Development of a method for the preparation of zirconium-89 radiolabelled chitosan nanoparticles as an application for leukocyte

trafficking with positron emission tomography.

M. Fairclough1*, B. Ellis2, H. Boutin1, A.K.P. Jones3 A. McMahon1, S. Alzabin4, A. Gennari5

and C. Prenant1

1. Wolfson Molecular Imaging Centre, Manchester, UK. 2. Nuclear Medicine Centre, Central Manchester University Hospital, NHS Foundation Trust, UK. 3. Clinical Sciences Building, Salford Royal NHS Foundation Trust, Manchester, UK. 4. Epistem Ltd, Manchester, UK. 5. Division of Pharmacy & Optometry, School of Health Sciences, University of Manchester, Manchester, UK.

*Correspondence to: M. Fairclough, Wolfson Molecular Imaging Centre, The University of Manchester, 27 Palatine Road, Manchester M20 3LJ, UK. Tel.: +44(0)1612750034E-mail: michael.fairclough@manchester.ac.uk

AbstractPositron Emission Tomography is an attractive imaging modality for monitoring the

migration of cells to pathological tissue. We evaluated a new method for radiolabelling

leukocytes with zirconium-89 (89Zr) using chitosan nanoparticles (CN, Z-average size 343 ±

210 nm and zeta potential +46 ± 4 mV) as the carrier. We propose that cell uptake of 89Zr-

loaded CN occurred in a two-step process; cell membrane interaction with 89Zr-loaded CN

was followed by a slower cell internalisation step.

KeywordsPET; Zr-89; chitosan; nanoparticles; leukocyte trafficking; inflammation imaging.

IntroductionNanoparticles (NPs) have been extensively used for in-vivo imaging of macrophages

using modalities such as Magnetic Resonance Imaging (MRI), Positron Emission

Tomography (PET), Single Photon Emission Computed Tomography (SPECT) or

fluorescence (Majmudar et al., 2013, Keliher et al., 2011, Jaffer et al., 2006, Devaraj et al.,

2009). Application of macrophage-targeting nanoparticles as diagnostic tools for cancer,

atherosclerosis or myocardial infarction has been actively investigated (Weissleder et al.,

2014).

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Among the nanomaterial-based imaging agents described, chitosan is attractive for

clinical applications due to its bio-compatibility, bio-degradability and apparent low toxicity

(Kean and Thanou, 2010). Chitosan has reached applications in a number of fields including,

but not limited to, drug and gene delivery systems (Nam et al., 2009, Prabaharan, 2015,

Sarvaiya and Agrawal, 2015), and wound dressing and as scaffolds for tissue engineering

(Jayakumar et al., 2010). Chitosan is a linear polysaccharide consisting of randomly

distributed β-1-4-linked D-glucosamine and N-acetyl-D-glucosamine units and has metal

chelating properties (Varma et al., 2004, Clark et al., 2016, Tshuva et al., 2001). It is

proposed that the amine and the secondary hydroxyl groups of the glucosamine moieties

contribute to the metal complexation as represented in Figure 1. The polymer is commercially

available and can be obtained at various molecular weights and with different degrees of

deacetylation (DD); furthermore nanoparticles of chitosan can be conveniently produced.

Figure 1: Postulated representation of the binding of zirconium-89 to chitosan

Different techniques are described for chitosan nanoparticles (CN) production

(Chandra Hembram et al., 2016, Nagpal et al., 2010). Ionotropic gelation is attractive owing

to the mild aqueous conditions used in comparison to other mechanisms of nanoparticle

formation such as micro-emulsification or complex coacervation (Nagpal et al., 2010). The

method of complex coacervation requires a negatively charged polyelectrolyte to be mixed

with positively charged chitosan and so is not applicable to CN labelling with positively

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charged 89Zr. Although micro-emulsion has the advantage of producing nanoparticles with a

narrow size distribution (< 100 nm), this method has major drawbacks in the context of cell

radiolabelling. The use of organic solvent as well as a time-consuming preparation process

and complex washing steps means that micro-emulsification is simply not appropriate for

imaging of leukocyte trafficking using PET. By using ionotropic gelation, nanoparticles are

formed by crosslinking the linear chitosan polymer chains using the nontoxic polyanion

pentasodium tripolyphosphate (TPP). The strong electrostatic interactions of TPP with the

polycationic chitosan results in a fast gel formation (Calvo et al., 1997, Gan et al., 2005,

Nagpal et al., 2010).

