doi.org/10.26434/chemrxiv.12730463.v1

[89Zr]Zr(oxinate)4 Allows Direct Radiolabelling and PET Imaging ofSmall Extracellular VesiclesAzalea Khan, Francis Man, Farid Faruqu, Jana Kim, Fahad Al-Salemee, Alessia Volpe, Gilbert O. Fruhwirth,Khuloud T. Al-Jamal, Rafael T. M. de Rosales

Submitted date: 28/07/2020 • Posted date: 29/07/2020Licence: CC BY-NC-ND 4.0Citation information: Khan, Azalea; Man, Francis; Faruqu, Farid; Kim, Jana; Al-Salemee, Fahad; Volpe,Alessia; et al. (2020): [89Zr]Zr(oxinate)4 Allows Direct Radiolabelling and PET Imaging of Small ExtracellularVesicles. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.12730463.v1

89Zr]Zr(oxinate)4 allows direct radiolabelling of exosomes/small extracellular vesicles (sEVs) and in vivoPET-CT imaging

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[89Zr]Zr(oxinate)4 allows direct radiolabelling and PET imaging of small extracellular vesicles

Azalea Khan1, Francis Man1,2, Farid Faruqu2, Jana Kim1, Fahad Al-Salemee1, Alessia Volpe1,3, Gilbert Fruhwirth1, Khuloud Al-Jamal2, and Rafael T. M. de

Rosales1

1 Department of Imaging Chemistry and Biology, School of Biomedical Engineering and Imaging Sciences, King’s College London, St. Thomas’ Hospital, London

2 Institute of Pharmaceutical Sciences, School of Cancer & Pharmaceutical Sciences, King’s College London, Franklin Wilkins Building, London

3 Molecular Imaging Group, Department of Radiology, Memorial Sloan Kettering Cancer Centre, New York

Abstract

[89Zr]Zr(oxinate)4 allows direct radiolabelling of exosomes/small extracellular vesicles (sEVs) and in vivo PET-CT imaging.

Graphical Abstract

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Introduction

Exosomes, better described as small extracellular vesicles (sEVs),1 are cell-derived

nanovesicles enclosed by a phospholipid bilayer, secreted by most cell types.2 They are formed

inside endosomal multivesicular bodies and are released into the extracellular space by

exocytosis. One of the common features of sEVs are their small size, with hydrodynamic

diameters in the 30–150 nm range. They are characterised by the presence of specific

membrane marker proteins such as CD63, CD9, Alix and TSG101.3 The role of sEVs is the

transport and exchange of cytosolic molecules, such as nucleic acids, lipids and proteins

between cells,4 thus acting as messengers in cell-cell communication and disease progression.5

For example, tumour cell-derived sEVs have been shown to promote tumour cell proliferation,6

metastasis,7 and induce anticancer drug resistance.8 Interestingly, both natural and drug-loaded

sEVs (derived from stem cells, immune cells or cancer cells) have shown therapeutic potential

in diseases such as cancer,9 Alzheimer’s disease,10 and type 2 diabetes.11 Furthermore, they

have the ability to cross the blood-brain barrier (BBB),12 and to selectively target different

tissues.13 Therefore, there is an increasing interest in the use of sEVs as nanotherapeutics.14 In

this context, it is important to develop imaging tools that track the in vivo behaviour of sEVs.

Doing so will not only improve our understanding of their biology, but also potentially

maximise their application as nanotheranostic tools.

Optical imaging has been employed to investigate the distribution of cell-derived

sEVs,15-18 but with associated challenges in quantification and signal tissue penetration.

Radionuclide imaging can easily overcome these limitations. In particular, positron emission

tomography (PET) imaging allows highly sensitive and quantitative whole-body imaging, with

no background signal and unlimited tissue penetration in both animals and humans.19 At the

time of writing, there are only a handful of peer-reviewed publications on the radiolabelling

and in vivo imaging of sEVs,18, 20-28 of which only 3 were aimed for PET imaging using two

different radioisotopes (64Cu and 124I).26-28 These PET radiolabelling methods rely on the

binding of these radionuclides to membrane proteins which, given the importance of these

surface components in the role of sEVs as messengers and cell-cell communication,29 may

result in altered biodistribution and function.20, 28, 30 Consequently, we believe that

radiolabelling within the intraluminal space of sEVs is preferable.

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Based on our previous work on cell and liposomal nanomedicine radiolabelling,31-33 we

hypothesised that radiometal complexes that are metastable, lipophilic, and neutral, such as

those based on ionophore ligands, would allow intraluminal sEV radiolabelling. In particular,

we have recently shown that the PET radionuclide 89Zr, complexed by 8-hydroxyquinoline (8-

HQ or oxine) allows direct radiolabelling of liposomes demonstrating intraluminal delivery of 89Zr across the lipid bilayer of vesicles.34 Here, we report a radiochemical synthesis method of

[89Zr]Zr(oxinate)4 that allows efficient radiolabelling of sEVs and in vivo tracking in mice using

PET imaging.

