The One Year Fate of Iron Oxide Coated Gold. Nanoparticles in Mice
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1 Supporting Information The One Year Fate of Iron Oxide Coated Gold Nanoparticles in Mice Jelena Kolosnjaj-Tabi, 1,2+ Yasir Javed, 3+ Lénaic Lartigue, 1,3 Jeanne Volatron, 1 Dan Elgrabli, 1 Iris Marangon, 1 Giammarino Pugliese, 5 Benoit Caron, 4 Albert Figuerola, 5 Nathalie Luciani, 1 Teresa Pellegrino, 5 Damien Alloyeau, 3 Florence Gazeau 1*
2 Figure S1: Characterization of polymer-coated gold/iron oxide heterostructures. The TEM images show a layer of polymer which surrounds heterostructures. The hydrodynamic diameter in water is peaked around 60 nm (as determined from DLS measurement, the distribution by number is shown). Electrophoresis in agarose gel at 1%, when a voltage of 100 V is applied for 1 hour, shows a migration of the heterostructure from the loading well (the red dotty line) towards the positive pole. This means that the anhydride groups of the polymer once hydrolysed in water confers a very negative surface to the water transferred heterostructures.
3 Figure S2: Characterization of PEG-coated gold/iron oxide heterostructures. The TEM images shows well dispersed NHs while the thin layer of PEG is not visible around each heterostructure. The hydrodynamic diameter measured by number is peaked around 65 nm and is very narrow. Although the PEG-coated NHs are individually coated nano-object, they have a zeta potential around -5 mv and therefore do not migrate on the agarose gel (data not shown).
4 Polymer-coated NHs PEG-coated NHs Water NaCl 0.9% PBS Figure S3: Stability of polymer-coated and PEG-coated gold/iron oxide heterostructures in water, saline (NaCl 0.9%) and PBS (10 mm iron). Both PEG-coated and polymer-coated particles are stable in water (tested over 48 hrs). Polymer-coated particles are also stable in 0.9% NaCl and in PBS, while PEG-coated particles tend to aggregate in NaCl 0.9% in one hour and more slowly in PBS. Here the optical absorption at 500 nm is plotted as a function of time. Protocol for absorption analysis The stability of polymer coated and ligand exchanged nanoparticles was studied in purified water, saline solution ( NaCl 0.9%), PBS (ph 7.4), DMEM (FBS free), DMEM 10%FBS and DMEM 50%FBS. The sample concentration was fixed at 56 µg/ml (in iron) and the absorption spectrum for each sample was recorded at defined intervals up to 60 minutes. For the samples in water, being more stable we have recorded the spectra up to 2 days. A Cary 50 spectrophotometer was used to record the spectrum from 1100 nm to 250 nm.
5 Polymer-coated NHs PEG-coated NHs DMEM DMEM +10% FBS DMEM +50% FBS Figure S4: Stability of polymer-coated and PEG-coated gold/iron oxide heterostructures (10 mm iron) in Dulbecco s Modified Eagle s Medium (DMEM) without fetal bovine serum (FBS)) and DMEM with 10% and 50 % FBS. Both PEG-coated and polymer-coated particles tend to aggregate in DMEM medium without serum, but they both recover long term stability (tested over 48 hrs) in DMEM with either 10% or 50% FBS. Here the optical absorption at 500 nm is plotted as a function of time.
6 Polymer-coated NHs PEG-coated NHs Water DMEM +10% FBS DMEM +50% FBS Figure S5: Hydrodynamic diameter distribution measured by DLS (distribution in number) for polymer-coated and PEG-coated gold/iron oxide heterostructures in DMEM without FBS and DMEM with 10% and 50 % FBS. Mean hydrodynamic diameters shift from around 60 nm in water to 140 nm and 117 nm in 10 % FBS and 91 nm and 62 nm in 50% serum, for PEG-coated and polymer-coated heterostructures, respectively.
7 Polymer-coated NHs PEG-coated NHs Water DMEM +10% FBS DMEM +50% FBS Figure S6: TEM micrographs of polymer-coated and PEG-coated gold/iron oxide heterostructures in water and in DMEM with 10% and 50 % FBS. The protein corona surrounding heterostructures in presence of FBS is clearly seen. It is more apparent on polymer-coated particles.
