DOI: 10.1038/NNANO.2014.62 Selective uptake of single ... · Selective uptake of single-walled carbon nanotubes by circulating monocytes for enhanced tumour delivery Bryan Ronain

Selective uptake of single-walled carbon nanotubes by circulating monocytes for enhanced tumour deliveryBryan Ronain Smith, Eliver Eid Bou Ghosn, Harikrishna Rallapalli, Jennifer A. Prescher, Timothy Larson, Leonore A. Herzenberg, Sanjiv Sam Gambhir

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2014.62

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 1

© 2014 Macmillan Publishers Limited. All rights reserved

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Supplementary Fig. S1. Intravital microscopy system used to image monocytes and nanoparticles inside living mice. Call-‐out: SCID mouse with dorsal window chamber imaged via intravital microscopy using customized motion stabilizer. Supplementary Fig. S2 Negative control for hyperspectral dark-‐field microscopy for Fig. 1c-‐d a. b.

c.

Supplementary Fig. S2. Negative control using darkfield hyperspectral microscopy. a. White blood cell harvested from mouse without SWNTs. We harvested blood from mice injected with vehicle (PBS) but no SWNTs. The SWNT hyperspectral library (Supp. Fig. S2c), generated using a solution of SWNTs only, was overlaid on the


image and no SWNTs were observed (thus the lack of red pseudocolored pixels, as observed in Fig. 1c when SWNTs are present). b. A screenshot of the pixel-‐by-‐pixel analysis, with a representative sampling of pixels, showing that no pixel out of the 277,704 pixels in the image from Supp. Fig. S2a matched the hyperspectral SWNT library. c. Illustrates how the SWNT spectral library was generated. On the left is a cluster of SWNTs on a slide. On the right is a spectral library taken from SWNTs all over the slide. Each curve is derived from a single pixel. Three of the spectra on the right are generated from the pixels to which the red arrows point on the left.


Supplementary Fig. S3 Uptake of SWNTs by Ly-‐6Chi monocytes does not activate them by 6h after injection

Supplementary Fig. S3. SWNT uptake does not activate monocytes based on surface marker expression analysis. Plots represent expression levels of CD11b (top) and Ly-‐6C (bottom) on the surface of monocytes 2h and 6h after SWNT uptake in living mice. The FACS analysis is reported as histograms indicating the fluorescence intensity of CD11b and Ly-‐6C surface expression on Ly-‐6Chi monocytes. Monocytes are known to up-‐regulate surface markers (e.g., CD11b and Ly-‐6C) within a few hours of activation1. However, our data show SWNT uptake does not alter the surface phenotype of monocytes 2h and 6h after intravenous injection, indicating a lack of cell activation after monocyte uptake of SWNTs.

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Supplementary Fig. S4 Monocyte interaction with endothelium over time a

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RAD vs RGD: p = 0.0103 Supplementary Fig. S4. The dependence of peptide (conjugated to the SWNT surface) on monocyte interaction with endothelium over time. a. Based on videos of monocytes in blood vessels in the RGD-‐SWNT, RAD-‐SWNT and plain SWNT conditions, counts were made of monocytes interacting with vascular endothelium, normalized by unit time and by unit vascular volume and plotted here as crude rates. The number of monocytes containing RAD-‐SWNTs and cy5.5-‐SWNTs that interact with the vasculature rise over time, while the number of interacting monocytes in the RGD-‐SWNT condition decreases slightly over a week. This corresponds with our observation of increased numbers of RGD-‐SWNT laden monocytes over time in tumour interstitium (Fig. 3b). Note also the much lower overall number of monocytes laden with RGD-‐SWNTs compared with RAD-‐SWNT and Cy5.5 conditions over the time range indicated. This may suggest that monocytes in the RGD-‐SWNT condition enter tumour interstitium more quickly than in the other conditions, perhaps due to RGD interaction with its integrin cognate, based on our data in Figure 3. This would explain our observation of more monocytes loaded with RGD-‐SWNTs in the tumour interstitium, yet fewer monocytes loaded with RGD-‐SWNTs on the vascular endothelium surface compared to controls RAD-‐SWNT and Cy5.5-‐SWNT. Thus the idea that RGD on SWNTs encourages interaction of monocytes with the tumour vascular endothelium surface and supports increased deposition of monocytes is consistent across our intravital imaging (e.g., Fig. 3 and Supplementary Fig. S4) and Raman imaging (Supplementary Fig. S5) results. Nevertheless, further validation experiments are necessary. b. We perform statistics on monocytes interacting with endothelium on day 1 and up to day 7 (integrated from day 2 to day 7). We computed crude rates for plain (no peptide) SWNTs, and computed the statistics for rate ratios between Plain vs. RAD-‐SWNT and Plain vs. RGD-‐SWNT conditions. On day 1, 927 observations were made of blood vessels; up to day 7, 1112 observations were made. n=11 mice were evaluated. On both day 1 and up to day 7, p-‐values show that Plain and RAD-‐SWNTs are not significantly different, but Plain vs. RGD-‐SWNTs display a significant difference as do RAD-‐ vs. RGD-‐SWNTs.

