Towards non-invasive diagnostic imaging of early- …...Towards Non-Invasive Diagnostic Imaging of Early-Stage Alzheimer’s Disease Kirsten L. Viola 1† , James Sbarboro 1† , Ruchi

Towards non-invasive diagnostic imaging of early-stage Alzheimer’s disease-Supplemental Materials

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Supplemental Materials

Towards Non-Invasive Diagnostic Imaging of Early-Stage Alzheimer’s Disease

Kirsten L. Viola1†, James Sbarboro1†, Ruchi Sureka1†, Mrinmoy De2, Maíra A. Bicca1,5, Jane Wang1,

Shaleen Vasavada1, Sreyesh Satpathy6, Summer Wu6, Hrushikesh Joshi2, Pauline T. Velasco1, Keith

MacRenaris4, E. Alex Waters4, Chang Lu1, Joseph Phan1, Pascale Lacor1, Pottumarthi Prasad3, Vinayak

P. Dravid2#*, William L. Klein1#*

Towards non-invasive diagnostic imaging of early-stage Alzheimer’s disease

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2014.254

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 1

© 2014 Macmillan Publishers Limited. All rights reserved.

http://dx.doi.org/10.1038/nnano.2014.254


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Supplemental Methods

Zeta Potential measurement

Zeta potential was measured by first sonicating the MNS for 5 min, then diluting 100µL of 1mg/mL Fe

concentration of iron oxide particles into 1mL of Milli-Q water and measuring the zeta potential using a Malvern

Sternacell (Zetasizer, Malvern Instruments).

CellTiter 96 MTS assay

MTS assays were conducted, according to manufacturer’s instructions (CellTiter 96 cell proliferation kit,

Promega), in triplicate using 96 well plates seeded at a density of 5000 cells/well and incubated for 24 hr. with

various MNS concentrations. Samples were then washed with PBS before addition of MTS reagents.

Relaxivity

MR relaxivity of the nanostructures was measured using a Bruker minispec 60 MHz (1.41T) relaxometer. T2

relaxation times for the stabilized MNS, in 5mM phosphate buffer, pH7.4, were measured as a function of Fe

concentration (mM), and r2 values were calculated based on a slope of 1/T2 versus Fe concentration.

NU4 antibody

Monoclonal antibody NU4 was developed against Aβ1−42 oligomers and characterized by Lambert et

al.19

and others. This antibody recognizes oligomers isolated from AD brain tissue, blocks binding of

both human-derived and synthetic oligomers to neurons, and shows preferential binding to high

molecular weight (>50 kDa) synthetic oligomers in competition dot blot assays. This antibody

recognizes epitopes dependent on the tertiary structure formed by self-association of Aβ1−42, does not

recognize Aβ1−40 and shows minimal binding to monomeric Aβ1−42.



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Preparation of NU4- and IgG1-Fluorescent conjugates

Non-Immune IgG1 (purchased from Invitrogen), and anti-AβO NU4 IgG were conjugated to fluorescent

dyes using an AlexaFluor 633 or an AlexaFluor 568 Protein Labeling Kit (Invitrogen) and eluted from

the purification resin with sodium azide-free buffer. Antibodies were stored at -80°C prior to

conjugation with MNS or stored at 4°C in 0.01% sodium azide. Sodium azide was removed using a

ZebaSpin Desalting column (Pierce) prior to conjugation.

Aβ Oligomer Preparation

Fluorescently-labeled Aβ oligomers were prepared essentially according to the protocol published by

Klein and colleagues.19

Briefly, Aβ1-42 (American Peptide) or FAM-Aβ1-42 (Anaspec) was dissolved in

hexafluoro-2-propanol (HFIP) and aliquotted into microcentrifuge tubes. Hexafluoro-2-propanol was

removed by evaporation and traces removed under vacuum; the tubes were stored at -80ºC. An aliquot

of each was dissolved in anhydrous dimethyl sulfoxide (DMSO) to ~5 mM, mixed 5:1 (mol: mol) Aβ:

FAM-Aβ, and diluted in ice-cold Ham’s F12 medium without phenol red (Caisson Laboratories) to 100

µM. This solution was incubated at 4ºC for 24 hr. and centrifuged at 14 000 g for 10 min. The

supernatant, defined as the FAM-AβO preparation, was transferred to a clean microfuge tube and stored

at 4ºC until use. Protein concentration was determined using Coomassie Plus protein assay kit (Pierce).

The same protocol was employed to prepare unlabeled A oligomers in the absence of FAM-Aβ1-42.

Cell Culture

Hippocampal cells were prepared and maintained for at least 18 days as previously described23

by using

(0.002%) poly-L-lysine coated coverslips plated at a density of 1.04 x 104 cells per cm

2 in Neurobasal

media (Invitrogen) with B27 supplements and L-glutamine (2.5 µM).



