Covalent assembly of nanoparticles as a peptidase-degradable platform for molecular MRI

Ligand-conjugated microparticles of iron oxide (MPIO) have the potential to provide high sensitivity contrast for molecular magnetic resonance imaging (MRI). However, the accumulation and persistence of non-biodegradable micron-sized particles in liver and spleen precludes their clinical use and limits the translational potential of MPIO-based contrast agents. Here we show that ligand-targeted MPIO derived from multiple iron oxide nanoparticles may be coupled covalently through peptide linkers that are designed to be cleaved by intracellular macrophage proteases. The synthesized particles possess potential characteristics for targeted MRI contrast agents, including high relaxivity, unappreciable sedimentation, clearance from circulation and no overt toxicity. Importantly, we demonstrate that these particles are rapidly degraded both in vitro and in vivo, and that the targeted probes can be used for detection of inflammation in vivo using MRI. This approach provides a platform for molecular MRI contrast agents that is potentially more suitable for translation to humans.

for the translational capacity that the mMPIO method may deliver through its degradability. In this way the advantages of SPIO and MPIO are combined in one system.

Supplementary Figures
Supplementary Figure 1. Schematic to illustrate the molecular basis of the mMPIO as a targeted MRI contrast agent.
Intravenously injected targeted mMPIO bind to their target on the diseased endothelial surface (i), but do not bind to healthy endothelium (v). The unbound mMPIO are rapidly cleared from blood (ii and vi).
Thus, negligible background contrast effects are evident with mMPIO shortly after injection. mMPIO are efficiently taken up by macrophages (ix), and after internalisation and fusion to lysosomes the internal peptide linkers are rapidly degraded to yield nanoparticles that are further degraded as widely described.
In contrast targeted USPIO are slowly cleared from blood, which increases the background level during MRI (iii, iv, vii, viii). USPIO   HPLC chromatogram of the stability of peptide 1 in human serum at times 1h (red), 2h (green), 4h (blue) and 24h (violet), and peptide 1 in PBS at 24h (orange).  The data show the mMPIOs assembled with some structural variance; however, larger particles appear to be pseudo-spherical. Scale bars 50-500 nm, as shown.
(b) Size and reproducibility of the mMPIO synthesis. NPs (building blocks) and mMPIO size distribution from crude reaction mixtures were measured by DLS (Zetasizer Nano ZSP) and analysed with Zetasizer Software (Malvern, version 7.11). The average resulting mean particle diameter is 743.01 ± 242.18 nm (mean±s.d, n=8) for the synthesized mMPIOs and 131.05 ± 40.01 nm (mean±s.d, n=8) for the NPs building blocks used. Each synthesis was measured at least 3 times and the standard deviation was plotted for each individual synthesis. The figure was generated with GraphPad Prism 5.01 software.
(c) Comparison of size, size distribution of commercial particles and mMPIOs as measured by DLS. Size distribution was measured by DLS (Zetasizer Nano ZSP) and analysed with Zetasizer Software (Malvern, version 7.11). Both commercial and mMPIOs were measured at least 3 times and the standard deviation was plotted for the particles. The figure was generated with GraphPad Prism 5.01 software.

Supplementary Figure 19. Spectrophotometric determination of particle sedimentation.
Sample light absorption was measured at λ = 500 nm for 24 hours in a closed cuvette. Dynabeads rapidly sediment within few hours, MPIO sedimentation is slightly slower but appreciable finally mMPIO remain in solution during the time of the experiment with negligible particle sedimentation. T 2 relaxivity plots for uniform dispersions of particles in a 6% agarose matrix. mMPIO (red circles) have a greater relaxivity (shown by a steeper slope) than Dynabeads (green triangles), but slightly lower than the MPIO (black diamonds). Errors are expressed as mean±s.d. of three samples.  Three masked and thresholded T 2 *-weighted images from a 3D dataset obtained from a mouse injected intrastriatally with 20 ng of IL-1β in 1µl of saline 3 h before intravenous injection of the control isotype IgG-mMPIO (4 mg Fe / kg body weight). Negligible contrast effects arising from the mMPIO are evident.

Supplementary
The masked and thresholded images were analysed in ImagePro (Media Cybernetic, UK). The same images as shown in Figure 5   [a] ratio between peptido-NPs and carboxy-NPs.  Table 5. Activity assay of peptides with cathepsin L.
The assay condition is described in the methods section. The reaction mixture was analysed by HPLC to detect the formation of products from the overnight reaction.

Suppl. Figs. 33-34
Biodistribution VCAM-mMPIO Suppl. Fig. 35 Antibody loading determination Suppl. Fig. 37 Supplementary mMPIO possess equivalent R 2 values than particles with similar iron core size but due the larger number of cores contained in each mMPIO the T 2 contrast effect per particle is orders of magnitude larger. [a] Name of the compound and reference from which the data has been taken. [b] Assuming 40.5 metal atoms/nm 3 of core. 20 [c] Relative to Sinerem.

Supplementary Table 11. Haematology analysis 2 days post-αhuVCAM-mMPIO injection.
Statistical analysis showed no significant differences between groups with unpaired, two-tailed t-tests Eosin -eosinophils; Baso -basophils.  Samples were flash-frozen on liquid nitrogen and were heated at 100 °C for 120 seconds prior to the analyses by HPLC (Supplementary Figures 14-17 and Supplementary Table 5).

Transmission electron microscopy (TEM)
5-10 µl of a sucrose-purified particle suspension (0.1 mg Fe/ml) in Milli-Q water were allowed to settle briefly (1 minute) onto pyroxylin and carbon-coated copper grids (400 mesh, Agar Scientific), and then blotted dry with filter paper. For negative staining 2% (w/v), uranyl acetate was used; the excess solution was removed with filter paper, and the grids left to air-dry until needed. Grids were viewed at 80 Kv in an FEI Tecnai12 TEM (FEI UK Ltd, Cambridge) and images were obtained using a bottom-mounted AMT XR60 CCD camera (Deben UK Ltd, Bury St. Edmunds).

Synthesis of 733 nm amino terminated particles (MPIO)
10 mmol of FeCl 3 ·6H 2 O and 3.0 g of dextran (MW 11000) were dissolved in 20 mL of water and deoxygenated thoroughly by repeated cycles of vacuum assisted by sonication and argon flushing. After the first deoxygenation cycle, 6 mmol of FeCl 2 ·4H 2 O in 5 mL of water was added and the solution was deoxygenated by the above procedure (4 times). Whilst being stirred with an overhead stirrer at 600 rpm, 8mL of NH 4 OH (25%) were added at a rate of 168 mL/h. The reaction was heated to 80 ºC and then stirred at that temperature for 1 hour. The solution was cooled, dialyzed against water in a Spectra/Por membrane (MWCO 100000) for 21 h.
10 mL of the above synthesized particles (5.7 mg/mL) were loaded into a 250 mL round flask equipped with a 30x16 mm oval stirrer bar. While the solution was stirred at 500 rpm, 5 mL of NaOH (aq. 10 M) were added at a rate of 168 mL/h. After that, 4 mL of epichlorohydrin was added at a rate of 96 mL/h.
The mixture was stirred at 1000 rpm for 7h and then 7 mL of NH 4 OH (25%) was added at a rate of 168 mL/h. The mixture was stirred at 1000 rpm for 14 hours. The mixture was then dialysed against water in a Spectra/Por 2 membrane and then concentrated on a Vivaspin 15 unit (30000 MCWO) to 15 mg Fe/mL. haematological and blood chemistry measurements at each time point, and consequently results were considered significant at p<0.0014.