Supplementary Figure 1: Nanoparticle characterization. (a) Absorbance profiles of gold nanoparticles post-modification with methoxy-terminated

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1 Supplementary Figure 1: Nanoparticle characterization. (a) Absorbance profiles of gold nanoparticles post-modification with methoxy-terminated poly(ethylene) glycol (mpeg) confirm the lack of aggregation. Transmission electron microscopy image of mpeg-coated gold nanoparticles (b) and quantum dots (d) validate that particles were monodisperse and spherical. (c) Fluorescent emission profiles of mpeg-functionalized quantum dots. Insets of (a) and (c) are electrophoretic shifts of gold nanoparticles and quantum dots respectively pre- and post-functionalization on 0.7% agarose gels. MAA denotes mercaptoacetic acid coated quantum dots. mpeg loading on particles was confirmed by band shift towards the negative electrode and demonstrates that electrophoretic mobility of mpegnanoparticles is dominated by electro-osmotic forces over zeta-potential 1. Scale bars in (B) and (D) denote 20 nm.

2 Supplementary Figure 2: Dose dependent changes in skin tone. Images depict the differences in mouse skin tone before and 24 hours post-tail vein injection (HPI) with 0.07 (a), 0.67 (b), and 6.67 (c) pmol/g body weight of mpeg-coated gold nanoparticles. Only at the highest dose was there a noticeable blue colour change (black arrow) in mouse skin tone.

3 Supplementary Figure 3: Effect of chronic gold nanoparticle dosing on mouse skin colour. Representative images of mouse skin tone tracked over 9 days. Mice were tail-vein injected with 0.07 pmol/gbw of gold nanoparticles every other day. Darkening of mouse skin tone occurred on the 5 th day of the study (black arrow). Results suggest that changes in skin tone is not solely related to the bolus dose concentration but can also occur after repeated systemic exposure to nanoparticles.

4 Supplementary Figure 4: Comparison of nanoparticle content in mouse blood versus skin. Histological sections acquired 24 hours post-injection were taken at 20x magnification illustrating the accumulation of gold nanoparticles (a) and quantum dots (b) in skin tissue. Nanoparticles do not appear to be confined to blood vessel-like structures. Scale bars denote 50 µm. Graphs show that gold nanoparticle (c) and quantum dot (d) content in the skin was statistically distinct from the quantity present in the blood. Results confirm that nanoparticle accumulation in skin samples was related to tissue accumulation versus presence in blood. DPI is defined as days post-injection of nanoparticles. Error bars denote standard error of the mean (n>3). Asterisk denotes statistical significance of p < calculated by student s t-test.

5 Supplementary Figure 5: Transmission electron microscopy image of skin. Skin was obtained from a mouse injected with 0.17 pmol/g body weight of 100 nm mpeg functionalized gold nanoparticles. Lower injection dose and sub-optimal particle size was chosen to verify that nanoparticles accumulating in the skin were un-degraded. (a) Depicts the overall cross-section of the epidermis and dermis of the skin. (b) Inset magnifying the epidermal layer of the skin. (c) Inset magnifying nanoparticles in the dermis (black). All images were taken at a fixed exposure setting. No nanoparticles were found in the epidermis. Speckles seen in (b) are common tissue artifacts of cutaneous fixation and are not nanoparticles as their size and electron density were less than those of the nanoparticles seen in (c). Scale bar in (a) denote 8 µm while those in insets (b) and (c) represent 1 µm.

6 Supplementary Figure 6: Mouse body weight analysis. Changes in mouse body weight were monitored over 21 days post-injection (DPI) of 6.64 pmol of gold nanoparticles (yellow) or phosphate buffered saline (black). Percent changes in mouse body weight did not vary significantly from their original weight (grey). Error bars denote standard error of the mean (n>3). Red dotted line depicts the ethical toxicity threshold whereby mice would have to be euthanized.

7 Supplementary Figure 7: Blood biochemistry results from treated mice. Samples were obtained from mice intravenously treated with 6.64 pmol of gold nanoparticles at 7 and 21 days post-injection (DPI). Results were compared against un-injected healthy mice and breeder specifications (Charles River Laboratories). Grey dotted lines denote the optimal ranges for female CD1 nude athymic mice as defined by Charles River Laboraories 2. Hematological readings associated with red blood cell (a f) and coagulation (g & h) markers were within healthy ranges indicating that nanoparticle treatments did not cause red blood cell or platelet lysis. Phagocytic cell markers (i l) were below breeder specifications for both treated and untreated animals. As treated and control mice were statistically similar (student s t- test p < 0.05), the low readings were attributed to factors unrelated to nanoparticle exposure (stress and age) and concluded that gold nanoparticles did not illicit infectious or inflammatory reactions systemically. Note: although our gold nanoparticles did not appear to cause inflammation or toxicity, our results should not be generalized to other nanoparticle types. Error bars denote standard deviation (n>3). Titles denote the parameter and units for values on the y-axis.