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Supplementary Fig. S5 Raman intensity of SWNTs in tumour over time

Supplementary Fig. S5. Raman intensity of SWNTs in tumour over time p.i. The Raman signal in the window of mice injected with RGD-‐SWNTs and RAD-‐SWNTs was measured from day 3 to day 9 post-‐injection, and the linear trendlines and data points are shown. a. In the RAD-‐SWNT condition, the total amount of SWNTs is slightly increasing within the tumour over time, despite the fact that increased numbers of SWNTs can not arrive from the vasculature, since the circulation half-‐life is on the order of 2-‐3 hours. Therefore the rise must be solely due to monocyte entry into tumour. Individual data points are plotted for n=4 mice. b. The total amount of RGD-‐SWNTs in tumour over the same time period also increases. It increases more rapidly than RAD-‐SWNTs, with a slope nearly 20-‐fold

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higher. Combined with Supplementary Fig. S4 and Fig. 3, these trends indicate that the RGD on the SWNTs may increase the affinity for RGD-‐SWNT laden monocytes to interact with vasculature and enter tumour interstitium. Individual data points for RGD-‐SWNT injected mice comprising (b) are plotted for n=6 mice. Because the mice were imaged with both intravital microscopy and Raman microscopy, at some time-‐points some mice were sometimes unavailable for Raman imaging for reasons of health maintenance (e.g., due to exposure to anesthesia).


Supplmentary Fig. S6 SWNTs require phospholipid-‐PEG for solubilisation

Supplementary Fig. S6. Top: Photo of SWNTs either with phospholipid-‐PEG (left) or in pure water without any PEG (right). In this experiment, SWNTs were sonicated either with PL-‐PEG (left) or without PL-‐PEG (right) (as shown) and then centrifuged. Bottom: Spectroscopic data of the supernatant solutions from each tube after centrifugation. No signal is observed from SWNTs without PEG. This indicates that PL-‐PEG is bound to the SWNT surface. O.D. Optical Density.

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Supplementary Fig. S7 Cy5.5 is attached to PL-‐PEG

Supplementary Fig. S7. Cy5.5 is attached to the amine on PL-‐PEG (which is attached to SWNTs, based on Supplementary Fig. S6). Top: Spectra of SWNT-‐aminePEG-‐Cy5.5 and control SWNT-‐methoxy-‐PEG-‐Cy5.5 (SWNT-‐mPEG-‐Cy5.5) after conjugation to Cy5.5 and filtration via centrifugal filter. Bottom: SWNT-‐subtracted spectra of the same samples. This demonstrates that while there appears to be some direct stacking of the dye onto the nanotube (no reaction should be expected between dye and the methoxy group), the majority of dye on the SWNT is attached via the amine functional group on amine-‐PEG. Note that the initial dye concentration was the same for the methoxy-‐ and amine-‐PEG groups. O.D. Optical Density.

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Supplementary Fig. S8 TEM of SWNTs

Supplementary Fig. S8. SWNTs were air dried onto a grid and imaged using high-‐resolution TEM (FEI Titan 80-‐300, Environmental TEM). The green arrows point to SWNTs, while the yellow arrow designates a nanoparticle catalyst.


