Visualisation of dual radiolabelled poly(lactide- co -glycolide) nanoparticle degradation in vivo using energy-discriminant SPECT Journal of Materials Chemistry B PAPER

The determination of nanoparticle (NP) stability and degradation in vivo is essential for the accurate evaluation of NP biodistribution in medical applications and for understanding their toxicological eﬀects. Such determination is particularly challenging because NPs are extremely diﬃcult to detect and quantify once distributed in a biological system. Radiolabelling with positron or gamma emitters and subsequent imaging studies using positron emission tomography (PET) or single-photon emission computerised tomography (SPECT) are some of the few valid alternatives. However, NPs that degrade or radionuclides that detach or are released from the NPs can cause artefact. Here, submicron-sized poly(lactide- co -glycolide) nanoparticles (PLGA-NPs) stabilised with bovine serum albumin (BSA) were dual radiolabelled using gamma emitters with diﬀerent energy spectra incorporated into the core and coating. To label the core, 111 In-doped iron oxide NPs were encapsulated inside PLGA-NPs during NP preparation, and the BSA coating was labelled by electrophilic substitution using 125 I. After intravenous administration into rats, energy-discriminant SPECT resolved each radioisotope independently. Imaging revealed diﬀerent fates for the core and coating, with a fraction of the two radionuclides co-localising in the liver and lungs for long periods of time after administration, suggesting that NPs are stable in these organs. Organ harvesting followed by gamma counting corroborated the SPECT results. The general methodology reported here represents an excellent alternative for visualising the degradation process of multi-labelled NPs in vivo and can be extended to a wide range of engineered NPs.