In-vivo imaging of leukocytes with PET can be used in immunological studies to track

the selective recruitment of specific immune cells during pathogenesis, to detect

infectious/inflammatory foci and also to devise rational therapeutic strategies based on

longitudinal studies. In a previous publication (Fairclough et al., 2016) we reported on the

use of CN for leucocyte labelling with 89Zr for inflammation imaging with PET. In the

present study we aimed at improving CN preparation for optimum 89Zr-loading and cell

uptake in order to optimize the leukocyte labelling method. Size and surface charge of

nanoparticles have been examined as determinant properties for the cell uptake efficiency.

The affinity of CN for 89Zr and the subsequent uptake and retention of 89Zr-loaded CN into

mixed human leukocyte cells have been investigated.

Experimental

Materials and Equipment89Zr-oxalate was purchased from Perkin Elmer (US)/BV cyclotron (Netherlands) and

chitosan (15 kDa, > 85% DD) was purchased from Tebu-bio (France). Acetic acid,

hydrochloric acid, sodium hydroxide, sodium tripolyphosphate (TPP) and fluorescein

isothiocyanate were all purchased from Sigma Aldrich (UK) and were used without any

further purification. For isolation of mixed leukocyte cells, Acid Citrate Dextrose (formula A)

was purchased from Huddersfield Pharmacy Manufacturing Unit (UK) and HES 6% solution

(hydroxyethyl starch) was purchased from Grifols (UK). Conjugated antibodies to human

CD11b, CD14, CD15, CD16 and CD68 were purchased from Thermo Fisher Scientific UK

(formerly eBioscience). FACS buffer was prepared using phosphate buffered saline,

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penicillin (1 %), streptomycin (Sigma Aldrich, UK), EDTA (Invitrogen, UK) and 5% Foetal

bovine serum (Gibco, UK).

CN were purified using Pur-A-Lyzer Maxi dialysis tubes (12 kDa molecular weight

cut off) from Sigma Aldrich (UK). A PK121R multispeed refrigerated centrifuge from

Thermo Scientific (UK) was used along with a Micro Centaur centrifuge (MSE, UK) for

mixed leukocyte isolation. A Thermo Shaker PHMT (from Grant Instruments UK) was used

to incubate solutions of leukocytes and CN with zirconium-89. Separation of mixed

leukocytes from whole blood was performed in a SafeFlow 1.2 safety cabinet from Bioair

Instruments (Italy). Measurement of radioactive samples was performed using an Isomed

2000 dose calibrator from MED (Germany). CN hydrodynamic diameter (Z-average size),

polydispersity (PDI) and zeta potential (ζ-potential) were measured at a temperature of 25 °C

using a Zetasizer Nano ZS instrument (Model ZEN3600, Malvern Instruments Ltd., UK)

equipped with a solid state HeNe laser (λ = 633 nm) and at a scattering angle of 173°. Size

measurement data were analysed by using the General purpose algorithm. The electrophoretic

mobility of the samples was converted in zeta potential by using the Smolikowski equation.

Fluorescence-activated cell sorting (FACS) analysis of labelled cells was performed

on a MACSQuant analyser (Miltenyi Biotec, Germany).

Preparation of CN and radiolabelling with 89Zr

Chitosan (15 kDa, > 85% degree of deacetylation, 14 mg) was solubilised in HCl (4.6

mM, 20 mL) to form a 0.7 mg/ml solution, and was left to stir for 24 hours. The pH of the

chitosan solution was then adjusted to 5 with the drop-wise addition of sodium hydroxide

(0.1 M). Meanwhile, a 1 mg/ml solution of TPP was prepared (10 mg TPP in 10 mL of

deionised water, pH adjusted to 5) and both the TPP and the chitosan solutions were filtered

through a 0.22 µm filter membrane. Next nanoparticles were produced with various

chitosan:TPP ratios (w/w) by the addition of 72 µL, 80 µL, 90 µL and 120 µL of TPP

solution to 928 µL, 917 µL, 903 µL and 860 µL of chitosan solution to obtain chitosan:TPP

ratios (w/w) of 9:1, 8:1, 7:1 and 5:1 respectively. The mixtures were allowed to stir for 30

minutes before being transferred to Pur-A-Lyzer Maxi dialysis tubes (12 kDa molecular

weight cut off) and were dialysed against de-ionised water for 24 hours (water was refreshed

every 3 hours) to remove the excess of TPP cross-linking agent. The CN solution was then

recovered and stored at 2-4 ºC (this temperature is reported to have no effect on size and

physical stability of the particles (Thakur et al., 1977). CN were characterised for size and ζ -

4

potential by DLS. From previous work we found that approximately 26% of chitosan

polymer forms nanoparticles by ionotropic gelation (Fairclough et al., 2016).