Scheme 1. Schematic representation of the intraluminal 89Zr radiolabelling strategy of sEVs. The lipophilic [89Zr]Zr(oxinate)4 complex is able to pass through the lipid bilayer of the vesicles where 89Zr dissociates from the oxine ligands (that presumably become protonated and are able to cross the lipid bilayer) and 89Zr binds to intravesicular metal chelating ligands, such as proteins and nucleic acids within the sEV.

Results and Discussion

[89Zr]Zr(oxinate)4 synthesis was optimised to allow sEV radiolabelling (Fig. 1A). In particular,

the final solution has to be isosmotic to avoid sEV damage and with a high 89Zr concentration

for in vivo PET imaging studies. To achieve this our synthesis involved the conversion of

[89Zr]Zr(oxalate)4 in 1M oxalic acid, as received from cyclotron production, into [89Zr]ZrCl4

(in 1M HCl) by ion exchange chromatography.35 This was followed by a drying step involving

gentle heating under a flow of N2 gas to remove HCl and H2O, allowing concentration of the

radioactivity. At this point, 80 μL of a pH 7 buffered (HEPES) solution containing 40 µg (0.3

µmol) of oxine and 1 mg/mL polysorbate-80 as a surfactant were added (Method 1). To confirm

the formation of [89Zr]Zr(oxinate)4, the solution was analysed using radiochromatography

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(Whatman® No 1 cellulose and ethyl acetate). Using this system, [89Zr]Zr(oxinate)4 migrates

to the solvent front (Rf = 1), whereas unreacted [89Zr]ZrCl4 stays at the origin (Rf = 0) (Fig.

1B). Performing the reaction at 4°C significantly improved the radiochemical yield (RCY)

compared to room temperature (95.1 ± 2.6% vs. 85.7 ± 4.5%; p <0.0446; n = 3). Partition

coefficient measurements (logD7.4) are consistent with the formation of a neutral lipophilic

[89Zr]Zr(oxinate)4 complex (Fig. 1C). [89Zr]Zr(oxinate)4 was also synthesised using an

alternative method (Method 2) involving reaction of [89Zr]ZrCl4 with oxine as a solution in

EtOH, followed by pH neutralisation (see supplementary methods). No significant difference

in the radiochemical properties were observed between the two methods, based on RCY and

logD7.4 assessments (Fig. S1). However, radiolabelling of sEVs using Method 1 was found to

be highly reproducible and stable. Hence, Method 1 was chosen for in vivo experiments.

Figure 1: (A) Schematic representation of the [89Zr]Zr(oxinate)4 synthesis. (B) Radiochromatogram showing presence of unreacted 89Zr when the reaction was performed at room temperature for 10 min, but not when at 4°C. (C) LogD7.4(PBS) of control 89Zr and [89Zr]Zr(oxinate)4 (n = 3).

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As the release of sEVs from tumour cells is considerably higher than from normal

cells,36-38 we isolated sEVs from the cell culture supernatant of three cancer cell lines (B16.F10-

GFP melanoma, MDA-MB-231.CD63-GFP breast cancer, and PANC1 pancreatic cancer cells)

by precipitation or ultracentrifugation. Upon isolation, sEVs were characterised by size (using

nanoparticle tracking analysis (NTA)) and their protein content was quantified (BCA assay).

Additionally, MDA-MB-231.CD63-GFP and PANC1 sEVs were analysed by dot blot for

expression of sEV markers CD63 and CD9. NTA revealed the average modal diameter for all

three sEVs to be <150 nm, in compliance with the size range for sEVs (Fig. S2). The amount

of protein detected in B16-F10.GFP sEVs was significantly higher than in both MDA-MB-

231.CD63-GFP and PANC1 sEVs (Fig. S3), which could be due to by precipitation isolation

employed to isolate the former sEV types.39 Presence of CD63 and CD9 biomarkers was

confirmed for both MDA-MB-231.CD63-GFP and PANC1 sEVs.

We then tested the sEV radiolabelling capabilities of [89Zr]Zr(oxinate)4. sEVs were

incubated with [89Zr]Zr(oxinate)4 for 20 min at 37°C (Fig. 2A). These conditions were chosen

based on our previous studies showing that [89Zr]Zr(oxinate)4 cell radiolabelling is

temperature-independent and rapid (<20 min).32 Following incubation, a small amount of the

Zr chelator desferrioxamine (DFO) was added to scavenge any potential free 89Zr4+ ions from

the reaction solution, including those that may be weakly bound to the phospholipid membrane,

as previously observed with liposomal vesicles.40 The same sEV radiolabelling procedure was

performed using non-chelated 89Zr as a control (89Zr-control). The reaction mixture was then

passed through a size-exclusion chromatography (SEC) column (Sepharose® CL-2B)20 that

effectively separated sEVs from smaller molecules, including DFO-bound 89Zr. The results,

shown in Fig. 2B, demonstrate significantly higher radiolabelling yields with

[89Zr]Zr(oxinate)4 compared to 89Zr-control for all different sEVs, supporting our hypothesised

radiolabelling strategy. Thus, [89Zr]Zr(oxinate)4 and not unchelated 89Zr is able to pass through

the lipid bilayer membrane into the intraluminal space of sEVs, where 89Zr exchanges ligands

and bind to intravesicular metal-chelating ligands, as we have previously demonstrated in cells

and liposomes.32-34 Importantly, there was no significant change in the hydrodynamic size of