Introduction
The pharmacokinetics (PK) and biological fate of nanoparticles (NPs) need to be known to accurately assess their toxicological effects 1 and therapeutic efficacy 2 in biomedical applications.However, NPs are extremely difficult to detect and quantify once distributed in a biological system.In preclinical studies, organs are often harvested at pre-determined time points after administration or incorporation of the NPs and, depending on their nature, are then directly visualised by imaging techniques such as transmission electron microscopy (TEM) 3 and confocal Raman microscopy 4 or indirectly quantified by ion beam microscopy 5 or inductively coupled plasma mass spectrometry (ICP-MS). 6These techniques require the sacrifice of large numbers of experimental animals to ensure statistical power, can only be applied to certain NPs, and not all of them are quantitative.New in situ methodologies are required for PK and fate studies in vivo.
NPs can be labelled with radionuclides (e.g., positron or gamma emitters) to reduce animal use and generate quantitative data in fate studies.Radiolabelled NPs are then commonly detected in vivo using ultra-high sensitivity imaging modalities such as positron emission tomography (PET) or single-photon emission computerised tomography (SPECT). 7A wide variety of procedures have been developed for the incorporation of positron or gamma emitters into NPs including direct ion [8][9][10] or neutron activation, [11][12][13] the attachment of bifunctional chelators (BFCs) followed by complexation with a radiometal, [14][15][16] the a Radiochemistry and Nuclear Imaging, CIC biomaGUNE, Paseo Miramo ´n 182, 20009, San Sebastia ´n, Guipu ´zcoa, Spain.E-mail: jllop@cicbiomagune.es;Tel: +34 943 00 53 33 b MOE Key Laboratory of Macromolecular Synthesis, Functionalization, incorporation of a pre-labelled tag into NPs, [17][18][19] or the incorporation of a labelled precursor during NP synthesis. 20Although the spatiotemporal data gathered by radiolabelling and subsequent imaging are useful, the radiochemical integrity or chemical stability of the NPs is not reported: NPs that degrade or radionuclides that detach or are released from the NPs can cause artefact.
Ideally, in vivo radiochemical integrity and NP stability should be determined when investigating NP biodistribution.Moreover, knowledge of NP stability or degradation is fundamental when assessing the biological fate of NPs, since NP degradation can significantly alter toxicological endpoints. 21or example, the degradation of an organic coating around a metal NP may lead to the exposure of the metal core to the cell interior.As a consequence, the metal cations release could catalyse biochemical reactions or increase intracellular ion concentrations which might negatively affect cell homeostasis.Additionally, NP degradation is directly related to the efficiency of NPs in carrying therapeutics and releasing them at their desired location if they are designed to deliver drugs, and the evaluation of their in vivo stability is critical for the successful translation of drug candidates into the clinic.
The assessment of the in vivo stability of small radiolabelled organic molecules is usually achieved by blood sampling followed by plasma isolation and analysis using instrumental analytical techniques. 22,23Although this strategy is also suitable for the assessment of the in vivo stability of radiolabelled NPs, they are not easily isolated from blood samples.Therefore, the problem is often simplified by assessing NP stability in vitro using model media such as water, buffers, or biological fluids [24][25][26] and extrapolating the results to in vivo conditions.Despite being valid in some cases, this strategy has severe limitations due to the inherent complexity of biological systems.
The incorporation of multiple imaging agents into a single NP can provide complementary information regarding their stability when used with a combination of imaging modalities.In one of the few reported examples of this approach, oleate-stabilised magnetic NPs with a DSPE-PEG coating were labelled with 64 Cu and investigated by combined PET and magnetic resonance imaging (MRI). 27However, simultaneous PET-MRI acquisition remains a challenge in the preclinical setting, with consecutive or parallel studies using both modalities required.Additionally, both modalities have very different intrinsic sensitivity, which complicates experimental set up and data analysis.More recently, iron oxide NPs stabilised with oleic acid and phospholipids were radiolabelled with 59 Fe, 14 C, and 111 In and the biodistribution of all three radioisotopes investigated in mice following sacrifice and organ harvesting. 28Although this study generated useful information about NP stability, 59 Fe and 14 C are inappropriate imaging agents for in vivo studies and animal sacrifice was required.
Very recently, strategies for the preparation of double-labelled functionalised gold NPs have been reported. 29,30Following a similar approach, we present here a strategy for the in vivo determination of NP stability, also based on dual radiolabelling using two gamma emitters with different emission energies.In our approach, the NP core and the surface were independently labelled.We exploited the favourable properties of poly(lactide-co-glycolide) NPs (PLGA-NPs) which, due to their submicron size, display excellent biodegradability and biocompatibility.Additionally, PLGA copolymers were approved by the Food and Drug Administration (FDA) for clinical drug delivery. 31Furthermore, their size and the use of emulsion techniques in their preparation allowed our novel approach to be implemented.First, we synthesised iron oxide NPs containing 111 In and stabilised with oleic acid.Iron oxide NPs were then encapsulated in the PLGA-NP core by mixing them with the PLGA phase during emulsification, overcoming the drawback of chemically modifying PLGA molecules for radiolabelling, which may alter their hydrophobicity and affect NP synthesis.Bovine serum albumin (BSA) was used as a stabilising agent for the emulsion droplets, facilitating the incorporation of 125 I, the second radioisotope, by electrophilic substitution on the tyrosine residues of the protein.Subsequent SPECT imaging with energy discrimination allowed quantitative assessment of the in vivo stability of NPs, minimising the use of experimental animals.This was achieved by monitoring the change of the intensities of 111 In and 125 I, after appropriate decay correction taking into consideration the half-lives for each isotope.Dissection through gamma counting experiments was conducted and correlated with SPECT images for validation.This general strategy can be extended to a wide variety of engineered NPs and is a valuable tool for in vivo evaluation of NP stability.

