Three- Dimensional Optical Mapping of Nanoparticle Distribution in Intact Tissues Shrey Sindhwani 1† , Abdullah Muhammad Syed 1† , Stefan Wilhelm 1 , Dylan R. Glancy 2 , Yih Yang Chen 1 , Michael Dobosz 3 , and Warren C. W. Chan 1,2,4,5,6* 1 Institute of Biomaterials and Biomedical Engineering, Rosebrugh Building, Room 407, 164 College Street, Toronto, Ontario M5S 3G9, Canada 2 Department of Chemistry, 80 St George Street, Toronto, Ontario M5S 3H6, Canada 3 Roche Diagnostics GmbH, PORDBJ 6164, Nonnenwald 2, Penzberg, 82377, Germany 4 Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Room 230, Toronto, Ontario M5S 3E1, Canada 5 Department of Chemical Engineering, 200 College Street, Toronto, Ontario M5S 3E5, Canada 6 Department of Material Science and Engineering, 160 College Street, Room 450, University of Toronto, Toronto M5S 3E1, Canada † These authors contributed equally to this work. * Corresponding author: Warren C. W. Chan ([email protected])
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Three- Dimensional Optical Mapping of Nanoparticle Distribution in Intact Tissues
Shrey Sindhwani1†, Abdullah Muhammad Syed1†, Stefan Wilhelm1, Dylan R. Glancy2, Yih Yang
Chen1, Michael Dobosz3, and Warren C. W. Chan1,2,4,5,6*
1 Institute of Biomaterials and Biomedical Engineering, Rosebrugh Building, Room 407, 164
College Street, Toronto, Ontario M5S 3G9, Canada
2 Department of Chemistry, 80 St George Street, Toronto, Ontario M5S 3H6, Canada
Figure S1: Characterization of quantum dots. (a) Transmission electron microscopy (TEM) and (b) size distribution of 590 nm emitting quantum dots (QDs) revealing a mean core diameter of 6.4 nm. (c) The absorption and emission spectra of these QDs with first exciton peak at 570
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nm and emission peak at 590 nm. (d) Surface modification scheme showing the conversion of mercaptoacetic acid coated QDs (QD-MAA) to HS-PEG(5 kDa)-methoxy coated QD-PEG. (e) Agarose gel electrophoresis shows the relative migration of QD-MAA (negatively charged) and QD-PEG (neutral). (f) Hydrodynamic diameter of QD-MAA and QD-PEG as measured by dynamic light scattering. (g) Post-clearing retention of quantum dots in vitro through cross linking with tissue proteins. Error bars indicate standard deviation of 3 replicates.
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Figure S2: In Vivo retention of nanoparticles with different sizes. Increasing the core size of nanoparticles from 6.4 nm (QD-PEG) to 100 nm (Gold Nanoparticles; Au NP) increases in vivo retention. Moreover, the processing is compatible across different material types and sizes, which is relevant for understanding the impact of physiochemical properties of nanomaterials in vivo. Error bars indicate standard deviation of 3 replicates.
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Figure S3: Parametric characterization of clearing rate in different organs. (a) The degree of clearing was quantitatively analyzed by measuring optical density (OD) of the tissue using a spectrophotometer. The readout is a spatial heatmap of the different OD signals for each tissue. This was subsequently processed in MATLAB to generate mean OD from the identified tissue area. (b and e) The rate of clearing for the brain, kidney, and liver was measured for 28 days. The data shows that brain hemispheres have the fastest rate of clearing in comparison to liver and kidney. Representative photographs show the increased transparency upon clearing for 28 days and subsequent index matching. (c and f) The rate of clearing for brain hemispheres decreased with increasing acrylamide concentration (2 – 8 %) in hydrogel monomer solution in both active and passive clearing. Photographs of cleared brains confirming the extent of clearing at 28 days with increasing acrylamide concentration. (d and g) Upon raising the temperature to 50°C, the time to reach the endpoint absorbance was decreased ~3 fold. The combined effect of clearing at 50°C with tissues embedded in 2% acrylamide hydrogel led to approximately 4.5-fold faster rate of clearing compared to conventional conditions of 4% acrylamide and 37°C. Error bars indicate standard deviation of 3 replicates. * Clearing was terminated to avoid tissue damage, which is expected around OD 0.33.
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Figure S4 Increasing SDS concentration from 4% to 8% does not affect rate of clearing. OD was measured in 4% acrylamide cross-linked kidneys. Profile of OD against time (days) for (a) kidney, active clearing at 50°C, and (b) kidney, active clearing at 37°C. Increasing SDS concentration did not change the rate of clearing for any tested parametric condition. Error bars indicate standard deviation of 3 replicates. SDS = sodium dodecyl sulfate. OD = optical density.