B16-F10.GFP and PANC1 sEVs before and after radiolabelling, unlike MDA-MB-231.CD63-

GFP sEVs (Fig. 2C). However, there was no significant change in the expression of sEV

markers CD63 and CD9 before and after radiolabelling of MDA-MB-231.CD63-GFP and

PANC1 sEVs (Fig. 2D). Taking all these results into account, and the higher RLY achieved

(Fig. S4), PANC1 sEVs were chosen for further in vitro and in vivo experiments.

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Figure 2. (A) Schematic representation of the sEV radiolabelling protocol using [89Zr]Zr(oxinate)4. (B) Radiolabelling yield (RLY) of 1x1010 B16-F10.GFP sEVs (blue) = 5.7 ± 1.3%, 1x1010 MDA-MB-231.CD63-GFP sEVs (green) = 6.2 ± 0.8%, and 1x1011 PANC1 sEVs (maroon) = 16.2 ± 4.0%; (n = 3). (C) NTA data showing the hydrodynamic diameter of respective sEVs before and after radiolabelling, analysed by Student’s unpaired t-test. (D) CD63 and CD9 expression of 231 and PANC1 sEVs by dot blot before and after radiolabelling.

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Radiochemical stability of 89Zr-PANC1 sEVs was assessed in human serum at 37°C

using SEC (Sepharose). However, we found that this system resulted in losses of ca. 80% of

vesicles in the column (data not shown). Hence, we took an alternative approach to evaluate

the in vitro stability that included testing the ability of 89Zr-PANC1 sEVs to be taken up by

cells. Thus, 89Zr-PANC1 sEVs, [89Zr]Zr(oxinate)4 or 89Zr-control were incubated at 37°C in

serum supplemented cell media with the following cells: PANC1 (parental cells), HEK-293T

(healthy cells with known nanoparticle uptake properties),41 MDA-MB-231 and DU-145 (non-

parental cancer cells). Interestingly, 89Zr uptake by both PANC1 cells and HEK-293T cells was

significantly higher for the 89Zr-PANC1 sEV group, compared to the two control groups (Fig.

3A-B). In contrast, there were very low levels of 89Zr-PANC1-sEV uptake by the non-parental

cancer cell lines (Fig. 3C-D). It is worth highlighting the higher uptake of 89Zr-PANC1-sEVs

in both PANC1 and HEK-293T cells compared to that achieved by [89Zr]Zr(oxinate)4, taking

into account that the latter has proven cell-radiolabelling properties.32, 42 Thus, these data

suggest that 89Zr-PANC1-sEVs are stable at 37°C in serum containing media, and demonstrate

quick uptake by both parental cells and HEK-293T cells, but not by other non-parental cancer

cells.

Figure 3: In vitro uptake of 89Zr-PANC1 sEVs by (A) parental PANC1 cells, (B) HEK 293T cells, (C) MDA-MB-231 cells, and D) DU145 cells after co-incubation in serum supplemented media for 4 h. The final cell uptake data were normalised for 50,000 cells. Data given as mean ± SD of n = 3 and analysed by one-way ANOVA with Turkey’s correction for multiple comparisons.

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Encouraged by these results, we performed an in vivo PET-CT imaging and

biodistribution study of PANC1 sEVs in healthy mice (C57BL/6). To assess the impact of

damaged vesicles on the imaging of sEVs, we evaluated two groups: (i) intact 89Zr-PANC1

sEVs and (ii) heat-damaged 89Zr-PANC1 sEVs. The heat damage protocol consisted of two

cycles of heating-cooling (90°C to 0°C) 89Zr-PANC1 sEVs, and aimed at denaturing the

vesicles, but avoiding complete breakdown. Indeed, the heat damage process resulted in a

significant increase in size and partial release of internal contents compared to intact 89Zr-

PANC1 sEVs (Fig. S5). 89Zr-PANC1 sEVs were prepared with a RLY of 32% (starting with 1

x 1012 sEVs). PET-CT imaging within 1 h post intravenous (iv.) injection (~1 x 1010

sEVs/mouse), showed short circulation times and rapid uptake of intact 89Zr-PANC1 sEVs in

the liver, spleen, bladder, as well as several lymph nodes (LN) and brain (Fig. 4A i). Short

circulation times, liver/spleen as well as bladder uptake have been observed in other imaging

studies of sEV biodistribution via iv. administration.16-18, 20, 21, 25-27, 43 However, to the best of

our knowledge, this is the first time LN uptake is observed using in vivo imaging. With the

help of CT imaging, the PET signals observed from the suspected LNs can be correlated with

their well-documented location in mice (e.g. cervical, brachial, pancreatic, renal, inguinal,

