Carbon nanotubes exhibit fibrillar pharmacology in primates

<div><p>Nanomedicine rests at the nexus of medicine, bioengineering, and biology with great potential for improving health through innovation and development of new drugs and devices. Carbon nanotubes are an example of a fibrillar nanomaterial poised to translate into medical practice. The leading candidate material in this class is ammonium-functionalized carbon nanotubes (fCNT) that exhibits unexpected pharmacological behavior in vivo with important biotechnology applications. Here, we provide a multi-organ evaluation of the distribution, uptake and processing of fCNT in nonhuman primates using quantitative whole body positron emission tomography (PET), compartmental modeling of pharmacokinetic data, serum biomarkers and ex vivo pathology investigation. Kidney and liver are the two major organ systems that accumulate and excrete [⁸⁶Y]fCNT in nonhuman primates and accumulation is cell specific as described by compartmental modeling analyses of the quantitative PET data. A serial two-compartment model explains renal processing of tracer-labeled fCNT; hepatic data fits a parallel two-compartment model. These modeling data also reveal significant elimination of the injected activity (>99.8%) from the primate within 3 days (t_1/2 = 11.9 hours). These favorable results in nonhuman primates provide important insight to the fate of fCNT in vivo and pave the way to further engineering design considerations for sophisticated nanomedicines to aid late stage development and clinical use in man.</p></div

Compartmental modeling analysis of [⁸⁶Y]fCNT pharmacokinetics in the nonhuman primate liver.

<p>A parallel two-compartment model is proposed to predict clearance and distribution of [⁸⁶Y]fCNT in the liver, a secondary tissue for processing fCNT. In our model, C₁ is speculated to be the hepatic tissue space, but excluding the liver sinusoidal endothelium, while C₂ is assumed to describe specifically the liver sinusoidal endothelium. C₂ is defined as the compartment having relatively slow clearance, whose correspondence to the sinusoidal endothelium is supported by our data in rodents [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183902#pone.0183902.ref008" target="_blank">8</a>]. Distribution and clearance of the tracer occurs from liver as described by the chart shown in (<b>A</b>) showing the blood compartment (C_B) and the two parallel tissue compartments (C₁ and C₂). The rate constants noted in (<b>B</b>) where k₁ denotes transport from blood into C₁ and k₃ indicates the rate constant for accumulation of the tracer in liver sinusoidal endothelium. The rate constants k₂ and k₄ describe the rate at which [⁸⁶Y]fCNT is returned to the blood and/or eliminated into the bile. Plots of the raw data from PET/CT imaging and the fitted data are presented in (<b>C</b>) and (<b>D</b>). Note that extrapolation of the fitted data (<b>D</b>) calls attention to significant elimination (99.9%) of the tracer from liver within a 72 hour period with biological half-life of ~12 hours. The standard deviation, coefficient of variance and the 95% confidence intervals for each k value are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183902#pone.0183902.s012" target="_blank">S2 Table</a> for liver.</p

Functionalized fibrillar nanocarbon.

<p>(<b>A</b>) An illustration of the key moieties covalently appended on the ammonium-functionalized carbon nanotube and the radiosynthetic steps to prepare [⁸⁶Y]fCNT (n.b. not drawn to scale). (<b>B</b>) Representative CryoTEM image of fCNT in water (100 μg/L) showing the fibrillar nature of this material in solution (scale bar, 2000 μm). (<b>C</b>) Radiochromatograph of [⁸⁶Y]fCNT.</p

Compartmental modeling analysis of [⁸⁶Y]fCNT pharmacokinetics in the nonhuman primate kidney.

<p>The kidney is the primary organ that accumulates and excretes [⁸⁶Y]fCNT in mammals (i.e., primate and rodent) and pharmacokinetically processes [⁸⁶Y]fCNT as described by a serial two-compartment model (<b>A</b>). In this model Compartment 1 (C₁) is assumed to be the renal tissue space <i>in toto</i> and Compartment 2 (C₂) is the proximal tubule cell population that serves as a hold up volume which delays washout from the kidney. This assignment is supported by our data herein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183902#pone.0183902.g002" target="_blank">Fig 2</a>) and in rodents [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183902#pone.0183902.ref006" target="_blank">6</a>]. (C_B) is the blood compartment. The fitted rate constants values are listed in (<b>B</b>) where k₁ denotes transport from blood into kidney; k₃ indicates the rate constant for accumulation of the tracer in proximal tubule cells; k₄ describes the fraction of [⁸⁶Y]fCNT that is not accumulated by these target cells, yet remains in kidney and is ultimately eliminated into the urine in a manner described by rate constant k₂. Plots of the data obtained from quantitative PET/CT imaging and the curve fits are presented in (<b>C</b>) and (<b>D</b>), where (<b>D</b>) draws attention to the significant elimination (99.8%) of the tracer from the kidney within a 72 hour period with a biological half-life of ~12 hours. The standard deviation, coefficient of variance and the 95% confidence intervals for each k value are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183902#pone.0183902.s011" target="_blank">S1 Table</a> for kidney.</p

Blood chemistries and hematology–animal 1.

