stm.sciencemag.org/cgi/content/full/11/506/eaaw7736/DC1

Supplementary Materials for

Imaging-assisted nanoimmunotherapy for atherosclerosis in multiple species

Tina Binderup, Raphaël Duivenvoorden, Francois Fay, Mandy M. T. van Leent, Joost Malkus, Samantha Baxter, Seigo Ishino, Yiming Zhao, Brenda Sanchez-Gaytan, Abraham J. P. Teunissen, Yohana C. A. Frederico, Jun Tang,

Giuseppe Carlucci, Serge Lyashchenko, Claudia Calcagno, Nicolas Karakatsanis, Georgios Soultanidis, Max L. Senders, Philip M. Robson, Venkatesh Mani, Sarayu Ramachandran, Mark E. Lobatto, Barbara A. Hutten, Juan F. Granada,

Thomas Reiner, Filip K. Swirski, Matthias Nahrendorf, Andreas Kjaer, Edward A. Fisher, Zahi A. Fayad*, Carlos Pérez-Medina, Willem J. M. Mulder*

*Corresponding author. Email: willem.mulder@mssm.edu (W.J.M.M.); zahi.fayad@mssm.edu (Z.A.F.)

Published 21 August 2019, Sci. Transl. Med. 11, eaaw7736 (2019) DOI: 10.1126/scitranslmed.aaw7736

The PDF file includes:

Materials and Methods Fig. S1. S-HDL characterization. Fig. S2. In vivo S-HDL cell specificity evaluation. Fig. S3. Pharmacokinetics and plaque targeting of S-HDL in rabbits and pigs. Fig. S4. S-HDL treatment effect in rabbits and pigs. Fig. S5. Histological evaluation of S-HDL treatment. Fig. S6. S-HDL treatment effect in individual rabbits and pigs. Table S1. Composition and size of different S-HDL batches prepared for this study.

Other Supplementary Material for this manuscript includes the following:

(available at stm.sciencemag.org/cgi/content/full/11/506/eaaw7736/DC1)

Data file S1 (Microsoft Excel format). Primary data.

Materials and Methods

HPLC and radio-HPLC. HPLC was performed on a Prominence HPLC system

(Shimadzu) equipped with two LC-10AT pumps and an SPD-M10AVP photodiode array

detector. Radio-HPLC was performed using a Lablogic Scan-RAM Radio-TLC/HPLC

detector. Size exclusion chromatography was performed on a Superdex 10/300 column

(GE Healthcare Life Sciences) using PBS as eluent at a flow rate of 1 mL/min.

Culturing of bone marrow-derived macrophages (BMDMs). Bone marrow was

harvested from C57BL/6 mice. Cells were washed with PBS and red blood cells were

lysed. Cells were cultured in RPMI supplemented with 10% FBS, 1% Pen-Strep, 1% L-

Glutamine, MEM non-essential amino acids and 1 mM HEPES buffer (Gibco by

Life Technologies). Recombinant murine GM-CSF and IL-4 were obtained from

Peprotech were added both at a final concentration of 20 ng/mL. Media was changed after three days

and after another three days BMDMs were used for in vitro experiments.

Confocal laser scanning microscopy of S-HDL uptake in BMDMs. BMDMs were

plated on coverslips at the bottom of 6-well plates at a density of 1.2x105 cells per well and

incubated with either benchtop- or microfluidizer-produced DiO-labeled S-HDL for 24

hours. The same concentration of DiO was used for both formulations: 2.2 μg/mL diluted in

complete RPMI medium. Cells were washed with Hank’s Balanced Salt Solution (HBBS)

and fixated with 4% paraformaldehyde for 15 minutes at 37 0C. After fixation, cells were

washed three times with HBBS and labeled with 5 μg/mL Wheat Germ Agglutinin (WGA)

Alexa Fluor 555 Conjugate antibody, from Life Technologies for 10 minutes at room

temperature. Slides were then mounted with VECTASHIELD Vibrance Antifade Mounting

Medium with DAPI, purchased from Vector Laboratories. Fluorescence was visualized with

a Leica SP5 DM confocal laser microscope using a 63x oil immersion objective lens (1.4

numerical aperture).

In vitro evaluation of S-HDL formulations in BMDMs. BMDMs were plated in a 96-well

plate at 5x104 cells/well and incubated with 10 μM of benchtop or microfluidizer S-HDL.