popliteal, and others; Fig. S6). sEV/exosome uptake in secondary lymphoid organs (i.e. spleen

and LNs) following iv. injection in the same mouse strain has been recently demonstrated, and

is mediated by CD169+ macrophages.44 Interestingly, sEVs are known to express α-2,3-linked

sialic acid, which is the preferred ligand of CD169 thus providing a plausible explanation for

the high spleen and LN uptake observed.45 The possibility of these imaging signals being due

to released free 89Zr seems improbable due to its significantly different biodistribution (same

mouse strain and timing; data courtesy of Ma et al.46) (Fig. 4A iii). In addition, 89Zr-PANC1

sEVs were visible within the brain (Fig. 4B), supporting the previously reported ability of sEVs

to cross the BBB.12, 47 Heat-damaged 89Zr-PANC1 sEVs showed a similar biodistribution to

intact 89Zr-PANC1 sEVs, with the major differences being a significantly lower spleen uptake

and a higher bone signal (Fig. 4A ii). These two findings can be explained by the bigger size

of the denatured vesicles and partial release of contents, as a result of the heat damaging

process. In both groups bone signal increased at 24 h post injection, as expected due to the

metabolic activity in the liver/spleen that will result in the release of bone-tropic ‘free’ 89Zr. In

addition, fewer lymph nodes were visible and no brain signal was observed.

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Figure 4. (A) Maximum intensity projection PET-CT images of (i) intact 89Zr-PANC1 sEVs, (ii) heat-damaged 89Zr-PANC1 sEVs, and (iii) neutralised 89Zr4+ biodistribution in a C57BL/6 mouse at 1 h and 24 h post-intravenous injection; white arrowheads = representative lymph nodes (see Fig S6), B = bladder, † = PET image scale for control 89Zr at 1 h is 10 times that of the other images; the scale had to be adjusted for image clarity. B) PET-CT images (axial, sagittal and coronal slices) of a mouse injected with intact 89Zr-PANC1 sEVs showing uptake within the brain; image scale is the same as in (A). (C) Ex vivo biodistribution showing uptake of “intact” (n = 3) and “heat-damaged” (n = 2) 89Zr-PANC1 sEVs. D) Ratio of liver:bone uptake (n = 3) and spleen:bone uptake (n = 2); data given as the geometrical mean ± SD of the n values. Statistical significances were calculated using Student’s unpaired t-test.

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The PET-CT imaging findings correlated well with the ex vivo biodistribution data at

24 h post injection, showing high liver/spleen signal and significantly higher uptake of intact 89Zr-PANC1 sEVs in the spleen (58.5 ± 10.5 %ID/g) compared to heat-damaged 89Zr-PANC1

sEVs (19.1 ± 10.3 %ID/g), p = 0.0258 (Fig. 4C). Liver uptake was also higher for intact 89Zr-

PANC1 sEVs, whereas bone uptake was higher for heat-damaged 89Zr-PANC1 sEVs. These

two latter findings are consistent with the imaging studies discussed above but the differences

were not statistically significant. However, differential uptake of intact vs. heat-damaged 89Zr-

PANC1 sEVs was observed when looking at the liver:bone (7.7 ± 2.5 vs. 2.4 ± 0.4, respectively)

and spleen:bone (8.6 ± 2.9 vs. 1.6 ± 0.4, respectively) uptake ratios, suggesting a potential role

of these as imaging biomarkers for assessing the in vivo stability of radiolabelled sEVs (Fig.

4D).

In summary, we have developed and optimised the synthesis of [89Zr]Zr(oxinate)4 for

sEV radiolabelling and demonstrated that this radiotracer allows for a simple, efficient and

direct radiolabelling method for sEVs. Unlike previously reported sEV PET radiolabelling

methods, [89Zr]Zr(oxinate)4 targets the internal components, avoiding potential modification of

surface biomolecules. PET-CT imaging of 89Zr-PANC1 sEVs showed 89Zr uptake in liver,

spleen, brain, as well as suspected accumulation in lymph nodes, according to their location.

We have also demonstrated that heat-damaged 89Zr-PANC1 sEVs show significant differences

in spleen uptake, and liver:bone and spleen:bone uptake ratios that have potential as imaging

biomarkers for sEV stability. Using PANC1 cell derived sEVs as a model, our results

demonstrate that [89Zr]Zr(oxinate)4 is an efficient radiolabelling agent for sEVs. Further work

will be aimed at understanding the nature of the extensive lymph node and brain 89Zr uptake,

and using PET imaging to study the potential role of sEVs as nanotheranostics. We believe this

radiochemical tool will help the sEV field to further investigate their in vivo behaviour, to

answer questions on their basic biology as well as explore their potential as disease biomarkers

and therapeutics.