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Figure S5: QD-PEG imaged and analyzed in multiple organs. (a) 3D maximum intensity projection (MIP) and 2D XZ projection of an image of cleared liver with nuclei labelled in blue (DAPI), blood vessels in red (GSL1-A647) and QD-PEG labelled in green. The corresponding 2D XY projection shows that most QD-PEG associated with blood vessels in apparent endosomal compartments. (b) 3D MIP and 2D XY projection of kidney show QD-PEG associated with vessels particularly in the glomeruli but absent from renal tubules. (c) 3D MIP and 2D XY projection show QD-PEG in spleen. Scale bars indicate 100 µm in 3D MIP of (a), (b), (c) and 30 µm for 2D XY projections in (a), (b), (c)
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Figure S6: Probing the protein loss due to clearing at different temperatures. The protein loss was measured in brains and kidneys at different temperatures using the Bicinchoninic acid assay. (a, b) Raising the clearing temperature to 50°C for clearing brains increased protein loss to 16% but had no effect for kidneys. (c) In addition to tissue disintegration (not shown) and loss of fluorescence, clearing brains at 55°C increased protein loss to more than 20%. Error bars reflect standard deviation. n=3 for all points.
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Supplementary Videos S1 - S2 3D distribution of quantum dots in mouse livers. Liver lobes from CD1 Nude mice injected with QD-PEG were cleared. The fluorescence of QD-PEG is retained and distribution with respect of vasculature, (red; GSL-1 lectin conjugated to Cy5) spanning several millimeters across lobes, can be easily visualized for mapping spatial relationships (Video S1). In addition of vessels, the spatial mapping of QD-PEG with respect to cells (DAPI) is visualized in Video S2. While most QD-PEG localize near or within vessels, their subcellular accumulation inside discrete puncti indicates cellular uptake and subsequent localization into cell organelles, perhaps endosomes. S3 - S4 3D distribution of quantum dots in mouse kidneys. These videos show low (Video S3) and high (Video S4) magnification spatial distribution of QD-PEG in mouse kidneys with respect to vasculature and cells. QD-PEG are not found in tubules as expected due to their larger size compared to excretion size cut-off. They were found to be localized within the glomeruli and blood vessels. S5 3-D distribution of quantum dots in mouse spleen. These video shows spatial distribution of QD-PEG in a mouse spleen with respect to vasculature and cells. S6 Probing fluorescence and tissue integrity in whole tumors. Orthotopic breast tumors were inducted and grown in mice using MDA-MB-231 cells. These tumors were resected and cleared using optimized protocol (50°C, 2% acrylamide). Tissue integrity, fluorescence of endogenous proteins (red; tdTomato expressing MDA-MB-231 cells), molecular labels for vessels (green; GSL-1 lectin conjugated to Alexa647) and nuclei (blue; SYTOX Green) are undamaged and easily visualized. Tumor vasculature is chaotic and heterogeneous in different regions of the tumor with varying vessel distribution, size and tortuosity. S7 Slice-by-slice view of whole tumor showing organization of tumor cells and vasculature. This video shows slice-by-slice emergence and distribution of vessels, cancer cells and nuclei in the orthotopic tumor described above in S6. S8 Light sheet microscopy of whole tumor vasculature. Similar to the tumor developed in Video S6, another optimized protocol cleared orthotopic breast tumor was imaged using light sheet microscopy. The tumor vasculature is heterogeneous, irregular and torturous when compared to surrounding muscle vasculature. S9 Slice-by-slice view of whole tumor showing organization of tumor vasculature post light sheet microscopy. This video shows slice-by-slice heterogeneity of vessels in the orthotopic tumor described above in S8. Supplementary Code S10: Matlab script used for calculating OD for an organ. Attached script converts the output from Tecan M200 Pro reader (measured with “15x15 circle filled” setting) into a mean absorbance for an organ. Organ must be measured in a 6-well plate. See “Instructions for ODcalc.txt” for details.
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Supplementary Code S11: Matlab scripts used to map nuclei and QDs against distance from vessels. Attached script generates the number of nuclei and total fluorescence of QDs at any given distance from blood vessels in an image. Requires images (all tif series): raw image of each channel, binary segmented image of each channel, image with labelled nuclei and the positive and negative distance transformation of the blood vessel segmentation. The raw imaged used for this analysis is available at: https://dx.doi.org/10.6084/m9.figshare.3167908.v1