Supplementary Movie Captions Supplementary Movie 1 SWNTs at a concentration of 0.068 mg/ml (400 nM, based on assumptions of 1.2 nm SWNT diameter and 150 nm average length) are injected via tail vein into the mouse. Within seconds of injection, SWNTs (grayscale) rush into blood vessels (red) in the tumour (green). Cells containing SWNTs (larger, discrete compartments in the vessels) are later observed moving through the blood vessels. Supplementary Movie 2 SWNT-‐loaded cells were collected from mouse blood and isolated using FACS. Here a live Ly-‐6Chi monocyte containing SWNTs is shown. Applying hyperspectral imaging, Figure 1c-‐d shows that SWNTs are contained within the monocytes. Supplementary Movie 3 SWNT-‐loaded monocytes (grayscale) can be observed rapidly flowing through blood vessels (red) and interacting with vascular endothelium in a tumour in a live mouse approximately 5 hours p.i. The tumour channel is removed from the video for ease of viewing. Supplementary Movie 4 In some blood vessels (red), RGD-‐SWNT-‐loaded monocytes (grayscale) are observed moving rapidly through blood vessels. The arrow designates an RGD-‐SWNT-‐laden monocyte crawling along the surface of the vascular endothelium with subsequent flattening along the vessel wall, as commonly observed in monocyte extravasation into tissues. Supplementary Methods Dorsal Skinfold Chamber Male retired breeder C.B-‐17 SCID mice (>28g body weight) were used for surgical implantation of dorsal skinfold chambers. Briefly, for the surgery, mice were anesthetized using an IP injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight). Two sides of a titanium chamber (APJ Trading, Ventura, CA) were used to sandwich the shaved dorsal skin of the anesthetized mouse. A ~12 mm diameter circle of skin was removed from one side of the sandwich and a 12 mm glass cover slip (Ted Pella, Redding, CA) was used to cover the skin. Animals were given carprofen at 3 mg/kg SC immediately prior to and 2-‐3 days post surgery to help animals recover from surgery. After 3 days, a tumour was inoculated underneath the cover slip. All animal procedures were approved by the Stanford University Institutional Animal Care and Use Committee.


Tumour Model To image tumours, a stable, bright green cell line was produced. U87MG, a human glioblastoma cell line (obtained from American Type Culture Collection, ATCC), was labeled with enhanced green fluorescent protein (EGFP). The stable EGFP expressing cell lines were established using a lentiviral vector (pRRLsin18.CMV-‐EGFP, a gift from Luigi Naldini, HSR-‐TIGET, Italy) with an EGFP transgene as described2. Cells were incubated overnight in media containing high titer virus, after which cells with very high EGFP expression were selectively sorted using FACS. Cells were grown in ATCC-‐recommended medium. C.B-‐17 SCID mice (Charles River, Wilmington, MA) were inoculated with ~500,000 U87MG cells into the dorsal window in low volume. Mice were injected with SWNTs and tumours were imaged 7–10 days after tumour inoculation, at which point they were 4–7 mm in diameter. Flow Cytometry Staining

Red blood cells were lysed using ammonium-‐chloride-‐potassium (ACK) lysing buffer. The remaining immune cells were resuspended at 25 x 106 cells/mL using custom RPMI-‐1640 medium deficient in biotin, L-‐glutamine, phenol red, riboflavin, and sodium bicarbonate (Invitrogen). Cell suspensions were pre-‐incubated with anti–CD16/CD32 mAb to block FcγRII/III receptors and stained on ice for 30min with the following fluorochrome-‐conjugated mAb in an 11-‐color staining combination: FITC-‐labeled anti-‐Ly-‐6C (AL-‐21); PE-‐labeled anti–I-‐Ad (AMS-‐32.1); PECy5-‐labeled anti-‐CD5 (53–7.3); PECy5.5-‐labeled anti-‐CD19 (1D3) or CD11c (N418); PECy7-‐labeled anti–Gr-‐1 (RB6-‐8C5); APC-‐labeled anti-‐CD49b (DX5); APCCy7-‐labeled anti-‐CD11b (M1/70); Pacific Blue-‐labeled anti-‐F4/80 (BM8) and biotin-‐labeled anti-‐CD11c (HL3), CD80 (16-‐10A1), or CD86 (GL-‐1) . The Cy5.5-‐labeled SWNTs (with or without functional peptides) occupy the Cy5.5 channel. Cells were then washed and stained again on ice for 15min with streptavidin Qdot605 (Invitrogen) to reveal biotin-‐coupled antibodies. Antibodies were either purchased (Invitrogen and BD Pharmingen) or conjugated in our laboratory. After washing, stained cells were resuspended in 10 μg/mL propidium iodide (revealed in the PE-‐Texas Red channel) to exclude dead cells. Cells were analyzed on flow cytometry instruments as described in the main text. To distinguish autofluorescent cells from cells expressing low levels of individual surface markers, we established upper thresholds for autofluorescence by staining samples with fluorescence-‐minus-‐one (FMO) control stain sets in which a reagent for a channel of interest is omitted3. Statistical analysis of monocytes flowing through vasculature To test the interaction of SWNT-‐loaded monocytes with tumour vascular endothelium, we recorded videos of n=11 mice in the RGD-‐SWNT, RAD-‐SWNT, or Cy5.5-‐SWNT conditions. Counts of non-‐interacting and interacting monocytes (“interaction” implies interaction with endothelium) were made from approximately one day up to one week post-‐injection of SWNTs using Olympus Fluoview FV300, version 4.3 (Olympus Corporation, Tokyo, Japan) software and