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Aβ Oligomer Incubation and Immunolabeling of Cells

Cells were incubated at 37°C in conditioned media collected from the cell cultures containing FAM-

AβOs or an equivalent dilution of vehicle. Following incubation with AβOs or vehicle for 60 min, the

cells were rinsed rapidly 3 times with warm media then fixed by adding an equal volume of warm 3.7%

formaldehyde (in PBS) to the third rinse in each well/dish and allowing it to sit at RT for 5 min. The

media/formaldehyde was completely removed and replaced with a volume of 3.7% formaldehyde for 5

min at RT. Cells were blocked in 10% normal goat serum (NGS) in PBS or HBSS for 45 min at RT then

incubated overnight at 4°C on an orbital shaker with 633-tagged antibody or 633-NU4MNS diluted in

blocking buffer. The cells were washed 3 times for 5 min each with PBS or HBSS. Coverslips were

mounted onto glass slides using ProLong Gold Anti-fade reagent with DAPI (Invitrogen) and imaged

using an epifluorescence (TE2000, Nikon) or confocal microscope (Leica SP2, Leica Corp).

Immunolabeling of slices

Human cortical brain tissue from AD or non-demented age-matched controls was obtained from the

brain bank at the Cognitive Neurology and Alzheimer’s Disease Center (CNADC) at Northwestern

University (Chicago, IL). Tissue was sliced at a thickness of 50μm, using a freezing microtome and

stored in a 20% sucrose solution in PBS. Floating slices were rinsed 2x10 min with TBS, incubated 5

min in endogenous peroxidase inhibitor solution (10% methanol, 3% H2O2 in TBS), and washed with

TBS, changing solution every 5 min until bubbles were no longer evident and slices stopped floating on

the surface. Slices were rinsed with 0.3% Triton X-100 in TBS (TBS-X) 3 times for 10 min each and

blocked with 10% NGS in TBS-X for 20 min at room temperature. Slices were incubated with Alexa

Fluor 633-NU4, 633-NU4MNS, or 633-IgGMNS in TBS-X at room temperature for 1.5 hr. and

overnight at 4°C with gentle rotation. Finally, slices were washed 5 times for 10 min each with TBS-X

and mounted with ProLong Gold Antifade reagent for examination by fluorescence microscopy.



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Thioflavin S counterstain.

Thioflavin-S counterstaining to NU4 immunofluorescence labeling was performed as previously

described49

with a few modifications. 5xFAD and WT brains were sliced at a thickness of 50μm and

immunolabeled following the same protocol described above (immunolabeling of slices). Slices were

incubated with Alexa Fluor 568-NU4 overnight 4°C with gentle agitation. The slices were then washed

with PBS for 5 times 5 min each and incubated with 0.002% of Thioflavin-S solution in TBS-T (diluted

from a stock solution 0.02% of Thioflavin-S in distillated water) for 10min. Slices were then washed 3

times for 1 min in 50% ethanol and 2 times in TBS-T for 5 min. The slices were mounted with ProLong

Gold Antifade reagent for examination by fluorescence microscopy. Images were acquired at 40x

magnification and analyzed by ImageJ software.

Cultured Cell Preparation and Magnetic Resonance Imaging

Cultured hippocampal cells were treated with NU4MNS as described above. Samples were kept

submerged in phosphate buffer for MR imaging with a 7T animal MRI scanner (Bruker). The MR

images were analyzed using ImageJ software (NIH). The images were uniformly thresholded and the

“particle measurement” function of the software was used to calculate the integrated density of the

images in each experimental group. The graphs were plotted using the mean and SEM of at least 5

coverslips from each experimental group from two independent experiments

Brain Slice Preparation for MR Imaging

Human cortical brain slices were treated with NU4MNS or IgGMNS as described above. Samples were

prepared for use with the Bruker PharmaScan 7T animal MR scanner. The sample vessel was prepared

by drilling an opening with a Dremel tool in a 50mL conical BD Falcon tube (115x30mm) for

longitudinal mounting of tissue samples. Samples were transferred onto a layer of low melting point

agarose (1%), where samples were placed side-by-side. A second layer of agarose was laid over the



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tissue slices and a second tissue layer was placed on the agarose with slices side-by-side. A final layer

of agarose was placed on top of the second tissue layer. In order to induce the agar to harden, the tube

was repeatedly refrigerated for several min at 4°C.