Synthesis, radiolabelling and characterization of NPs
The synthesis of dual labelled NPs ([ 125 I/ 111 In]NP1, see Fig. 1) consisted of two steps.In the first step, the PLGA core was labelled by including 111 In-doped iron oxide NPs stabilised with oleic acid during NP fabrication; in the second step, the BSA used as a surface stabiliser was labelled with 125 I. Radiolabelling of the NP core might be envisioned via attachment of a radiotag to lactic-coglycolic polymers.This process is, however, challenging.Hence, the entrapment of pre-labelled iron oxide NPs was considered as a convenient strategy.
Iron oxide NPs were prepared by a slight modification of a previously published co-precipitation method. 32The addition of 111 InCl 3 during co-precipitation yielded mono-disperse iron oxide magnetic NPs coated with oleic acid, with a diameter of 12 AE 3 nm as determined by TEM (Fig. 2a).NPs were further dispersed in an organic PLGA solution using a well-established protocol. 33An aqueous BSA solution was transferred to the organic phase, and the solution was sonicated to form organic phase droplets stabilised with BSA and containing iron oxideloaded PLGA-NPs ([ 111 In]NP1).Finally, the incorporation of 125 I by electrophilic aromatic substitution was carried out to yield [ 125 I/ 111 In]NP1.
The selection of the correct combination of radioisotopes was of paramount importance to enable subsequent imaging studies using energy discriminant SPECT.Ideally, the energy spectra should not overlap, and the energy peaks should fall within the energy range of the SPECT system used (in our case 25-250 keV).Additionally, both radioisotopes should have a sufficient half-life to investigate biodistribution for long periods of time after administration. 111In (T 1/2 = 2.8 days, maximum energy emission at 171 and 245 keV) is a radioisotope with good in vivo imaging characteristics, which has been widely used in both the pre-clinical and clinical settings.It is usually attached to the molecule of interest by the formation of a radiometalchelator complex; however, previous studies have suggested that it can be readily incorporated into the crystal lattice of iron oxide NPs, 34 as here.For BSA labelling, 125 I was chosen for several reasons: first, it has been widely used for the radiolabelling of peptides, proteins, and antibodies via electrophilic substitution by taking advantage of the presence of tyrosine residues; second, 125 I has an energy maximum at 35.5 keV and a long half-life (close to 60 days) and is reasonably cheap and widely available.Hence, both isotopes have a relatively long half-life, appropriate emission characteristics, and established radiolabelling routes.
The hydrodynamic diameter of [ 125 I/ 111 In]NP1 NPs was determined by dynamic light scattering (DLS): the mean NP size was 300 AE 40 nm (B75%), with a minor population (B25%) of 460 AE 70 nm (Fig. 2b) also observed in the intensity distribution.
TEM analysis performed after radioactive decay showed that iron oxide NPs were properly entrapped in the polymeric matrix core (Fig. 2c and d).TEM imaging under dry conditions revealed PLGA-NP sizes of about 250 AE 50 nm.The PLGA-NPs had a negative x-potential of À15 mV at neutral pH.Gamma spectrometry of the final NPs showed the presence of peaks corresponding to 111 In and 125 I (Fig. 2e and f).The ratio of both isotopes was always maintained in the range 125 I/ 111 In = 1.3-1.6.
The stability of [ 125 I/ 111 In]NP1 was measured in physiological saline (0.9% NaCl) and mouse plasma at 37 1C.The NPs showed good stability in physiological saline (Fig. 3), with both isotopes remaining attached to the NPs up to 48 hours.At t = 6 days, significant release of 125 I and 111 In was observed, although 64% and 75% of the radioactivity, respectively, remained attached to the NPs.