After 24 or 48 hours, cell viability was measured using CellTiter-Glo Luminescent Cell

Viability Assay following the manufacturer’s instructions. To asses proliferation, BMDMs

were plated at 750.000 cells/mL and incubated with 10 μM of benchtop or microfluidizer S-

HDL. After 4 hours, BrdU was added at 10 μM and incubated for another 20 hours. Cells

were further processed following the instructions of the manufacturer (BD Pharmingen

BrdU flow kit).

Fluorescent labeling of S-HDL. To study potential biological differences between S-HDL

generated by traditional benchtop methods or high-pressure homogenization, batches

were produced that included DiO. For NIRF imaging, DiD-S-HDL was prepared, as

previously reported (6, 7), by adding DiD to the phopholipid/simvastatin mix at a 1.5% w/w

during the lipid film formation process. Finally, DiD-S-HDL was mixed with unlabeled S-

HDL to achieve the desired simvastatin dose.

Evaluation of DiO-S-HDL cellular specificity in mice. Female Apoe-/- mice (n=9, 18

weeks on Western diet; weight: 36.9 ± 4.0 g) were injected with benchtop- or

microfluidizer-produced DiO-labeled S-HDL at 0.5 mg/kg DiO. Twenty-four hours after

injection, mice were euthanized and extensively perfused with saline, and the aortas and

spleens were carefully excised for ex vivo evaluation by flow cytometry.

Near-infrared fluorescence imaging. Shortly after euthanasia, perfused tissue samples

were placed on thick black paper and imaged on a Xenogen IVIS Spectrum Preclinical

Imaging System (Perkin Elmer). Fluorescence images were acquired with selected

excitation and emission band-pass filters: DiD (λExc = 640 nm, λEm = 680 nm) and Evans

Blue (λExc = 605, λEm = 680 nm). Exposure time for each image was 4 s. Data were

processed and analyzed using Living Image software (Perkin Elmer) by drawing a region

of interest around the arteries of interest and quantified as radiant efficiency using Living

Image Software.

Autoradiography. Following euthanasia, animals were perfused and their aortas (mice

and rabbits) or femoral arteries (pigs) were excised and blotted. To determine radiotracer

distribution, digital autoradiography was performed by placing tissue samples in a film

cassette against a phosphorimaging plate (BASMS-2325, Fujifilm) for 2 hours (mouse

aortas) or 48 hours (rabbit aortas, pig femoral arteries) at -20 °C. Phosphorimaging plates

were read at a pixel resolution of 25 μm with a Typhoon 7000IP plate reader (GE

Healthcare). Quantification was carried out using ImageJ software.

Histology and immunohistochemistry. Tissue samples from mice, rabbits and pigs were

placed in paraformaldehyde overnight and subsequently embedded in paraffin. Sections

were taken from the aortic arch in mice, from the abdominal aorta of rabbits, and from the

abdominal aorta just proximal to the iliac bifurcation and from the largest palpable plaque

in the right femoral artery in pigs. Samples were cut in 5-μm sections, mounted on slides

and stained with Hematoxylin & Eosin (rabbits), Masson trichrome (mice, rabbits and pigs),

CD68 (mice) and RAM-11(rabbits) according to standard protocols.

Blood tests in pigs. Blood was collected by vena puncture from all pigs at the end of the

study. Serum was sent to IDEXX Laboratories and analyzed using an Olympus AU400

Chemistry Analyzer. Whole blood was collected in EDTA-containing tubes and analyzed

using an IDEXX procyte DX Hematology Analyzer for complete blood count analysis.

Fig. S1. S-HDL characterization. (A) Representative transmission electron microscopy (TEM) images of reconstituted high-density lipoprotein (rHDL, left), benchtop-produced simvastatin-HDL (S-HDL, middle), and microfluidizer-produced S-HDL (right). Scale bar = 10 nm. (B) Murine BMDM cell viability in the absence (control, C) and presence of benchtop- (B) and microfluidizer-produced (M) S-HDL, at 24 and 48 hours of incubation (n = 8 per condition). (C) Gating procedure used in the flow cytometric evaluation of murine BMDM proliferation assay. (D) Murine BMDM proliferation in the absence (control, C) and presence of benchtop- (B) and microfluidizer-produced (M) S-HDL, at 24 and 48 hours of incubation (n = 4 per condition). (E) Confocal microscopy images of murine BMDMs that were incubated with benchtop- or microfluidizer-produced DiO-S-HDL, or were left

untreated (control). Scale bar = 30 m. DAPI: nuclei; DiO: S-HDL; WGA: wheat germ agglutinin, cell surface stain. (F) HPLC size exclusion chromatograms demonstrating co-elution of [89Zr]-S-HDL (blue trace, radioactivity signal) and unlabeled S-HDL (black trace, UV absorption at 220 nm). Data are presented as mean ± SD from one experiment, and were analyzed using ANOVA (Kruskal-Wallis) followed by multiple comparisons. * P < 0.05, **** P < 0.0001.

