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Supplemental Figure 1. Characterization of physicochemical properties of glycol chitosan coated gold nanoparticles (GC-AuNPs). (a) A transmission electron microscograph (a) to show monodispersed and spherical GC-AuNPs with a diameter of approximately 20 nm. Scale bar=200 nm. (b) UV-visible light absorption spectra of GC-AuNPs. (c) The surface plamson resonance peak of GC-AuNPs appears at 533 nm. Size distribution of GC-AuNPs measured with dynamic light scattering. By dynamic light scattering, the diameter is 99.4±16.8 nm, which is different from the transmission electron microscopy measurement because of the hydrophilic coat (i.e. GC) expanding in solution.

Supplemental Figure 2. In vitro experiment to show a tight correlation between GC-AuNPs of known concentrations and mean microCT (mCT) densities (Pearson correlation, P<0.001, R=0.995).

Supplemental Figure 3. In vivo mCT imaging to allow an assessment of thrombus burden 5 min after GC-AuNP injection and 2 h after carotid thrombosis in mice. Longest length, volume of thrombus, and AuNP amount of thrombus, but not thrombus mean mCT density, were proportional to thrombotic insult: application of 1 mm vs. 2 mm FeCl3-pledgets to the left carotid artery. Box plots show median and mean as the horizontal mark and asterisk in the box; upper and lower quartiles define the upper and lower limits of the box. Whiskers define largest and smallest values not defined as outliers. P values were obtained by Mann-Whitney tests.

Supplemental Figure 4. In vivo microCT imaging to allow detection of recurrent carotid thromboses up to 48 h after an initial single-dose injection of GC-AuNPs intravenously into the tail vein of mice (n=6). Error bars represent mean±SEM. Red- or black-colored #P≤0.1 and *P<0.05 by Friedman tests for the imaging data of thrombosis recurrences over 48 h. Red error-bars show quantification of each thrombus-image acquired at each of the four corresponding time points. Black error-bars show quantification of all four thrombi as measured at the final 48 h time point. Please note that thrombus mCT-densities and AuNP amounts in thrombus were higher initially compared to later time points, probably due to less circulating gold nanoparticles being available in the blood pool as the body slowly eliminates circulating particles over time. By contrast, the longest length and volume of thrombus was similar, regardless of time after nanoparticle injection. Between the paired datasets of baseline values (red error-bars) and the corresponding follow-up values at 48 h (black error-bars) in each thrombus (i.e. the first thrombus at 0 h vs. 48 h, the second thrombus at 6 h vs. 48 h, the third thrombus at 24 h vs. 48 h, and the fourth thrombus at 48 h vs. 48 h), there was no significant difference.

Supplemental Figure 5. In vivo microCT imaging to allow detection of recurrent carotid thromboses up 3 weeks (w) after an initial single-dose injection of GC-AuNPs intravenously into the tail vein of mice (n=3). Error bars represent mean±SEM. Red- or black-colored #P≤0.1 and *P<0.05 by Friedman tests for the imaging data of thrombosis recurrences over 3 w. Red error-bars show quantification of each thrombus-image acquired at each of the four corresponding time points. Black error-bars show quantification of all four thrombi as measured at the final 3 w time point. Please note that thrombus mCT-densities were higher initially compared to later time points, probably due to less circulating gold nanoparticles being available in the blood pool as the body slowly eliminates circulating particles over time. Between the paired datasets of baseline values (red error-bars) and the corresponding follow-up values at 3 w (black error-bars) in each thrombus (i.e. the first thrombus at 0 w vs. 3 w, the second thrombus at 1 w vs. 3 w, the third thrombus at 2 w vs. 3 w, and the fourth thrombus at 3 w vs. 3 w), there was no significant difference. When all four thrombi were quantified at the final 3 w time point, the longest thrombus length tended to be lower initially compared to later time points, probably due to spontaneous thrombolysis.

Supplemental Figure 6. In vitro experiments to show the binding of GC-AuNPs to fibrin clot. After fibrin clot was formed (bottom-up, B) in GC-AuNP colloids by adding fibrinogen and thrombin (top-down, T) or fibrin clot that had been preformed outside was immersed in GC-AuNP colloids (bottom-up, B), UV absorbance of GC-AuNP colloids was decreased, suggesting that some GC-AuNPs were bound to the clot. When the same amount of either fibrinogen or thrombin was simply added to the colloids, UV absorbance of GC-AuNP colloids was not changed. The binding of GC-AuNPs to fibrin clot may have occurred due to interactions between the negative charges of carboxyl groups of amino acids such as glutamate of fibrin and the positive charges of amine groups of chitosan polymers.2

Supplemental Figure 7. Quantification of in vivo microCT images to show significant thrombolytic effects of tPA treatment (initiated 30 min after carotid thrombosis) in the 1-mm-FeCl3-pledget subgroup mice (upper two rows, n=32), but not in the 2-mm-FeCl3-pledget subgroup mice (lower two rows, n=32). Box plots show median and mean as the horizontal mark and asterisk in the box; upper and lower quartiles define the upper and lower limits of the box. Whiskers define largest and smallest values not defined as outliers. P values were obtained by Kruskal-Wallis ANOVA with post-hoc Mann-Whitney tests (vs. saline control group, i.e. animals without tPA administration).

Supplemental Figure 8. Quantification of in vivo microCT images to show no significant thrombolytic effects of tPA treatment when initiated 2 h after carotid thrombosis in mice (n=46). Box plots show median and mean as the horizontal mark and asterisk in the box; upper and lower quartiles define the upper and lower limits of the box. Whiskers define largest and smallest values not defined as outliers. P values were obtained by Kruskal-Wallis ANOVA tests.

Supplemental Figure 9. A scatter plot to correlate the baseline mean AuNP-mCT density of thrombus with post-tPA reduction of the thrombus volume in the mice treated with tPA at 30 min after carotid thrombosis (n=54; Pearson correlation, P=0.076, R=−0.244).

Supplemental Fig 10. In vivo AuNP-microCT imaging-based quantitative characterization of spatiotemporal thrombus-evolution II (continued from Fig 3). (a and b) Cross-correlation between spontaneous or post-tPA thrombus area ratio (After / Before) in various parts of the thrombus (proximal, middle, or distal segment) vs. thrombus area in the pre-proximal (a; proximal-to-the-proximal) or post-distal (b; distal-to-the-distal) common carotid artery (CCA) region in animals with (pink color) or without (green color) clot extension beyond the initial thrombus. (a) In the animals with thrombus growth into the pre-proximal CCA, there is a trend of proximal-to-distal decrease in the median (pink arrow-heads) and mean (with pink error-bars) thrombus area ratios. Thrombus area reduction is relatively low in the proximal segment but high in the distal segment of the animal, compared to the animals without the thrombus growth (green-error-bars). (b) In the animals with newly-appeared post-distal thrombus, there is a trend of proximal-to-distal increase in the median (pink arrow-heads) and mean (with pink error-bars) thrombus area ratios. Thrombus area reduction is relatively high in the proximal segment and weak in the middle segment; and, thrombus area is not reduced but increased in the distal segment. This is not the case in the animals without a post-distal thrombus (green-error-bars). Green- or pink-colored **P<0.01 and pink-colored #P=0.088 by Friedman's exact test (within-group analyses). Blue-colored **P<0.01 by Mann-Whitney tests (inter-group analyses).

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