CN (300 µL) were incubated with 89Zr (approximately 17 MBq as 89Zr-oxalate) in a

Thermo-shaker at room temperature for 45 minutes. Following incubation, the 89Zr-loaded

CN were separated from free 89Zr by centrifugation (11600 g for 10 min) and washed twice

with deionised water. The supernatant (containing free 89Zr) and the pellet of 89Zr-loaded CN

were measured for radioactivity; radiolabelling efficiency is expressed as percentage of the

ratio between radioactivity associated with the CN and total radioactivity. The pellet of 89Zr-

loaded CN was then re-suspended in saline (200 µL) for further experiments.

Isolation of mixed human leukocytes and measurement of leukocyte

radiolabelling efficiency and retention of [89Zr]-loaded CN

Each experiment was carried out using mixed leukocytes freshly isolated from whole

blood following erythrocyte sedimentation according to a reported procedure (Ellis, 2011,

Fairclough et al., 2016). In order to evaluate leukocyte radiolabelling efficiency, the pellet of

mixed leukocytes was divided into 8 samples at a density of 3-5 x 107 cells in 200μl of saline

per sample. Next 200 µL of the 89Zr-loaded CN re-suspended in saline was added to each

sample with gentle mixing. Next the samples were incubated in a thermo-shaker at 37 ˚C, at

1400 rpm for 30 minutes (n=2), 1 hour (n=2), 2 hours (n=2), 3 hours (n=1) and 24 hours

(n=1). Following incubation, leukocyte radiolabelling was terminated at each time interval by

the addition of cell free plasma (CFP, 1 mL). The radiolabelled leukocytes were separated

from the mixture by centrifugation (150 g for 5 minutes) to leave a pellet of 89Zr-labelled

leukocytes. The radioactivity in the supernatant and the pellet was measured and

radiolabelling efficiency was expressed as a percentage of the ratio between radioactivity

associated with the leukocytes and total radioactivity. To ensure co-precipitation of 89Zr-

loaded CN with leukocytes was not occurring; suspensions of 89Zr-loaded CN were spun by

centrifugation at 150 g for 5 minutes. In these conditions no 89Zr-loaded CN was found to

precipitate as a pellet.

To assess the retention of 89Zr in leukocytes, cells (3-5 x 107 in 200μl saline) were

radiolabelled with 89Zr-loaded CN as described above (2 hours incubation) followed by

centrifugation (150 g for 5 minutes). The recovered pellet of 89Zr-labelled leukocytes was

then re-suspended in CFP and allowed to incubate for 24 hours at 37˚ C. At intervals of 1, 2,

3, and 24 hours (n=2 for all time-points), the mixture was centrifuged (150 g for 5 minutes),

5

the supernatant was removed and the pellet containing 89Zr-labelled leukocytes was re-

suspended in fresh CFP. Removed CFP fractions and leukocyte pellets were measured for

radioactivity and the radioactivity efflux rate determined over 24 hours. The results for both

uptake and efflux of 89Zr in leukocytes cell can be seen in Figure 4 and Figure 5.

Fluorescent labelling of CN, labelling of mixed leukocyte cells with FITC-CN and flow cytometry

Chitosan was labelled with fluorescein isothiocyanate (FITC) according to a

previously reported method (Huang et al., 2002) with some adaptions. Briefly, chitosan (150

mg) was added to a 1% acetic acid (60 mL) and left to stir overnight. Next FITC (7 mg) was

dissolved in anhydrous methanol (60 mL) and was combined with the chitosan solution. The

reaction was allowed to proceed at room temperature for 4 hours under stirring in darkness.