Interestingly, incubation in plasma resulted in the progressive release of 125 I.At 48 h of incubation, approximately 50% of the 125 I was released from the NPs.This value increased up to 85% at t = 6 days.The decrease in the 125 I signal observed in mouse serum hints at the removal of labelled BSA from the NPs.BSA can be replaced by unlabelled proteins from the serum or simply released as a consequence of their interaction with serum components.BSA molecules are linked to the PLGA only by hydrophobic interactions, which may not be strong enough to guarantee their attachment to the NP surface in the presence of serum components.On the other hand, the 111 In percentage remained almost constant up to 2 days, with a progressive decrease afterwards to reach values around 65% at t = 6 days.These values suggest that iron oxide NPs remain PLGA encapsulated over the first 48 h of the experiment, and the release occurs very slowly and only at long times after incubation.This was expected, since degradation (weight loss) of PLGA in vitro is reported to only occur after at least one week in phosphatebuffered saline (pH 7.4). 35,36 vivo imaging studies Stability studies were followed by in vivo SPECT imaging; static images were acquired for each radioisotope at different time points after administration using a SPECT camera with energy discrimination (Fig. 4a); pure 125 I-labelled BSA was used as a control (Fig. 4b).
SPECT imaging after administration of the NPs showed that the biodistribution of the two radiotags could be followed independently.Three hours after administration, the majority of the radioactivity of both radioisotopes (red for 125 I, green for 111 In) was located in the liver and lungs with 125 I/ 111 In ratios of 1.12 AE 0.21 and 0.92 AE 0.32, respectively (Fig. 4a), suggesting that the majority of the NPs remained intact in these organs.Accumulation in the lung was probably due to the size of the NPs, which may form micro-emboli by occlusion of the small lung capillaries. 37Sequestration by the reticulo-endothelial system (RES) may also contribute to lung accumulation.At this time point, there was selective accumulation of 125 I in the bladder and thyroid glands, suggesting that the NPs had already undergone some degradation.One and two days after administration, a fraction of 125 I and 111 In still co-localised in the liver and lungs ( 125 I/ 111 In ratio = 1.07 AE 0.14 and 0.92 AE 0.18 in the liver at t = 24 and 48 h, respectively; 125 I/ 111 In ratio = 0.67 AE 0.22 and 0.42 AE 0.16 in the lungs at t = 24 and 48 h, respectively), but the 125 I signal in the thyroid gland progressively increased with time.Six days after NP administration, 125 I was almost cleared from the body (except the thyroid gland), with the majority of detected radioactivity originating from 111 In in the liver.This suggests that the NP core had not fully degraded.
In order to obtain comparative data, pure 125 I-labelled BSA was also administered to the animals and SPECT scans were conducted at three, 24, and 48 hours (Fig. 4b).Pure 125 I-labelled BSA rapidly accumulated in the lungs, liver, thyroid gland, and urine. 125I was detected in the liver after 24 h and was almost cleared from this organ by 48 h, at which point radioactivity was only detected in the bladder and thyroid gland.
Altogether, the results suggest that [ 125 I/ 111 In]NP1 biodistributes due to the attachment of the protein to the NPs, and at initial time points the BSA follows the biodistribution of the core.It has previously been reported that 125 I-labelled BSA undergoes rapid clearance from mouse livers after intravenous administration (o0.2% of injected dose after 24), with the labelled metabolite [ 125 I]mono(di-)iodotyrosin detected in hepatocytes. 38This radiometabolite can easily cross biological membranes, which might explain the low uptake at later time points.The elimination of 125 I was much less dramatic from dual labelled NPs ( 125 I remained detectable in the liver six days after administration), suggesting that the radiolabelled BSA remained attached to NPs in the liver.