8 Supplementary Figure 8: Hematological analysis of gold nanoparticle treated mice. Blood enzyme levels of female CD1 nude athymic mice at 7 and 21 days post-injection (DPI) of 6.64 pmol of gold nanoparticles were compared to healthy, un-treated mice for hepatotoxicity. Grey dotted lines denote the optimal health conditions as defined by the breeder (Charles River Laboratories) 2. Low bilirubin levels (a) and alkaline phosphatase (b) for treated and untreated conditions were below breeder specifications but indicate that no bile duct or gall bladder obstruction was caused by nanoparticle presence. Similarly tests for nitrogen metabolism (c) and elimination (d) for injected animals were within error (student s t-test, p < 0.05) of control conditions. These results indicate that low levels were related to dietary conditions and that no muscular or hepatic tissue damage was resultant from nanoparticle exposure. Y-axis indicates the unit as listed in the title of each chart. Error bars denote the standard deviation between replicate animals (n>3).

9 Supplementary Figure 9: Quantum dot-related changes in skin tone. Panel (a-d) depicts the differences in mouse skin tone at 0.17, 24, 48, 72 hours post-injection (HPI). Presence of quantum dots in the histological image of skin (e) at 96 HPI confirms the dermal retention of quantum dots despite the lack of skin fluorescence. Yellow arrow identifies a phagocytic cell post-uptake of nanoparticles. The dissected mouse (f) shows the whole-animal distribution of nanoparticles at 96 HPI under ultraviolet lamp excitation. White arrows denote the location of lymph nodes that accumulate quantum dots (orange) over time. Mouse hunching in (d) vs (a)-(c) was the result of inconsistent imaging technique.

10 Supplementary Figure 10: Effect of host species on nanoparticle measurement with ICP-AES. Limit of detection of quantum dots (a & b) and gold nanoparticles (c & d) in the presence of skin samples from various animal models was measured using ICP-AES. Line graphs (a & c) identify that nanoparticle number in the presence of skin was linearly related to readings of gold and cadmium for over 3 orders of magnitude. Bar graphs (b & d) identify that ICP-AES measurements of nanoparticle number is statistically (Two-way ANOVA, p<0.05) independent of species of origin.

11 Supplementary Figure 11: Analysis of nanoparticle distribution in mouse skin. Graph (a) shows that gold nanoparticle accumulation of skin was statistically (student s t-test p < 0.05) similar for skin samples that were acquired over the entire body. Representative mouse image in figure (b) summarizes the approximate location of where skin samples were taken for (a). Error bars denote standard deviation (n>3).

12 Supplementary Table 1: Comparison of current nanoparticle (NP) detection modalities. Whole animal imaging 3,4 Confocal microscopy 4,5 Two-photon microscopy 6,7 Raman spectroscopy 8 ICP-AES 9 Detection Scheme Fluorescence Fluorescence Fluorescence Light Scattering Atomic Absorbance Measurement Method Macroscopic photo-excitation & imaging Surface photoexcitation & imaging Surface photoexcitation & imaging Surface photoexcitation & spectral analysis Tissue biopsy Elemental analysis Quantitation Semiquantitative Semiquantitative Semiquantitative Semiquantitative Quantitative Detectable Tissue Depth Skin depth < 20 µm Skin depth < 150 µm Skin depth < 800 µm Skin depth < 300 µm No Limit Limitations of approach - Susceptible to photo-bleaching - Signal attenuated by tissue inhomogeneity & depth - Requires emission properties to be known - Susceptible to photo-bleaching - Signal attenuated by tissue inhomogeneity & depth - Requires emission properties to be known - Susceptible to photo-bleaching - Signal attenuated by tissue inhomogeneity & depth - Requires emission properties to be known - NP detection is modulated by interacting biomolecules & aggregation. - Signal affected by tissue inhomogeneity - Measurement is destructive to analyte - Detection limited by size of biopsy - Detects inorganic materials

13 Supplementary Table 2: Description of blood biochemistry markers. Marker Red Blood Cells Hemoglobin Hematocrit Mean Corpuscular Volume Mean Corpuscular Hemoglobin Platelet/Thrombocyte Count White Blood Cell Count Mean Platelet Volume Red Blood Cell Distribution Width Neutrophils Lymphocytes Monocytes Function Carries oxygen to the organs. Synthesized in the spleen and bone marrow Metalloprotein in red blood cells that is responsible for transporting oxygen Volume of red blood cells in the whole blood. Average size of red blood cells Average amount of haemoglobin in each red blood cell Involved in haemostasis by inhibiting bleeding at site of trauma Involved in the immune and inflammatory responses Average size of platelets Coefficient that indicates the variation in red blood cell volumes Circulating phagocyte in response to foreign material Antibody and antigen production. Immune surveillance and antigenic memory. Differentiate into macrophages at sites of inflammation

14 Supplementary Table 3: Description of hematological parameters. Parameter Bilirubin ALP (alkaline phosphatase) ALT (alanine aminotransferase) AST (aspartate aminotransferase) Function By-product of haemoglobin breakdown. Requires conjugation to albumin to be excreted from body. Measures haemolytic rate and inability of liver to conjugate albumin to bilirubin. General function of dephosphorylating compounds in liver, kidney, and bone. Measurement of acute liver cell damage. Involved in alanine synthesis in the liver. Measurement of acute liver damage. Involved in the conversion of aspartate to glutamate in liver, heart, kidney, and other organs. Indication of inflammation and acute damage to these major organs.

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