Upon completion, NaOH (1 M) was added to the reaction mixture to raise the pH and

precipitate FITC labelled chitosan from the solution. Precipitated FITC-chitosan was

collected by centrifugation at 3,025 g for 10 minutes at 4 ˚C. The solid was washed with

70:30 ethanol:water until there were no traces of free FITC (by HPLC analysis). After FITC-

chitosan had been washed sufficiently, the precipitate was allowed to dry overnight. FITC

labelled chitosan nanoparticles (FITC-CN) were prepared from FITC-chitosan in the same

way as the non-fluorescently tagged chitosan nanoparticles. Mixed leukocytes suspended in

saline (200 µL) were incubated at 37 ˚C with FITC-CN (300 µL) for 2 hours after which

radiolabelling was terminated by the addition of CFP (1 mL). The FITC-CN labelled

leukocytes were separated from the mixture by centrifugation (150 g for 5 minutes) to leave a

pellet of FITC-labelled leukocytes which was washed again in CFP and then again with

FACS buffer (1XPBS with 2mM EDTA, 5%Foetal bovine serum and 1% penicillin and

streptomycin). Next FITC-CN-labelled cells were incubated for 20 minutes with conjugated

antibodies to human CD11b, CD14, CD15, CD16 and CD68 in order to assess the updatake

of FITC-CN on monocytes. Following this, the cells were washed again with FACS buffer

and FITC-CN uptake was measured using MACSQuant analyser.

Results

CN properties

Table 1 shows how CN physical properties vary when different chitosan:TPP weigh

ratios are used to construct CN. Nanoparticles obtained from 8:1 chitosan:TPP weight ratio

6

were used for leukocyte radiolabelling, these conditions were selected as they yielded the

smallest CN hydrodynamic diameter (343 ± 210 nm), with a polydispersity index (pdi) of

0.38 (Figure 2). These pdi values and size distribution plots are consistent with those

reported in the literature (Ing et al., 2012, Zhang et al., 2010). In addition, the positive

surface charge of CN produced from 8:1 chitosan:TPP weight ratio reached +46 ± 4

mV(Figure 3).

CS:TPP ratio

(w/w)

CN Hydrodynamic

diameter (nm)

pdi CN ζ-potential

9:1 545 ± 360 0.44 +48 ± 3

8:1 343 ± 210 0.38 +46 ± 4

7:1 412 ± 263 0.40 +43 ± 4

5:1 451 ± 256 0.32 +40 ± 4Table 1: Effect of chitosan:TPP weight ratio on the hydrodynamic diameter, polydispersity index (pdi)

and ζ-potential of CN.

1 10 100 1000 100000

2

4

6

8

10

12

Size (d.nm)

Inte

nsity

(%)

Figure 2: CN size distribution by intensity of scattered light for CN produced from a 8 :1 chitosan:TPP

weight ratio. The average size of CN produced from this chitosan:TPP ratio was 343 nm with a pdi of

0.38.

7

-148

-131

-115 -99 -82

.7-66

.5-50

.2-33

.9-17

.7-1.

4314

.831

.147

.363

.679

.996

.1 112

129

0

50000

100000

150000

200000

250000

300000

350000

Zeta Potential (mV)

Tota

l Cou

nts

Figure 3: CN zeta potential distribution of CN produced from a 8:1 chitosan:TPP weight ratio.

Affinity of 89Zr for CN

The fraction of 89Zr associated with the CN after 45 minutes incubation at room

temperature in the Thermo-shaker at 1400 rpm attained 31 ± 6 % (n = 15) following washing.

These conditions were found to be optimal in our previous work in which 74 % incorporation

of 89Zr into CN was reported (Fairclough et al., 2016).

Uptake and Retention of 89Zr-loaded CN in mixed leukocytes

Figure 4 and Figure 5 show, respectively, 89Zr-loaded CN uptake into and the egress

from leukocyte cells (monocytes and neutrophils). The plots shown in Figure 4 and Figure 5

are consistent with a two-step uptake mechanism. We suggest a mechanism in which 89Zr-

loaded CN is initially attracted to the cell surface, resulting in adhesion-dependent fast

accumulation to the cell membrane. It is postulated that this is then followed by a slower

internalisation step.

8

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 250%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

14.7%

21.9%

33.8%

44.9%

73.3%

Time (hours)

Upt

ake

of 8

9Zr

into

the

cell

Figure 4: Uptake of 89Zr-loaded CN in mixed human leukocyte cells.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

32.2%

50.2%

64.4%

78.8%

Time (hours)

% E

gres

s of 8

9Zr

from

leuk

ocyt

e ce

lls

Figure 5: Egress of 89Zr-loaded CN in mixed human leukocyte cells. Rate of efflux was measured in

leukocytes which were radiolabelled with 89Zr-loaded CN following 2 hour incubation.

