Quantum dot interactions and flow effects in angiogenic zebrafish (Danio rerio) vessels and human endothelial cells  Xiao-Yu Jiang, MSc, Christopher D. Sarsons, BSc, M. Juliana Gomez-Garcia, BSc, David T. Cramb, PhD, Kristina D. Rinker, PhD, Sarah J. Childs, PhD  Nanomedicine: Nanotechnology, Biology and Medicine  Volume 13, Issue 3, Pages 999-1010 (April 2017) DOI: 10.1016/j.nano.2016.12.008 Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Intravascular injection of carboxy-terminated QDs. (A) Microinjection setup for nanoparticle injection. (B) View of holding and injection needles. (C) Placement of holding capillary and injection needle for intravascular injection into the sinus venosus. (D-K) Images of the caudal venous plexus (CVP; green) of carboxy-QD injected 52 hpf embryos (red) at 1 hpi=54 hpf and at 18 hpi=74 hpf. The Dorsal Aorta (DA) vessel wall is outlined (dashes). (X) marks autofluorescent iridophores. Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 999-1010DOI: (10.1016/j.nano.2016.12.008) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 In vivo loss of QD fluorescence over time. (A) Example of locations of sampled areas (white boxes) in tissue parenchyma (t), dorsal aorta lumen (l) and background (z). (B) The ratio of the average pixel brightness of tissue parenchyma to the dorsal aorta lumen in four independent fish at 18 hpi compared to 1 hpi (*P<0.03). Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 999-1010DOI: (10.1016/j.nano.2016.12.008) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 In vivo behavior of amino- and carboxy-QDs. Comparison of the distribution pattern of amino-conjugated QDs (A, D, G) with carboxy-conjugated QDs (B, E, H) and control uninjected embryos (C, F, I) in the caudal venous plexus at 1 hpi=54 hpf (A, B, C), 4 hpi=58 hpf (D, E, F) and 18 hpi=72 hpf (G, H, I). Boxes mark location of enlarged insets. (X) marks autofluorescent iridophores. Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 999-1010DOI: (10.1016/j.nano.2016.12.008) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 QD accumulation and uptake in slow flowing vessels. (A, B) 52 hpf embryos were injected with amino- and carboxy-QDs. The DA, a large, high pressure, shows no evidence of accumulation of QDs at 30 mpi time point. (C, D) The small, low pressure vessels of the CVP show accumulation of QDs along the vessel walls (white arrows) and endothelial uptake of QDs (blue arrows). Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 999-1010DOI: (10.1016/j.nano.2016.12.008) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Schematic of in vitro flow chamber apparatus used for HUVEC studies. Illustration of the flow loop and chamber used in the in vitro studies, with key components labeled. The dotted red lines demarcate the area of the microscope slides on which HUVECs were seeded. Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 999-1010DOI: (10.1016/j.nano.2016.12.008) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Endothelial association with QDs after flow pre-conditioning. (A) Representative HUVEC cells (white) exposed to amino- or carboxy-QDs (red) for 18 h, following 20 h of culture under flow. Scale bars represent 100 μm. QD channel images are brightened for display although the image quantification was performed on raw images. (B) Quantification of QD-exposed HUVEC (mean±standard deviation). Significance calculated by one-way ANOVA with Tukey's HSD (**P<0.01, ****P<0.0001). Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 999-1010DOI: (10.1016/j.nano.2016.12.008) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 In vivo instability of Amino-PEG QD fluorescence. (A, B) At 1 hpi amino- and carboxy-QDs (red) are homogenously dispersed throughout the vasculature. (C, D) At 18 hpi, the carboxy QDs are punctate in blood vessels while amino QDs show a strongly diminished QD signal. (E, F) When sectioned at 1 hpi (E), QD are localized both outside of blood vessels and within the lumen of the CVP in amino-QD injected embryos. (F) At 18 hpi, QD signal is absent in amino-QD injected embryos. (X) marks autofluorescent iridophores. Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 999-1010DOI: (10.1016/j.nano.2016.12.008) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 8 Effect of blocking blood flow on endothelial uptake of quantum dots and effect of QD injection on animal viability. Comparison of nanoparticle distribution in the head (A-B) and trunk (C-D) in cross section and (E-F) in lateral section at 1 hpi (A, C, E) and 18 hpi (B, D, F) shows initial localization of particles within the vessel lumen (A′, C′, E′) at 1 hpi, and localization within the endothelial cell wall at 18 hpi (B′, D′, F′; green cells marked by yellow arrows where fluorescence overlaps). Blue marks nuclei. (G) TUNEL staining highlights cell death at 18 hpi. (H) Survival of treated embryos to 50 dpi. Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 999-1010DOI: (10.1016/j.nano.2016.12.008) Copyright © 2016 Elsevier Inc. Terms and Conditions

Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 999-1010DOI: (10.1016/j.nano.2016.12.008) Copyright © 2016 Elsevier Inc. Terms and Conditions