Acknowledgements:

The authors would like to thank Dr Oskar Timmermand for assistance during dissection, and

Dr Tim Witney for a loan of a 4-mouse hotel for preclinical PET/CT imaging. Azalea Khan is

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supported by the UK Medical Research Council (MRC) [MR/N013700/1] and King’s College

London MRC Doctoral Training Partnership in Biomedical Sciences. This work was also

funded by EPSRC programme grants EP/S032789/1 and EP/R045046/1 and the

Wellcome/EPSRC Centre for Medical Engineering [WT/203148/Z/16/Z]. We also

acknowledge support from the KCL and UCL Comprehensive Cancer Imaging Centre funded

by CRUK and EPSRC in association with the MRC and DoH (England). PET scanning

equipment at KCL was funded by an equipment grant from the Wellcome Trust under grant

number WT 084052/Z/07/Z. Radioanalytical equipment was funded by a Wellcome Trust

Multi User Equipment Grant: A multiuser radioanalytical facility for molecular imaging and

radionuclide therapy research. The authors finally acknowledge support the National Institute

for Health Research (NIHR) Biomedical Research Centre based at Guy's and St Thomas' NHS

Foundation Trust and KCL [grant number IS-BRC-1215-20006]. The views expressed are

those of the authors and not necessarily those of the NHS, the NIHR or the Department of

Health.

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Supplementary Information for:

[89Zr]Zr(oxinate)4 allows direct radiolabelling and PET imaging of small extracellular vesicles

Azalea Khan1, Francis Man1,2, Farid Faruqu2, Jana Kim1, Fahad Al-Salemee1, Alessia Volpe1,3, Gilbert Fruhwirth1, Khuloud Al-Jamal2, and Rafael T. M. de Rosales1

1 Department of Imaging Chemistry and Biology, School of Biomedical Engineering and Imaging Sciences, King’s College London, St. Thomas’ Hospital, London

2 Institute of Pharmaceutical Sciences, School of Cancer & Pharmaceutical Sciences, King’s College London, Franklin Wilkins Building, London

3 Molecular Imaging Group, Department of Radiology, Memorial Sloan Kettering Cancer Centre, New York

Experimental methods

Synthesis of [89Zr]Zr(oxinate)4 (method 1)

89Zr (10 – 100 MBq) in 1M oxalic acid (Perkin Elmer) diluted to 300 µL with deionised water

(pre-treated with Chelex resin, 50–100 mesh size), was loaded onto a pre-conditioned QMA

light cartridge (Sep-Pak, Waters) (conditioned with 5 mL ethanol, 10 mL saline and 10 mL

deionised water). Trapped 89Zr4+ was eluted with 500 µL of 1M HCl, and [89Zr]ZrCl4 was

collected between 150 and 500 µL. [89Zr]ZrCl4 was dried at 60°C under N2 in a Wheaton (V-

bottom) glass vial, followed by addition of 80 µL of aqueous buffered oxine (8-

hydroxyquinoline, 8HQ) solution containing 0.5 mg/mL 8HQ, 1 mg/mL Tween-80 and 1M

HEPES at pH 7.8 was added. [89Zr]Zr(oxinate)4 was then incubated at 4°C or at room

temperature (RT) for 10 min.

Alternative method (method 2): To aqueous [89Zr]ZrCl4, 40 µg 8HQ in ethanol (3M) was

added, and neutralised to pH ~7.2 with 1M NaHCO3. Control 89Zr was prepared by adding

ethanol to [89Zr]ZrCl4 and neutralising to pH ~7.2 with 1M NaHCO3.

[89Zr]Zr(oxinate)4 quality control

Radiochromatography: [89Zr]Zr(oxinate)4 complex formation was confirmed by instant thin

layer chromatography; stationary phase = Whatman N°1 paper (GE healthcare), mobile phase

= 100% ethyl acetate. The chromatograms were analysed on LabLogic Mini-Scan MS-1000F

(Eckert & Ziegler) connected to a β detector and Pearl software, or on Cyclone Plus Storage

Phosphor Imager (PerkinElmer) equipped with OptiQuant software.

Partition coefficient, logD7.4 (PBS): Lipophilicity of [89Zr]Zr(oxinate)4 was assessed using a

biphasic solvent system of PBS in octanol. [89Zr]Zr(oxinate)4 and control 89Zr (10 – 20 µL, 1

MBq) obtained by both formation methods were added to separate tubes, containing 500 µL

of both PBS and octanol. Triplicate samples were prepared. The mixtures were vortexed at

maximum speed for 3 minutes, followed by centrifugation at 16,000g for 3 minutes. Aliquots

from each phase were transferred to separate Eppendorf tubes and activities were measured

using a gamma counter (Wallac Wizard 1282 CompuGamma, Perkin Elmer).

Cell culture

For sEV isolation, all cells were cultured in cell media supplemented by 10% exo-depleted

foetal bovine serum (FBS). FBS was depleted of exosomes by ultracentrifugation at 100,000g

for 18 h at 4°C in Beckman L60 Ultracentrifuge with SW41 Ti rotor (Beckman Coulter),

followed by sterile filtration of the top two layers through a 0.22 µm PES membrane filter

(Merck).