Ex vivo studies
The biodistribution and chemical stability of the NPs were also investigated by dissection and gamma counting, which also provided accurate, organ-level biodistribution information and corroborated the in vivo data.The accumulation of 125 I and 111 In in different organs expressed as the % of injected dose per gram of tissue (%ID g À1 ) is shown in Fig. 5.
For both radioisotopes, the highest radioactivity concentrations were detected in the lungs at early time points after administration (%ID g À1 B40 at t o 2 h).The liver had the next highest concentrations (%ID g À1 between 20 and 30%), with a non-significant increasing trend observed between 5 and 30 min and a progressive decrease thereafter.After 48 h, the %ID g À1 were 11.6 AE 3.2 and 3.3 AE 1.8 for 125 I and 16.6 AE 4.6 and 9.6 AE 3.2 for 111 In in the liver and lungs, respectively.The accumulation of 125 I in the thyroid gland increased over time, reaching values close to 100%ID g À1 at 48 h.Interestingly, the accumulation of 111 In in the thyroid glands was very low (o3%) at all time points, which can be considered insignificant given the very small size of the thyroid gland (between 4-5 mg).The accumulation of 125 I was extremely high in the urine soon after administration (131 AE 33% ID g À1 at t = 30 min), with accumulation of 111 In below 5% throughout the experiment.Values below 0.1% ID g À1 were obtained for both radioisotopes in the brain (results not shown).Blood radioactivity was similar for both isotopes, with initial %ID g À1 values in the range of 1.5-2%, which progressively decreased to below 1% at t 4 2 h.
125 I/ 111 In %ID g À1 ratios at different time points for different organs after intravenous administration of the dual-labelled NPs are shown in Fig. 6: values close to 1 denote that the amounts of both isotopes (expressed as %ID g À1 ) are equivalent, suggesting that the two labels remain together and that the NPs have not degraded.At early time points after administration, the 125 I/ 111 In ratio was always close to 1, irrespective of the organ analysed.This ratio was maintained in the liver up to 24 h, with a slight, non-significant decrease thereafter (0.70 AE 0.27 at t = 48 h).A similar trend was observed in the lungs, with values very close to 1 at early time points after administration.However, a decreasing trend was readily observed by 24 h (0.52 AE 0.24 at t = 24 h) that became more pronounced at t = 48 h (0.34 AE 0.22).These values are concordant with those obtained in vivo using SPECT and energy discrimination.The 125 I/ 111 In ratios in the urine, intestine, and thyroid glands were very high even at early time points after NP administration, confirming selective accumulation of radioiodine in these tissues.As observed in the in vitro stability studies and in vivo SPECT experiments, these results suggest that labelled proteins slowly detached from the NP core in the presence of plasma proteins and this phenomenon was likely to have been even quicker in the in vivo circulation.
The abovementioned degradation of the protein coating which might have been replaced by other proteins during circulation was previously reported in in vitro studies, 39 and is an important issue since it can alter the biodistribution of NPs.Most coatings are not only used to protect the NP core or stabilise them in aqueous media, but also to direct the NPs to specific organs or tissues or to carry specific functions.The loss of these functionalities may lead to modification of the properties of NPs.In the case considered here, BSA is not covalently bonded to the NPs but interacts with PLGA by hydrophobic interactions, since it was used as a surfactant to stabilise the PLGA emulsion that led to the formation of the NPs.Under these conditions, the NP-PLGA core interaction is weak, which probably makes the removal of BSA easy.Covalently attaching the protein to the NP would most likely increase protein stability.This knowledge about the stability of the BSA coating is helpful for the design of surface coatings of NPs and how to engineer them to produce longer circulation times or improve their stability.