Fig. S2. In vivo S-HDL cell specificity evaluation. (A) DiO mean fluorescence intensity (MFI) in different cell types from Apoe-/- mouse aortas 24 hours post administration of benchtop- (B) and microfluidizer-produced (M) DiO-S-HDL (n = 4). MFI shown in normal scale (left) and log scale (right). (B) Gating procedure used in the flow cytometric evaluation of DiO-S-HDL’s cell specificity in Apoe-/- mouse spleens. (C) DiO mean fluorescence intensity (MFI) in different cell types from Apoe-/- mouse spleens 24 hours post administration of benchtop- (B) and microfluidizer-produced (M) DiO-S-HDL (n = 4). MFI shown in normal scale (left) and log scale (right). C = control. Lin+ = lineage positive; Neu = neutrophils; MØ = macrophages; Ly6Chi Mo = monocytes; DC = dendritic cells. Data are presented as mean ± SD from one experiment, and were analyzed using ANOVA (Kruskal-Wallis) with multiple comparisons.

Fig. S3. Pharmacokinetics and plaque targeting of S-HDL in rabbits and pigs. (A) Blood time–activity curve for [89Zr]-S-HDL in rabbits (top, n=2) and pigs (bottom, n=2) with atherosclerosis. (B) Representative Evans Blue near-infrared fluorescence imaging (left) and [89Zr]-S-HDL autoradiography (right) performed on arterial samples from one rabbit (abdominal aorta, top) and one pig (femoral artery tree, bottom). (C) Representative fluorescence microscopy images of arterial sections from one rabbit (top, scale bar = 500 µm) and one pig (bottom, scale bar = 500 µm) injected with DiD-S-HDL. The yellow boxed region is shown at higher magnification below top image (scale bar = 100 µm).

Fig. S4. S-HDL treatment effect in rabbits and pigs. (A) Variation in the monitored parameters in rabbits treated with PBS (placebo, left) or S-HDL (right) between baseline (B) and terminal (T) scans. (B) Variation in the monitored parameters in pigs treated with PBS (Placebo, left) or S-HDL (right) between baseline (B) and terminal (T) scans. Dots are color-coded for individual animals. Two data points are represented per pig, corresponding to each of the femoral arteries. VWA = vessel wall area; IAUC = intensity area under the curve; TBR = target-to-background ratio. Data were obtained in one experiment and analyzed using a linear mixed model (see Statistical analysis for details).

Fig. S5. Histological evaluation of S-HDL treatment. (A) Rabbit aortic sections stained with hematoxylin & eosin (H&E, top), Masson trichrome (middle) and RAM11 (macrophages, bottom) from animals treated with PBS (placebo, left) or S-HDL (right). (B) Porcine femoral artery sections (right) stained with Masson trichrome from animals treated with PBS (Placebo) or S-HDL. Sections were taken from the iliac bifurcation (top) and largest plaque in the femoral artery (bottom) of the same pig. Scale bar = 500 µm.

Fig. S6. S-HDL treatment effect in individual rabbits and pigs. (A) Combined representation of the variation in the monitored vessel wall parameters in individual rabbits treated with PBS (Placebo, top) or S-HDL (bottom), expressed as % change between baseline and terminal scans. (B) Combined representation of the variation in the monitored vessel wall parameters in individual pigs treated with PBS (Placebo, top) or S-HDL (bottom), expressed as % change between baseline and terminal scans. For FDG and FLT, data represent variation in TBRmax; VWA = vessel wall area; VWP = vessel wall permeability.

Table S1. Composition and size of different S-HDL batches prepared for this study. *Based on simvastatin. d.nm: diameter (in nanometers). Đ: dispersity index.

Composition (g) Size

Batch # APOA1 Simvastatin DMPC MHPC Recovery* (%) d.nm Đ

1 0.704 1.235 6.683 0.690 57.8 21.8 0.255

2 0.692 1.214 6.569 0.678 70.8 30.8 0.294

3 0.704 1.235 6.683 0.690 65.8 21.1 0.261

4 1.500 2.631 14.239 1.469 75.5 24.5 0.224

5 1.250 2.192 11.866 1.225 61.8 22.3 0.239

6 0.441 0.773 4.186 0.432 60.8 20.4 0.241

7 0.900 1.578 8.543 0.882 71.3 28.0 0.215

8 0.700 1.228 6.645 0.686 71.8 22.0 0.156