B16-F10.GFP mouse melanoma cells were cultured in high-glucose DMEM

supplemented with 10% exo-depleted FBS, 1% penicillin-streptomycin and 1% L-glutamine in

75-cm2 flasks at 37°C and 5% CO2. Cell supernatant was collected every 2 days and replaced

with fresh exo-depleted cell media. MDA-MB-231.CD63-GFP, human metastatic breast

cancer, and PANC1, human metastatic pancreatic cancer, cells were cultured in CELLine

AD1000 bioreactor flasks (Wheaton) at 37°C and 5% CO2, as described by Mitchell et al..(1)

Cells were cultured in 15 mL low glucose DMEM and RPMI 1640, respectively, supplemented

with 10% exo-depleted FBS, 1% penicillin-streptomycinand 1% L-glutamine (all supplied by

Sigma Aldrich) in the bottom cell chamber; with 500 mL of the same medium as before, except

with 10% standard FBS in the top reservoir chamber of the bioreactor flask. Cell supernatant

was collected weekly and replaced with fresh exo-depleted cell media. Medium in the

reservoir chamber was also replaced weekly.

The collected supernatants were subjected to centrifugation at 500g for 5 minutes

twice followed by at 2000g for 15 min, then filtration through a 0.22 µm PES filter. This filtered

conditioned medium (CM) was stored at 4°C for up to 6 weeks until used for sEV isolation.

sEV isolation

B16-F10.GFP sEVs were isolated by precipitation using ExoQuick-TC (System Biosciences)

following manufacturer’s protocol. Briefly, CM from B16-F10.GFP cells was incubated with

ExoQuick-TC overnight at 4°C, and centrifuged at 1500g for 30 min at 4°C. The exosome pellet

was washed in PBS by centrifuging at 1500g for 5 min at RT. Finally, the supernatant was

discarded, and the exosome pellet was suspended in 500 µL filtered PBS and stored at 4°C.

MDA-MB-231.CD63-GFP and PANC1 sEVs were isolated by following a protocol

described previously.(2) Briefly, 22.5 mL CM was layered on 3 mL 25% (w/w) sucrose cushion

in D2O (Sigma Aldrich) in a thick-walled polycarbonate centrifuge tube (Beckman Coulter), and

ultracentrifuged at 100,000g for 1.5 h at 4°C; SW48 Ti rotor. The sucrose layer was transferred

to another thick-walled centrifuge tube containing PBS, followed by another

ultracentrifugation step at 100,000g for 1.5 h at 4°C; 70.1 Ti rotor. Finally, the supernatant

was discarded, and the sEV pellet was suspended in 200 µL PBS and stored at 4°C.

Nanoparticle Tracking Analysis (NTA)

The hydrodynamic diameter and concentration of sEVs were measured by Nanoparticle

Tracking Analysis (NTA) using NanoSight LM10, equipped with a 488 nm blue laser and NTA

software v3.2 (Malvern Panalytical). The stock sample was diluted to achieve about 20–

80 particles/viewing frame. Measurements were made in triplicates of 60 s recording

duration for up to 3 serial dilutions of the sample. Parameters used to capture and analyse

data: screen gain = 2, camera level = 13, FPS = 25, viscosity = water and detection threshold =

5.

BCA Protein assay

The protein content of the sEVs was analysed in duplicates of up to 3 serial dilutions using

Pierce Rapid Gold BCA protein assay (Thermo Fisher), according to the manufacturer’s

microplate protocol. Absorbance was measured at 480 nm on SPECTROstar Nano (BMG

Labtech).

Dot blot

Expression of sEV biomarkers was detected by dot blot. 40 µL of sEVs (1x1010 particles/mL)

was spotted on nitrocellulose membranes (0.45 µm; Bio-Rad) and incubated at RT for 1 h in

blocking buffer (3% milk in TBS-T). Mouse anti-human CD63, CD81 and CD9 antibodies (1:1000

dilution in blocking buffer; BioLegend) were added to separate membranes, and incubated

overnight at 4°C. Staining was performed with HRP conjugated goat anti-mouse IgG antibody

(1:10,000 dilution in blocking buffer; BioLegend) for 1 h at RT. Chemiluminescence signal was

detected using an ECL detection system (Thermo Fisher).

Radiolabelling of sEVs

MDA-MB-231.CD63-GFP sEVs, ca. 1x1010 vesicles, or ca. 1x1011 PANC1 sEVs in 160 µL PBS,

were incubated with 20 µL [89Zr]Zr(oxinate)4 or 89Zr-control for 20 min at 37°C with frequent

shaking, followed by addition of 100 µL 1% DFO in PBS to trap any unbound 89Zr. Radiolabelled

sEVs were purified from unchelated radiotracer by size exclusion chromatography (SEC) using

Sepharose CL-2B resin (GE Healthcare). The column was self-packed under gravity according

to the dimensions of commercially available G-25 minitrap columns (GE Healthcare). The

reaction mixture was loaded onto the column, followed by 200 µL PBS and eluted with 700–

800 µL PBS. Radioactivity of the eluate and the column was measured using gamma counter

to calculate radiolabelling yield (RLY).