Reagents
All chemical reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were of analytical grade purity unless otherwise specified.Nanopure water (18.2MO cm) was obtained using a Thermo Scientific Barnstead NANOpure Diamond Water System (Thermo Fisher Scientific, Waltham, MA, USA).Na[ 125 I]I (solution in 0.1 M NaOH) was obtained from Perkin Elmer (Beaconsfield, UK), and 111 InCl 3 (solution in HCl) was purchased from Covidien (Dublin, Ireland).

Preparation of 111 In-labelled iron oxide NPs
Iron(II) chloride tetrahydrate (50 mg) and iron(III) chloride hexahydrate (60 mg) were dissolved in ultrapure water (25 mL).A fraction of this solution (0.5 mL) was withdrawn using a pipette and diluted with ultrapure water (4.5 mL) in a vial. 111InCl 3 solution (780 mL, 512 MBq) was immediately added, and the vial was capped and flushed with nitrogen gas (solution A).In parallel, ammonia (29% aqueous solution, 2.34 mL) was added to a 100 mL flask and diluted with ultrapure water to a final volume of 20 mL.The solution was stirred for 30 min under nitrogen gas flow (solution B).Solution A was then slowly  added to solution B with constant stirring (500 rpm) at room temperature.The solution turned yellow-black due to the formation of iron oxide NPs.After 30 min, the NPs were collected using a magnet, washed with water three times, and finally dispersed in purified water (5 mL).

Preparation of oleic acid-modified 111 In-labelled iron oxide NPs
Radiolabelled iron oxide NPs prepared as above were sonicated (20% power, 2 s + 2 s cycles) for 30 min (total running time).Oleic acid (100 mL) was then added dropwise under sonication.To prevent a temperature rise in the reaction mixture, a water bath (20 1C) was used.After 2 h, the NPs were collected using a magnet.To recover NPs attached to the sonication tip and to the walls of the vial, the aqueous solution was removed by decantation, hexane (1 mL) was added, and the mixture was further sonicated.Finally, the collected NPs were dispersed into hexane and the solution was concentrated to a final volume of 25 mL.

Preparation of PLGA-BSA particles
Oleic acid-modified 111 In-labelled iron oxide NPs as prepared above (25 mL) were added to a PLGA (PLGA 85:15) solution (2% in dichloromethane, 125 mL) and the resulting mixture was sonicated for 10 min.BSA solution (2% in purified water, 600 mL) was added, and the mixture was again sonicated for 20 s.The resulting solution was immediately poured into purified water (15 mL) and stirred for 2 h.PLGA NPs were then collected by centrifugation and washed twice with purified water.The resulting NPs were finally dispersed in PBS (50 mL).
Radiolabelling of PLGA-BSA NPs with 125 I Iodination was carried out using Pierce s iodination beads.The beads were washed with PBS (1 mL), dried with filter paper, and introduced to a vial with PBS (100 mL, pH = 7.2) and Na 125 I (ca.20 mL, 37 MBq).The mixture was incubated at room temperature for 5 min with occasional vigorous shaking.PLGA-BSA NPs (PBS solution, see above) were added to this solution, and the mixture was incubated for 15 min at room temperature.The NPs were finally isolated by centrifugation and washed twice with PBS.

NP characterisation
Size distributions and x-potentials were determined for all samples.Transmission electron microscopy (TEM) was performed using a JEOL JEM-1400 plus microscope (Jeol, Tokyo, Japan) working at 120 kV.For TEM examinations, a single drop (10 mL) of the aqueous solution (B0.1 mg mL À1 in milliQ water) of the NPs was placed onto a carbon film on 400 square mesh copper grids (CF-400-Cu, Electron Microscopy Sciences, Hatfield, PA, USA).The grid was left to dry in air for several hours at room temperature.
DLS and x-potential measurements were performed using a Malvern Zetasizer Nano ZS system (Malvern Instruments, Malvern, UK).For particle size determination, ten measurements for 10 s were carried out at a temperature of 25 1C at a 1731 scattering angle.A refractive index of 1.52 for PLGA was used.For x-potential measurements, a minimum of 15 runs were carried out.NP effective surface charge in 10 mM PBS was measured at pH 7.4.Measurements were performed in quintuplicate.The Smoluchowski approximation was used to convert the electrophoretic mobility to x-potential values.
Radiolabelled NPs were also characterised by g-spectrometry using a multi-channel analyser g-spectrometer (MUCHA, Raytest, Straubenhardt, Germany) calibrated with standard energy calibration sources.A small fraction of the NPs was placed into a vial and the energy spectrum was recorded.The areas under the peaks at 35 kV (maximum emission for 125 I) and 171 keV (energy peak for 111 In) were used to determine the absolute amounts of both radioisotopes.