ImageJ (NIH) software. Counts were adjusted by vessel volume (in cubic microns) and duration of observation period (in seconds). Adjusted counts of each type of interacting cell for the three agents were compared by nonparametric Kruskal-‐Wallis tests for overall differences, Wilcoxon tests for pairwise differences, and Jonckeere-‐Terpstra tests for trend (either RGD>RAD>Cy5.5 or RGD<RAD<Cy5.5). All tests were corrected for ties. For monocyte interactions up to one week after injection, incidence rate ratios among agents were estimated for non-‐interacting and interacting cells by negative binomial regression, with time from injection (expressed as log10 days) as a covariate. Robust standard errors were calculated to adjust for clustering within animal. Statistical analyses were done with Stata Release 9.2 (StataCorp LP, College Station, TX). Raman imaging of tumours Mice were imaged using a Raman Microscope (Renishaw Inc., Gloucestershire, UK). The mouse dorsal skinfold chamber was positioned beneath the microscope objective, with the skin-‐side of the chamber facing up toward the objective4. The microscope has a laser operating at 785 nm with a power of 6 mW. A computer-‐controlled translation stage was used to create a two-‐dimensional map of the SWNT signal across the entire tumour with a 250 μm step size using a 12X open field lens. Quantification of the Raman images was performed using the Nanoplex™ SENSERSee software (Oxonica Inc.), where the mean Raman signal detected from the tumours was calculated. Inductively-‐Coupled Plasma SWNTs (0.068 mg/ml) were heated in a solution containing concentrated nitric acid for 16h and in a solution containing concentrated sulfuric acid, nitric acid, and hydrochloric acid. We analyzed the solutions for iron content via Inductively-‐Coupled Plasma-‐Optical Emission Spectrometry (ICP-‐OES) using a Thermo Scientific ICAP 6300 Duo View Spectrometer. Equipment and settings For Fig. 1a, an Olympus intravital microscope (IV100, Olympus, Center Valley, PA) was used to acquire the image with a 10X Olympus objective lens (NA of 0.4) with 4X digital zoom. The image size was 640 X 640 pixels at 12 bits/pixel with 0.359 μm per pixel in both x and y directions. The full image size was 229.4 μm X 229.4 μm and sampling speed was 2.0 μs/pixel. EGFP from tumour cells was excited with a 488 nm laser line at 10% laser transmissivity with emission collected around 520 nm using an SDM560 dichroic mirror. The PMT (photomultiplier tube) voltage was 340 V. The nanoparticle (cy5.5) channel was collected with an excitation of 633 nm at 80% transmissivity and used a dichroic mirror of SDM750 to collect emission around 693 nm. The PMT voltage was 600 V. In the third channel, Angiosense 750 long-‐circulating dye was excited at 748 nm at 80% transmissivity. The PMT voltage was 632 V. All PMTs were in analog detection mode. The LUT (lookup table) for channel 1 was linear and cut-‐off at a minimum of 204 and maximum of 3220. The LUT for channel 2 was linear and cut-‐off at a minimum of 204. The LUT for channel 3 was linear and cut-‐off at a minimum of 204 and a maximum of 3400. The software