B16-F10.GFP sEVs, in 100 µL PBS, were incubated with 50 µL of [89Zr]Zr(oxinate)4 or 89Zr-

control at 37°C for 1 hour. Unbound [89Zr]Zr(oxinate)4 was separated from the radiolabelled

sEVs by centrifugation in hydrated MW3000 spin columns (Invitrogen) at 750g for 2 min at

RT.

In vitro PBS stability of MDA-MB-231.CD63-GFP sEVs

MDA-MB-231.CD63-GFP sEVs were incubated in PBS at 37°C for 24 h. At 0, 2 and 22 h sEVs

were analysed by SEC using Superose 6 increase column connected to AKTA Purifier (GE

healthcare). sEVs were eluted with PBS at 0.5 mL/min flow rate in 1 mL fractions. Using this

system, sEVs elute between 7 and 10 mL. Fluorescence of each fraction was measured in

triplicate using a fluorescence plate reader (GloMax® Multi Detection System, Promega). At

24 h, sEVs were also analysed using NTA.

In vitro serum stability of 89Zr-PANC1 sEVs

89Zr-PANC1 sEVs were incubated in PBS (n=1) or in human serum (n=2; 1:1 v/v) at 37°C for

24 h. Serum stability was assessed by SEC using Sepharose CL-2B column at 0, 2 and 24 h.

Radioactivity of the eluate and the column was measured using gamma counter to calculate

radiolabelling yield (RLY).

In vitro cell uptake of 89Zr-PANC1 sEVs

Uptake of 89Zr-PANC1 sEVs were assessed using 4 different cells types: (1) PANC1, (2)

HEK293T, (3) MDA-MB-231, and (4) DU145. In a 24-W plate 50,000 cells/well were seeded

and maintained in serum supplemented growth media at 37°C and 5% CO2. After 24h, 89Zr-

PANC1 sEVs, 89Zr-oxinate or 89Zr-control were added to each cell type in triplicate. Cell uptake

was assessed at 4 h. Supernatant was collected in a tube. Cells were trypsinised and collected

in a separate tube. Radioactivity of the supernatant and the cells were measured, and cell

uptake of radiotracer was calculated.

Heat-damaging of 89Zr-PANC1 sEVs

After radiolabelling, sEVs were damaged by x2 heat/cool cycle – heating to 90°C for 20 min

followed by incubation in ice for 10 min, repeated once more. To evaluate the damage, sEVs

were passed through ExoSpin miniHD SEC column (Cell Guidance Systems) for

characterisation by NTA, BCA protein assay and radiolabelling yield.

PET-CT imaging of 89Zr-PANC1 sEVs

Animal studies were carried out in accordance with the UK Home Office regulations under

The Animals (Scientific Procedures) Act 1986. Immunocompetent C57BL/6 male mice (8 – 10

weeks) were anaesthetised with 2 – 2.5% isoflurane in 100% oxygen. 89Zr-PANC1 sEV (0.2 – 1

MBq, ~1x1010 sEVs in 104 – 140 µL PBS/mouse), either intact (n = 3) or heat-damaged (n = 2),

were injected intravenously via the tail vein at t = 0. PET-CT imaging was performed on a

nanoScan PET-CT preclinical imaging system (Mediso Medical Imaging System) using an air-

heated standard single bed or a 4-bed mouse hotel;(3) anaesthesia was maintained

throughout the scans. PET imaging was started at t = 0.5 h for 2 h, and at t = 24 h for 1 h

followed by a CT scan. All PET/CT data were reconstructed in Nucline v.0.21 (Mediso Medical

Imaging System) using Monte Carlo based Tera-Tomo 3D PET reconstruction (400–600 keV

energy window, 1–3 coincidence mode, 4 iterations and 6 subsets) at an isotropic voxel size

of 0.4mm; images were corrected for scatter an attenuation and were decay corrected to the

time of injection. Reconstructed images were analysed using VivoQuant® (inviCRO Inc).

At the end of imaging session at t = 24 h, mice were culled by cervical dislocation whilst

under anaesthesia. Blood, urine and organs of interest were collected and weighed for ex vivo

biodistribution study. Standards of injected radiotracer were prepared by serial dilutions.

These standards along with the collected tissues were gamma counted to calculate

percentage injected dose (%ID/g).

Statistical analysis

All numerical data were analysed on GraphPad Prism 8 or Microsoft Excel 2016. All values

are given in 1 decimal place. Data are presented as mean ± standard deviation (SD), unless

stated otherwise. Unless otherwise stated, Student’s unpaired t-test was used to calculate

statistical differences between groups with P value <0.05 considered significant. Exact

significance values are reported in each figure.

References

1. J. P. Mitchell, J. Court, M. D. Mason, Z. Tabi, A. Clayton, Increased exosome production from tumour cell cultures using the Integra CELLine Culture System. Journal of Immunological Methods 335, 98-105 (2008).

2. F. N. Faruqu et al., Membrane Radiolabelling of Exosomes for Comparative Biodistribution Analysis in Immunocompetent and Immunodeficient Mice - A Novel and Universal Approach. Theranostics 9, 1666-1682 (2019).

