Convection-enhanced delivery of nanodiamond drug delivery platforms for intracranial tumor treatment Guifa Xi, MD, PhD, Erik Robinson, PhD, Barbara Mania-Farnell,

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Convection-enhanced delivery of nanodiamond drug delivery platforms for intracranial tumor treatment Guifa Xi, MD, PhD, Erik Robinson, PhD, Barbara Mania-Farnell, PhD, Elio Fausto Vanin, PhD, Kyu-Won Shim, MD, PhD, Tsurubuchi Takao, MD, PhD, Elise Victoria Allender, MS, Chandra Shekhar Mayanil, PhD, Marcelo Bento Soares, PhD, Dean Ho, PhD, Tadanori Tomita, MD Nanomedicine: Nanotechnology, Biology and Medicine Volume 10, Issue 2, Pages 381-391 (February 2014) DOI: 10.1016/j.nano.2013.07.013 Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Conjugation with NDs enhanced and prolonged DOX uptake and retention in C6 and U251MG cell lines. (A) Intracellular distribution of FITC, ND-FITC, DOX and ND-Dox in C6 and U251MG cells after 4h incubation. (B) and (C) 4h quantitative profiles of DOX uptake by C6 and U251MG cells treated with Dox(1μg/ml) or ND(5μg/ml)-Dox(1μg/ml). (D) Both cell lines showed longer retention of ND-Dox, at 6h and 24h after 2h treatment with DOX(2μg/ml) or ND(10μg/ml)-Dox(2μg/ml). (Photo exposure time: DAPI 100ms; DOX, ND-Dox 1600ms). (E) and (F) 24h quantitative DOX retention profiles in C6 and U251MG cell lines treated with DOX or ND-Dox. Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 381-391DOI: (10.1016/j.nano.2013.07.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 ND-Dox inhibited proliferation and induced apoptosis in U251MG cells. (A) Percentages of apoptotic (Annexin V-FITC +/PI−) and necrotic (Annexin V-FITC+/PI+) control cells or cells treated with NDs(20μg/ml), DOX(4μg/ml) or ND(20μg/ml)-Dox(4μg/ml) (UL: upper left; UR: upper right; LL: lower left, LR: Lower right). (B) Rates of apoptosis for treatments from multiple samples. Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 381-391DOI: (10.1016/j.nano.2013.07.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Distribution of DOX and ND-Dox immediately after CED. (A) Top: DOX and ND-Dox infused in normal Fisher 344 rats. Middle: Fluorescence generated by DOX and ND-Dox. Bottom: Higher magnification of highlighted area. (B) Top: Sections generating peak fluorescence. Bottom: Peak fluorescence measured by IVIS Xenogen 200. (C) Peak fluorescence intensity. (D) Distribution volume calculated from fluorescence emitted from DOX and ND-Dox. Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 381-391DOI: (10.1016/j.nano.2013.07.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Retention of DOX (1mg/ml) and ND(5mg/ml)-Dox(1mg/ml) in normal brain tissue. (A) Light microscopic images (upper rows) and fluorescence images detecting fluorescence generated from DOX (lower rows). Images show distribution of unmodified DOX and ND-Dox (right hemisphere) after CED at 6, 16, 24 and 72h. (B) Intensity of peak fluorescence in DOX and ND-Dox infused brain slides. (C) Retention volume calculated by detecting fluorescence emitted from DOX and ND-Dox. Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 381-391DOI: (10.1016/j.nano.2013.07.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 Effect of ND-Dox treatment on tumor growth and survival of U251MG-Luc human glioma-bearing NIH nu/nu nude rats. (A) Experimental design testing the effect of 5μl of CON (0.9% saline), NDs(5mg/ml), DOX(1mg/ml) or ND(5mg/ml)-Dox(1mg/ml) on tumor growth and survival of glioma-bearing rats. (B) Bioluminescence images showing tumor growth in xenograft rats inoculated with U251MG-Luc cells in response to each treatment. (C) Quantitative BLI showing tumor response to CON, ND, DOX and ND-Dox. (D) Survival of U251MG-Luc glioma bearing rats analyzed by a log-rank test based on the Kaplan–Meier method. (E) Representative images of H&E staining from each treatment group showing tumor size regression at the indicated day after tumor inoculation (Top Panel); 200× magnification of boxed area (Middle Panel); apoptotic cell death, measured by TUNEL staining of the boxed area (Bottom Panel). Nuclei were counter stained with DAPI (blue). Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 381-391DOI: (10.1016/j.nano.2013.07.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 381-391DOI: (10.1016/j.nano.2013.07.013) Copyright © 2014 Elsevier Inc. Terms and Conditions