Radiochemical integrity of labelled NPs
To assess the radiochemical stability, NPs were prepared and purified as described above and subsequently resuspended in 300 mL of PBS or mouse plasma.The suspension was then divided into five different aliquots containing 50 mL of the NPs each.The aliquots were kept at 37 1C for five and 30 min, and two, 24, 48 and 144 hours, respectively.The samples were then centrifuged and the NPs were separated by decantation and washed twice with PBS.The energy spectra of the supernatant and the NPs were then recorded, and the absolute amount of 125 I and 111 In in both fractions was determined.

Animal experiments
Animals.All the animal procedures were performed in accordance with the Spanish policy for animal protection (RD53/2013), which meets the requirements of the European Union directive 2010/63/UE regarding the protection of animals used in experimental procedures.The guidelines were approved by the Ethical Committee of CIC biomaGUNE and authorised by the regional government.Mice (BALB/cJRJ, 8-12 weeks of age, Janvier Labs, France) were housed in a controlled environment (12:12 light/dark cycle with dawn and dusk transitional periods, room temperature 22 1C, and 55% relative humidity) and maintained on commercially available pelleted diet and sterilised water ad libitum.Three animals per labelled species were used for in vivo SPECT/CT experiments; dissection and gamma counting experiments were performed on three animals at each time point.
SPECT/CT images.SPECT/CT images were acquired using the eXplore speCZT CT preclinical imaging system (GE Healthcare, Little Chalfont, UK).This system uses a stationary, full ring of cadmium zinc telluride (CZT) detectors and interchangeable rotating cylindrical collimators.The CZT detectors offer substantially better energy and spatial resolution than the NaI detectors used in conventional SPECT systems.The improved energy resolution is particularly important for applications such as dual isotope imaging since it allows for better energy peak separation and rejection of scattered photons to yield higher contrast images.
Three hours prior to examination, mice (three per labelled species) received an intravenous administration of either (i) [ 125 I/ 111 In]NP1 or (ii) 125 I-labelled BSA.With the mouse under isoflurane anaesthesia (1.5-2% in oxygen), whole-body SPECT/ CT scans were acquired at 3 h, 24 h, 48 h, and 6 days postinjection (p.i.).Using a full ring detector, 3601 of data were acquired by rotating the collimator at 451 (45 steps, 11 per step).

Fig. 2
Fig. 2 Characterisation of dual-labelled NPs: (a) TEM images of oleic acid-stabilised iron oxide NPs; (b) intensity distribution for NP1 in water as determined by dynamic light scattering; (c and d) TEM images of NP1; (e) gamma spectra corresponding to 111 In (red line) and 125 I (blue line); (f) gamma spectrum of NP1.

Fig. 3
Fig. 3 Stability of [ 125 I/ 111 In]NP1 in physiological saline (top) and mouse plasma (bottom) expressed as % radioactivity attached to the NP at different time points of incubation (T = 37 1C).Results are expressed as mean AE standard deviation (n = 3 per time point).

Fig. 4
Fig. 4 Coronal SPECT images of the biodistribution of (a) [ 125 I/ 111 In]NP1 and (b) [ 125 I]BSA after intravenous administration into mice; red colour: 125 I; green colour: 111 In.Images were co-registered with a CT image of the same animal to anatomically localize the SPECT signal.CT images were adjusted in the Y axis at each time point for appropriate fitting with the tracer distribution image.(c) A schematic of the different organs.

Fig. 5
Fig. 5 Accumulation of 125 I and 111 In in different organs as determined by dissection and gamma counting and expressed as the % of injected dose per gram of tissue (%ID g À1 ).Results are expressed as mean AE standard deviation (n = 3 per time point).

Fig. 6
Fig. 6 125 I/ 111 In ratios in different organs as determined by dissection and gamma counting.Results are expressed as mean AE standard deviation (n = 3 per time point).