3. H. E. Greenwood, Z. Nyitrai, G. Mocsai, S. Hobor, T. H. Witney, High-Throughput PET/CT Imaging Using a Multiple-Mouse Imaging System. J Nucl Med 61, 292-297 (2020).

4. K. Hummel, F. Richardson, E. Fekete, in Biology of the Laboratory Mouse, E. Green, Ed. (Dover Publications, New York, 1968), chap. 13.

5. H. Suami, D. W. Chang, K. Matsumoto, Y. Kimata, Demonstrating the Lymphatic System in Rats With Microinjection. The Anatomical Record 294, 1566-1573 (2011).

Supplementary figures

Figure S1

Figure S1: Comparison of [89Zr]Zr(oxinate)4 radiochemical properties synthesised by method

1 and method 2.

A) Radiochemical yield of [89Zr]Zr(oxinate)4 formulations (n = 5). B) LogD7.4 (PBS) of control 89Zr and [89Zr]Zr(oxinate)4 (n = 3), analysed by one-way ANOVA with Turkey’s correction for

multiple comparisons. Data presented as mean ± SD of the specified n values.

Figure S2

Figure S2: Characterisation of sEVs using NTA.

Representative size distribution data from NanoSight for the three different types of sEVs. Red

areas represent the standard error on the mean of the triplicates (see methods for details).

Modal average hydrodynamic diameter of respective sEVs are shown; n = number of sEV

isolations. Data given as mean ± SD of the isolations.

Figure S3

Figure S3: Quantification of protein content in equal amount of sEVs (1 x 1010 vesicles) as

measured by BCA protein assay

Amount of protein detected per 1x1010 B16-F10.GFP (n = 3), MDA-MB-231.CD63-GFP (n = 9),

and PANC1 sEVs (n = 8); analysed by one-way ANOVA with Turkey’s correction for multiple

comparisons. Data given as mean ± SD of the given n values.

B16-F10.GFP

MDA-MB-231.CD63-GFP

PANC10

20

40

60

Protein content per 1x1010 vesicles

Prot

ein

cont

ent /µ

g

0.6046

0.0071

0.0275

Figure S4

Figure S4: Comparison of radiolabelling yields of the three different types of sEVs

Compared to both B16-F10.GFP (n = 3) and MDA-MB-231.CD63-GFP (n = 3), PANC1 sEVs (n =

1) were radiolabelled with higher RLY.

Figure S5

Figure S5: Validation of 89Zr-PANC1 sEV damage.

After radiolabelling, sEVs were subjected to heat (90°C for 20 min) and cool (0°C for 10 min)

twice, and purified by SEC. sEVs diameter, concentration, protein content and radiolabelling

yield was compared to control (intact sEVs). Data presented here were normalised to the

control group and is mean ± SD of n = 2.

B16-F10.GFP

MDA-MB-231.CD63-GFP

PANC10

5

10

15

Radiolabelling yield of 1x1010 sEVs

Rad

iola

belli

ng y

ield

/ %

Size

Concentra

tion

Protein co

ntent

RLY0.0

0.5

1.0

1.5

Validation of sEV damage

Fold

diff

eren

ceco

mpa

red

to in

tact

sEV

s

Figure S6

Figure S6: 89Zr-PANC1 sEVs signal in several lymph nodes.

PET-CT images of a mouse injected with intact 89Zr-PANC1 sEVs and showing apparent uptake

in lymph nodes, based on well-documented anatomical location of mouse lymph nodes.(4, 5)

Table 1

Table 1: Biodistribution data for intravenously administered intact 89Zr-PANC1 sEV and heat-damaged 89Zr-PANC1 sEV 24 h post injection

89Zr Uptake (%ID/g)

Organs Intact 89Zr-PANC1 sEV

(n = 3) Heat-damaged 89Zr-PANC1 sEV

(n = 2)

Blood 0.74 ± 0.09 0.83 ± 0.37

Brain 0.05 ± 0.01 0.08 ± 0.06

Skin + Fur 0.44 ± 0.10 0.62 ± 0.35

Liver 54.34 ± 14.14 29.97 ± 18.28

Stomach 0.35 ± 0.05 0.37 ± 0.28

Large intestine 0.36 ± 0.07 0.56 ± 0.36

Small intestine 0.40 ± 0.07 0.59 ± 0.46

Heart 0.80 ± 0.03 0.50 ± 0.24

Pancreas 0.75 ± 0.40 0.50 ± 0.34

Spleen 58.46 ± 10.50 19.09 ± 10.34

Kidneys 2.36 ± 0.57 1.40 ± 0.69

Lungs 2.31 ± 0.68 4.96 ± 5.71

Muscle 0.28 ± 0.20 0.20 ± 0.15

Bone 7.18 ± 1.64 13.25 ± 9.79

Tail 1.07 ± 0.11 1.58 ± 0.84

Urine 1.27 ± 0.57 0.46 ± 0.17

Liver-to-Bone ratio 7.75 ± 2.48 2.41 ± 0.40

Spleen-to-Bone ratio

8.55 ± 2.88 1.59 ± 0.39

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