9

FACS analysis of FITC-CN labelled mixed leukocytesFACS analysis was used to determine the uptake of FITC-CN by the neutrophil

population of peripheral blood mononuclear cells. PBMCs were isolated from a healthy

donor as described above and cells were stained with a cocktail of antibodies prior to analysis

using the MACSQuant FACS analyser. The neutrophil population was visualised by gating

first on cells with a high granularity and cell size (Figure 6A) followed by exclusion of

monocytes and macrophages by conjugated antibodies to CD68, CD16 and CD11b. Figure 6

shows the density dot plots of VioBlue-CD15+ cells (neutrophil marker) against CN (Figure

6B) fluorescent intensity of FITC-CN.

Figure 6: CN uptake by neutrophils. FACS dot plots showing A) Gating strategy on the neutrophil population by granularity and size inclusion and B) Cell expression analysis of a neutrophil surface receptor CD15 in cells which have not been incubated with CN and C) Expression of CN in CD15 expressing neutrophils which have been incubated with CN.

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Discussion

CN properties

Size and surface charge of nanoparticles are critical parameters determining cell

uptake mechanism and efficiency. The surface charge of NPs is crucial to the initial

electrostatic attraction of the NPs to the negatively charged cell membrane and the size of the

NP is important to determining the mechanism of cell uptake. Both of these CN parameters

are very adaptive to changes in chitosan and polyanion (TPP) ratio (Ing et al., 2012).

Initial addition of the TPP poly-anion to solvated, positively charged chitosan chains

produced intra and intermolecular cross-linking which initiated the formation of

nanoparticles. Increasing the amount of TPP further increased the degree of reticulation in the

nanoparticles, expelling water molecules and producing more compact, smaller size CN.

Further TPP addition resulted in CN size increase while the ζ-potential continued to decrease.

This could be attributed to the neutralisation of the nanoparticle positive surface charge by

excess TPP causing particle build-up by accretion of free chitosan chains. Alternatively, a

large excess of TPP might promote CN aggregation due to TPP acting as an inter-particle

cross linker.

The high ζ–potential of CN produced from an 8:1 chitosan:TPP ratio (Figure 3)

confirms the absence of excess TPP at the CN surface and ensures CN stability by preventing

inter-particle attraction and formation of aggregates. Additionally the high ζ-potential favours

CN interaction with the leukocyte cell membrane.

Affinity of 89Zr for CN

The incorporation of 89Zr by CN was previously measured by us as 74 % (Fairclough

et al., 2016) but here the efficiency of 89Zr loading by CN was lower. The difference can be

attributed to the CN purification technique used. The centrifugation technique used in our

previous report has the drawback of leaving an excess of TPP on CN surface. Since TPP has

metal chelation properties, it is important to eliminate it from the CN formulation. In this

work, the use of dialysis instead of centrifugation ensures the total removal of TPP from CN

formulations. Further, optimisation of the radiolabelling of CN with 89Zr may be possible by

altering the pH of the 89Zr-oxalate solution.

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Uptake and Retention of 89Zr-loaded CN in mixed leukocytes

Cell uptake of charged NPs is commonly described as a two-step process, the

internalization proceeds interaction of the NP with the cell membrane (Lesniak et al., 2013).

The initial interaction with the membrane is determined by the physico-chemical features of

the NPs and particularly their surface charge. It has been shown that higher positive charge

on NPs enhance cellular uptake which is attributed to the electrostatic interaction with the

negatively charged cell membrane (Foged et al., 2005, Gustafson et al., 2015, Forest et al.,

2015, Roser et al., 1998, He et al., 2010, Tabata and Ikada, 1988).

Membrane-wrapping of the NPs by the cell could be the limiting step in the kinetics

of the uptake (He et al., 2010). The broad size dispersion of the CN (343 ± 210 nm) might

result in a variable internalization time and involve different, size-dependant, endocytosis

processes such as phagocytosis, macropinocytosis and clathrin-dependent and -independent

mechanisms, (Reno et al., 2013, Kuhn et al., 2014, Zhao et al., 2011, Nam et al., 2009).

