenic blood clots in the rat carotid artery in vivo by Near Infra-Red (NIR) monitored by In Vivo Imaging System (IVIS). PURPOSE: The objective of the present research was to assess the in vivo biocompatibility of FNDP-(NV)-Z~800nm infused intravenously to rats. Multiple biological variables were assessed along this 12 week study commissioned in anticipation of regulatory requirements for a long-term safety assessment. METHODS: Rats were infused under anesthesia with aforementioned dose of the FNDP-(NV), while equal number of animals served as control (vehicle treated). Over the 12 week observation period rats were tested for thriving, motor, sensory and cognitive functions. At the termination of study, blood samples were obtained under anesthesia for comprehensive hematology and biochemical assays. Furthermore, 6 whole organs (liver, spleen, brain, heart, lung and kidney) were collected and examined ex vivo for FNDP-NV) via NIR monitored by IVIS and histochemical inspection. RESULTS: All animals survived, thrived (no change in body and organ growth). Neuro-behavioral functions remain intact. Hematology and biochemistry (including liver and kidney functions) were normal. Preferential FNDP-(NV) distribution identified the liver as the main long-term repository. Certified pathology reports indicated no outstanding of finding in all organs. CONCLUSION: The present study suggests outstanding biocompatibility of FNDP-(NV)-Z~800nm after long-term exposure in the rat.

Pilot study on biocompatibility of fluorescent nanodiamond-(NV)-Z~800 particles in rats: safety, pharmacokinetics, and bio-distribution (part III).

INTRODUCTION: We hereby report on studies aimed to characterize safety, pharmacokinetics, and bio-distribution of fluorescent nanodiamond particles (NV)-Z~800 (FNDP-(NV)) administered to rats by intravenous infusion in a single high dose. METHODS: Broad scale biological variables were monitored following acute (90 minutes) and subacute (5 or 14 days) exposure to FNDP-(NV). Primary endpoints included morbidity and mortality, while secondary endpoints focused on hematology and clinical biochemistry biomarkers. Particle distribution (liver, spleen, lung, heart, and kidney) was assessed by whole organ near infrared imaging using an in vivo imaging system. This was validated by the quantification of particles extracted from the same organs and visualized by fluorescent and scanning electron microscopy. FNDP-(NV)-treated rats showed no change in morbidity or mortality and preserved normal motor and sensory function, as assessed by six different tests. RESULTS: Blood cell counts and plasma biochemistry remained normal. The particles were principally distributed in the liver and spleen. The liver particle load accounted for 51%, 24%, and 18% at 90 minutes, 5 days, and 14 days, respectively. A pilot study of particle clearance from blood indicated 50% clearance 33 minutes following the end of particle infusion. CONCLUSION: We concluded that systemic exposure of rats to a single high dose of FDNP-(NV)-Z~800 (60 mg/kg) appeared to be safe and well tolerated over at least 2 weeks. These data suggest that FNDP-(NV) should proceed to preclinical development in the near future.

Vascular thrombus imaging in vivo via near-infrared fluorescent nanodiamond particles bioengineered with the disintegrin bitistatin (Part II).

The aim of this feasibility study was to test the ability of fluorescent nanodiamond particles (F-NDP) covalently conjugated with bitistatin (F-NDP-Bit) to detect vascular blood clots in vivo using extracorporeal near-infrared (NIR) imaging. Specifically, we compared NIR fluorescence properties of F-NDP with N-V (F-NDPNV) and N-V-N color centers and sizes (100-10,000 nm). Optimal NIR fluorescence and tissue penetration across biological tissues (rat skin, porcine axillary veins, and skin) was obtained for F-NDPNV with a mean diameter of 700 nm. Intravital imaging (using in vivo imaging system [IVIS]) in vitro revealed that F-NDPNV-loaded glass capillaries could be detected across 6 mm of rat red-muscle barrier and 12 mm of porcine skin, which equals the average vertical distance of a human carotid artery bifurcation from the surface of the adjacent skin (14 mm). In vivo, feasibility was demonstrated in a rat model of occlusive blood clots generated using FeCl3 in the carotid artery bifurcation. Following systemic infusions of F-NDPNV-Bit (3 or 15 mg/kg) via the external carotid artery or femoral vein (N=3), presence of the particles in the thrombi was confirmed both in situ via IVIS, and ex vivo via confocal imaging. The presence of F-NDPNV in the vascular clots was further confirmed by direct counting of fluorescent particles extracted from clots following tissue solubilization. Our data suggest that F-NDPNV-Bit associate with vascular blood clots, presumably by binding of F-NDPNV-Bit to activated platelets within the blood clot. We posit that F-NDPNV-Bit could serve as a noninvasive platform for identification of vascular thrombi using NIR energy monitored by an extracorporeal device.

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor αIIbß3 and activated platelets.

Thromboembolic events (TEE) underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs) functionalized with bitistatin (Bit), a disintegrin that specifically binds to the αIIbß3 integrin, platelet fibrinogen receptor (PFR) on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP-Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, αIIbß3 receptor antagonist, specifically blocked F-NDP-Bit-PFR complex formation. Moreover, F-NDP-Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP). Our results suggest that engineered F-NDP-Bit particles could serve as noninvasive, "real-time" optical diagnostics for clots present in blood vessels.