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Supplemental Materials

Towards Non-Invasive Diagnostic Imaging of Early-Stage Alzheimer’s Disease

Kirsten L. Viola1†

, James Sbarboro1†

, Ruchi Sureka1†

, Mrinmoy De2, Maíra A. Bicca

1,5, Jane Wang

1,

Shaleen Vasavada1, Sreyesh Satpathy

6, Summer Wu

6, Hrushikesh Joshi

2, Pauline T. Velasco

1, Keith

MacRenaris4, E. Alex Waters

4, Chang Lu

1, Joseph Phan

1, Pascale Lacor

1, Pottumarthi Prasad

3, Vinayak

P. Dravid2#*

, William L. Klein1#*



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Supplemental Figure Legends

Supplemental Fig. 1. Attachment of NU4 antibody to hippocampal neurons is stringently AβO

dependent. Hippocampal cells, treated with 100nM FAM-AβOs (Left) or vehicle control (Right), were

probed with fluorescent NU4 (633-NU4). Findings demonstrate that NU4 is highly oligomer specific

and oligomer dependent. (FAM-AβOs are in green, oligomers detected by NU4 are in red, and co-

localization is seen in yellow) with nearly 100% of the fluorescent oligomers detected by the NU4

antibody (boxed area shown as detailed enlargement at bottom of panel). NU4 thus affords an excellent

targeting antibody for development of an AβO-specific MRI probe.

Supplemental Fig. 2. Characterization of MNS: MNSs are PEG-carboxylate ligand stabilized,

uniform in size, and non-toxic. The stability and uniformity in size and charge was characterized by

gel electrophoresis, dynamic light scattering (DLS) and zeta-potential measurement. Toxicity was

measured using ViaCount cell viability assay. (a) Gel electrophoresis in 1% agarose of 12 nm MNS

indicate the negative charge, stabilization and size distribution similar to conventionally used 2 nm and

20 nm gold nanoparticles stabilized with similar thiol-PEG-carboxylate ligands. (b) Dynamic light

scattering (DLS) measurement of MNS (and NU4MNS, not shown), show narrow size distribution of

hydrodynamic radii consistent with physical measurements by TEM. (c) The zeta potential of MNS,

determined by Malvern Zetasizer, was a high negative value (-40 mV), suggesting better aqueous

stability. (d) Hippocampal neuron viability after 24 hour MNS treatment using ViaCount assay with a

flow-cytometry cell counter.

Supplemental Fig. 3. MNS-antibody conjugate retains oligomer specificity and affinity. a) Antibody

conjugation to MNS does not diminish specificity for Aβ oligomers. Hippocampal cells were treated with

FAM-Aβ oligomers (pink, left column) and then probed with NU4, NU4MNS, or non-specific IgGMNS



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(detected by fluorescent secondary antibodies; turquoise, middle column). The coincidence of FAM-

AβOs and NU4MNS (purple, right column) was found to be virtually identical to that seen using

unmodified NU4. Non-specific IgGMNS did not detect Aβ oligomers or bind cells non–specifically,

showing MNS itself was not mediating the binding. b) Immunoprecipitation experiments show NU4MNS

maintains affinity for AβOs. NU4MNS were incubated with increasing concentrations of AβOs. Bound proteins

were eluted from the MNS, separated by SDS-PAGE and transferred to nitrocellulose. The blots were probed with

monoclonal anti- AβO antibody. The data show increasingly intense trimer and tetramer bands isolated as the

AβO concentration increases. The non-specific bands detected at the top of the blot are due to antibody and cross-

linked antibody being released from the nanostructures with the SDS extraction. c) Western blot of IPed AβOs

shows that NU4MNS but not IgGMNS bind AβOs in solution. AβOs in solution were immunoprecipitated with

either NU4MNS or IgGMNS, eluted and separated by SDS-PAGE. Blots were probed with a polyclonal anti-

AβO antibody. Data show that only the NU4MNS isolate 2 Aβ oligomer bands, identical in molecular weight to

the bands detected in the non-IPed AβOs control lane. Taken together, these results demonstrate that the

NU4MNS bind AβOs with specificity and high affinity, that there is no loss of affinity or specificity for AβOs

when NU4 is conjugated to MNS, and show that AβO binding by MNS is NU4 dependent.

Supplemental Fig. 4. NU4MNS probe characterization by MRI. a) Serial dilutions of probe were

imaged by the 7T MR imager to determine the relaxivity of the probe. b) Hippocampal cells, grown on

glass coverslips, were treated with Aβ oligomers or vehicle and probed with NU4MNS before imaging

in a 7T MR scanner. The magnetic nanostructures, detected as dark black, dense spots, were quantified

for each coverslip. The MRI signal for Aβ oligomer-treated neurons showed three times greater density

than vehicle-treated neurons. c) In vivo analysis by MRI of the change in distribution of the intranasally

delivered probe shows that NU4MNS binds to the hippocampus of Tg mice within 4 hours of

inoculation. The probe has significantly reduced MR signal 96-hours later, but is still evident in the



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hippocampus of the Tg mice. Re-inoculation with the probe on day 5 shows that AβOs are still prevalent

in the hippocampus, suggesting that the probe is not depleting AβOs as it clears the brain.






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Towards non-invasive diagnostic imaging of early- …...Towards Non-Invasive Diagnostic Imaging of Early-Stage Alzheimer’s Disease Kirsten L. Viola 1† , James Sbarboro 1† , Ruchi