<p>Blood chemistries and hematology–animal 1.</p

Kidney histology of the two primates that received fCNT and an untreated control.

<p>Animal 1 was evaluated at 190 days for chronic effects and Animal 2 was evaluated at 14 days for acute effects (both received [⁸⁶Y]fCNT) and a third animal that did not receive fCNT is an untreated control. Tissue was harvested at necropsy, fixed, sectioned, and stained with (<b>A-C</b>) H&E, (<b>D-F</b>) anti-CD31, (<b>G-I</b>) anti-Iba1, (<b>J-L</b>) TUNEL, and (<b>M-O</b>) Cleaved caspase 3. All scale bars are 100 μm.</p

Dynamic PET/CT images of [⁸⁶Y]fCNT in a cynomolgus monkey showing renal and hepatic processing activity.

<p>(<b>A</b>) Maximum intensity projection (MIP) image immediately following intravenous injection shows rapid kidney accumulation (blue arrows) and elimination of urine into the bladder (yellow arrow); (<b>B</b>) MIP of the same animal at 1.5 hours; (<b>C</b>) Axial image of the kidneys (blue arrows) immediately following intravenous injection; and (<b>D</b>) Axial image of the kidneys at 1.5 hours. (The scale bar is the same for panels <b>A</b>-<b>D</b>.) (<b>E</b>) MIP image immediately following intravenous injection shows diffuse blood compartment and heart activity (yellow arrow) as well as kidney (blue arrow) and liver (white arrow) accumulation of activity; (<b>F</b>) MIP of the same animal at 1.5 hours after the blood compartment activity clears and all of the activity has distributed or been eliminated. (The scale bar is the same for panels <b>E</b> and <b>F</b>.) Time activity curve (TAC) data for (<b>G</b>) kidney, urine in bladder, liver, and blood.</p

Body mass of animals 1 and 2.

<p>Body mass of animals 1 and 2.</p

Blood biomarkers, hematology and clotting parameters for primates that received [⁸⁶Y]fCNT.

<p>Each cynomolgus monkey received an intravenous dose of [⁸⁶Y]fCNT (1 mg/kg) on day 0 (green arrow indicates time of injection and imaging). Biomarkers for renal and hepatic functions in Animal 1 (red circles) and Animal 2 (blue triangles) were measured at baseline (day minus 4) and longitudinally thereafter until euthanasia and necropsy. Hematology and clotting parameters were also assayed. Additional data can be found in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183902#pone.0183902.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183902#pone.0183902.t002" target="_blank">2</a>.</p

Expression of the Carboxy-Terminal Portion of MUC16/CA125 Induces Transformation and Tumor Invasion

<div><p>The CA125 antigen is found in the serum of many patients with serous ovarian cancer and has been widely used as a disease marker. CA125 has been shown to be an independent factor for clinical outcome in this disease. In The Cancer Genome Atlas ovarian cancer project, MUC16 expression levels are frequently increased, and the highest levels of MUC16 expression are linked to a significantly worse survival. To examine the biologic effect of the proximal portion of MUC16/CA125, NIH/3T3 (3T3) fibroblast cell lines were stably transfected with the carboxy elements of MUC16. As few as 114 amino acids from the carboxy-terminal portion of MUC16 were sufficient to increase soft agar growth, promote matrigel invasion, and increase the rate of tumor growth in athymic nude mice. Transformation with carboxy elements of MUC16 was associated with activation of the AKT and ERK pathways. MUC16 transformation was associated with up-regulation of a number of metastases and invasion gene transcripts, including IL-1β, MMP2, and MMP9. All observed oncogenic changes were exclusively dependent on the extracellular “ectodomain” of MUC16. The biologic impact of MUC16 was also explored through the creation of a transgenic mouse model expressing 354 amino acids of the carboxy-terminal portion of MUC16 (MUC16^c354). Under a CMV, early enhancer plus chicken β actin promoter (CAG) MUC16^c354 was well expressed in many organs, including the brain, colon, heart, kidney, liver, lung, ovary, and spleen. MUC16^c354 transgenic animals appear to be viable, fertile, and have a normal lifespan. However, when crossed with p53-deficient mice, the MUC16^c354:p53^+/- progeny displayed a higher frequency of spontaneous tumor development compared to p53^+/- mice alone. We conclude that the carboxy-terminal portion of the MUC16/CA125 protein is oncogenic in NIH/3T3 cells, increases invasive tumor properties, activates the AKT and ERK pathways, and contributes to the biologic properties of ovarian cancer.</p></div