used for acquisition and manipulation for all intravital microscopy was Olympus Fluoview FV300, version 4.3. Animals were maintained at 37°C using a temperature-‐controlled plate and all images were taken in air through a dorsal window chamber implanted on the mice. For Fig. 3a, the IV100 was used to acquire the image with a 20X Olympus objective lens (NA of 0.75). The image size was 640 X 640 pixels at 12 bits/pixel with 0.718 μm/pixel in both x and y dimensions. The full image size was 458.8 μm X 458.8 μm and the sampling speed was 2 μs/pixel. The image is a depth projection of 10 slices at 5.94 μm/slice. EGFP was excited with a 488 nm laser line at 80% transmissivity using an SDM560 dichroic mirror. The PMT voltage was 210 V. The nanoparticle (cy5.5) channel was collected using an excitation of 633 nm at 60% transmissivity and a dichroic mirror of SDM750 to collect emission. The PMT voltage was 584 V. In the third channel, Angiosense 750 was excited at 748 nm with 40% transmissivity. The PMT voltage was 726 V. All PMTs were in analog detection mode. The LUT for channel 1 was linear and cut-‐off at 3666 maximum. The LUT for channel 2 was cut-‐off at 316 minimum, with gamma nearly linear at 1.12. The LUT for channel 3 was linear and covers the full range of data. Supplementary Discussion SWNTs have been employed in many studies over the past several years, showing immense potential for diagnostic and therapeutic purposes. Insights into their uptake into circulating cells upon intravenous injection could substantially improve the use of SWNTs in medical applications and potentially engender novel uses for them. On a side note, there can often be worries about contamination, particularly bacterial, in the nanoparticle solutions that are injected into animals. All aqueous solutions that we use are de-‐ionized, ultra-‐filtered (or Milli-‐Q) water. We sonicate the SWNT solution for an hour, which with SWNTs creates a great deal of local heat; this combination likely destroys a majority of bacteria. The sonication energy that helps to exfoliate the SWNTs through cavitation also kills bacteria around the sonication frequency we apply (significant antimicrobial action has been observed even after 15 minutes, while we sonicate for an hour)5. Then later filtration steps, in which the SWNTs are washed repeatedly, purge the SWNT solution of the small molecules, proteins, and endotoxins released from bacteria. In agreement with this, in general we do not find acute toxicity when injecting our SWNT solutions into mice6. Molecular contaminants within the SWNT solution are expected to be predominantly metal growth catalyst in the form of small iron-‐based nanoparticles. Using ICP-‐OES, we showed that iron was present in our solutions at <20 ppm (19.8 ppm). Note that iron is considered safe in the body – it has been approved by regulatory agencies for decades, for instance as an MRI contrast agent. Furthermore, because the iron is not functionalized with PEG, it likely clears from the bloodstream to the mononuclear phagocyte system (such as the liver and spleen) very quickly.


The goal of most nanoparticle studies in oncology is for the nanoparticles to directly target cancer, and targeting ligands are meant to increase uptake or to increase intracellular localization. Yet targeting cancer directly may not always be the most efficient approach. One reason to target immune cells instead (or additionally) is their innate homing ability to particular tissues, even cancer tissues. However, the natural homing properties of the cells may vary broadly by specific immune cell subset and specific tissue type and site; it may even be desirable to increase the homing properties of certain immune cell subsets. In our case, monocytes already home to tumour tissue more than normal tissues7. Yet further increasing the homing of monocytes to cancer may be useful, for instance to increase the amount of nanoparticles to hypoxic regions as delivered by hypoxia-‐attracted tumour-‐associated macrophages (see the discussion in the main text). Hypoxic regions are typically otherwise inaccessible to potentially therapeutic nanoparticles8. We have evidence that, for the first time, nanoparticles that target circulating cells (monocytes) can be exploited to increase the number of monocytes (and nanoparticles) at the tumour site (and because Ly-‐6Chi monocytes differentiate into tumour associated macrophages, they will be attracted to hypoxic tumour regions). We note that it is possible that our observation of peptide-‐dependent targeting of monocytes to tumour could also be affected by the fact that RGD has anti-‐angiogenic and tumour growth inhibition properties9, and further study is required. Nevertheless, our finding that RGD on SWNTs enhances SWNT-‐loaded monocyte uptake into tumour compared with controls makes this phenomenon a ligand-‐mediated targeting mechanism. This result may suggest that, if an appropriate “vascular zip code” endothelial cell surface protein can be found for a tissue of interest, an appropriate cognate ligand attached to SWNTs could increase Ly-‐6Chi monocytes in that tissue. In the RGD-‐SWNT condition, up to 25% of SWNTs in the tumour were due to monocyte uptake on Day 1 (Fig. 3, main text). Interestingly, while the amount of RGD-‐SWNTs that are bound to tumour cells decreases from day 1 to day 7 p.i.4, a trend is observed in which the total amount of SWNTs in the tumour increases (Supplementary Fig. S5) over the same time period based on the intrinsic signal of SWNTs via Raman imaging. Additionally, the number of monocytes in tumour containing RGD-‐SWNTs increases over the same time period (Fig. 3b, main text). Therefore, while we did not measure the fraction of SWNT uptake due to monocytes on day 7 p.i., these facts integrate to strongly suggest that the amount of RGD-‐SWNTs in the tumour due to monocyte delivery by day 7 is well above 25%. Such a substantial, previously undescribed targeting mechanism may portend a range of novel (immuno-‐)therapeutic and diagnostic applications in oncology.


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DOI: 10.1038/NNANO.2014.62 Selective uptake of single ... · Selective uptake of single-walled carbon nanotubes by circulating monocytes for enhanced tumour delivery Bryan Ronain