Figure 5 demonstrates that after two hours incubation of the 89Zr-loaded CN with

leukocytes, the 89Zr-loaded CN may only be partially internalized, and the rest remain

membrane bond. This could account for the relatively fast initial loss of 89Zr from the cell

(Figure 5) which we hypothesise may be due to the activation of the leukocyte population

following their exposure to 89Zr-loaded CN. Upon cellular activation, endocytosis is triggered

which causes a change in pH and leads to a change in the charge on the cell membrane,

therefore, it is possible that during these changes, the affinity towards 89Zr-loaded CN is lost.

The slow internalisation of 89Zr-loaded CN by leukocytes and the residual 89Zr-loaded CN

bound to the cell membrane appears to make this technique for leukocyte tracking with PET

less favourable to routine clinical applications, although it would be adequate for research

purposes. The stability of the internalised radioactivity in this technique of leukocyte

radiolabelling is attractive for preclinical studies of inflammation. Furthermore the technique

could be refined on two levels; first removal of 89Zr-loaded CN bound to the cell membrane

could be made more effective by using a more dynamic CFP wash. Secondly a more optimal

CN size could be produced for a faster cell uptake step. In a study of phagocytosis of latex

particles by leukocytes, Kawaguchi et al showed that latex particles with a diameter ranging

between 0.6 and 1.0 µM are more easily phagocytosed than particles outside of this range

(Kawaguchi et al., 1986). CN with sizes ranging between 0.6 and 1.0 µM can be produced by

12

using a higher chitosan:TPP ratio and it should also be possible to select CN of a specific

diameter by filtration.

FACS analysis of FITC-CN labelled mixed leukocytesFACS analysis of CD15+ neutrophils revealed a strong signal in the FITC-channel,

corresponding to FITC labelled CN (Figure 6C). This confirms an uptake of our CN by the

neutrophil population although the method of uptake i.e. cells surface adhesion; phagocytosis

or a receptor based mechanism has not been addressed in this study. The uptake of FITC-CN

by neutrophils is an encouraging result as these cells are central to any inflammatory response

in the body. Neutrophils are often the first cells to respond to inflammatory stimuli and

migrate to sites of inflammation where they can phagocytose foreign/self-antigens (Botelho

et al., 2002). Ultimately, our aim is to develop a new imaging tool to monitor the migration of

white blood cells using PET. Whilst this study has assessed CN uptake by neutrophils as the

carrier cell type, other phagocytes may also be used. Neutrophils are useful to image early

states of inflammation, but they also do possessed the shortest life span of all leucocytes.

Therefore, optimising uptake with other cell types will be useful for imaging later stages of

inflammation. The timing and duration of uptake will differ on a cell to cell basis and should

be considered in the design of future studies

Conclusion This work described the preparation of CN as a carrier of 89Zr into human leukocytes.

The production of CN by process of ionic gelation has benefits of using low concentration of

both chitosan and TPP which will therefore lower any possible cytotoxicity effects, in

addition, the process negates the use of organic solvents. CN with an average size of 343 ±

210 nm and surface charge of +46 ± 4 mV were obtained from 8:1 chitosan:TPP weight ratio

yielding the lowest pdi (0.38). CN produced by this method were successfully loaded with 89Zr with 31 ± 6 % (n = 15) of 89Zr being loaded. With CN produced by this method, a two-

step mechanism of 89Zr-loaded CN uptake by leukocytes was proposed. A cell membrane

interaction with 89Zr-loaded CN was followed by a slower internalisation step.

We have shown that these 89Zr-loaded CN can be used to radiolabel mixed human

leukocytes and the relative stability of 89Zr in leukocyte cells from 3 hours to 24 hours is

encouraging for pre-clinical PET imaging of leukocyte cells. The technique could also be

amendable for clinical application by optimising the CN size and using an appropriate

13

chitosan:TPP ratio. The cell uptake mechanism of CN detailed here may be useful to

understand the mechanism of cationic chitosan-mediated drug delivery or gene transport

across cell membranes for cell transfection. Additionally, the versatility of this method

means that it could be transferable to radiolabelling of other cell types such as macrophages

or stem cells for in vivo tracking with PET.

Acknowledgements Funding: This work was funded by the NIHR Manchester Musculoskeletal Biomedical

Research Unit (Grant number: R114999). MF PhD studentship was funded by the Wolfson

Molecular Imaging Centre, University of Manchester.

Competing interests: The authors declare that they have no competing interests.

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