8 Polymer-coated NHs PEG-coated NHs Figure S7: Gel electrophoresis of polymer-coated and PEG-coated heterostructures particles that were magnetically separated from the full media (DMEM, DMEM + 10% FBS and DMEM + 50 % FBS) in which they were incubated for 1 hour. The presence of proteins on separated particles is attested by the bands at the same position to that of FBS. Protocol for Gel electrophoresis After having dispersed nanoparticles (56µg of Fe) in either DMEM 10% FBS or DMEM 50% FBS, the sample was recovered by applying a magnet for 8 hours, and after removal of the supernatant, the nanoparticles collected at the magnet, were dissolved in 50 µl of DMEM (FBS free). Per each sample, to 20 µl of this solution, 5 µl of loading gel (glycerol and orange G in tris borate EDTA, TBE, buffer) were added; 18 µl of this new solution was loaded in the loading well of agarose gel (0.5% agarose in TBE). The electrophoresis was conducted on 7 cm agarose gel for 30 min at 100V. To visualize the protein, the gel was staining with Coomassie blue dye. The gel was stained in 10% acetic acid in water, containing 60mg/L of Coomassie Blue R-250, for 15 minutes; until the gel was of uniform blue color. The de-staining was carried out by keeping the sample for 2 hours in aqueous solution of 10% acetic acid and 40% MeOH. This washing step was repeated twice.
9 Figure S8. TGA analysis of a dried sample of heterostructures PEG-coated (upper panel) and polymer coated (lower panel). The sample was heated up under nitrogen at 10 C/min and the weight loss, which corresponds to the organic component of the material, as a function of temperature, was recorded. In the case of PEG-NH a loss of 9% (while the amount of NHs corresponds to 91%) is found with respect to a 23.5 % found for the PC-NH (in this case the amount of NHs corresponds to 76.5%). Notice that for the PC-NH, at higher temperature, we can also see another loss of about 18% that might correspond to the loss of the surfactants present at the NH surface. By considering the molecular weight of the gold-iron oxide dimer (4.9529*10 6 g/mol, that is calculated as the sum of the MW of a gold sphere of 4 nm in size and that of an iron oxide sphere of 14 nm in size, considering the unit cell and the size of the gold and iron oxide sphere) and that of the gallol-peg (3125,12 g/mol) or of the polymer (30000 g/mol) we can estimate the respectively average number of GA-PEG or polymer molecules grafted per nanostructures as: N GA PEG molecules (or polymer) N NH = 9% (23.5%) g/mol (30000 g/mol) = 157 PEG molecules (61 polymer)/nh g/mol 91% (76.5%)
10 Well covered surfaces Weakly covered surfaces Dimers embedded in polymer matrix Weakly protected dimers Figure S9: HR-TEM micrographs of polymer-coated NHs showing the heterogeneity of polymer coverage on NHs.
11 0 min 30 min 60 min 0min 30min 60min Figure S10: HR-TEM monitoring of PEG-coated NHs immersed in the lysosome-like medium for different time-lapse. The iron-oxide moieties are gradually eroded leaving resilient gold nanoparticles. We observe that the iron oxide comprises several crystalline lobes that are degraded independently with different kinetics. The arrow indicates the area between the lobes at which dissolution takes place.
12 Figure S11. Characterization of hollow heterostructures obtained by the polymer-coated NHs after having bleached the gold by using an iodine solution. Under TEM, the cavities left in most of the heterostructures confirms the gold dissolution, although a small fraction of NHs still maintain the gold domains. The shadows around the hollow NHs are due to the polymer coating shell. By comparing the migration on agarose gel of heterostructures before and after the gold removal, it could be observed a more violet band for the gold/iron oxide heterostructures which moves slightly more than when the gold has been removed. In this latter case the band is also more brownish as only a small gold fraction is associated to the sample. Possibly with the gold removal a partially loss of the polymer can occur thus reducing the overall charge of the empty dimers. This might explain the slightly lower migration. A part of polymer may also collapse in holes left by the dissolution of gold.
13 0 min 30 min 60 min Figure S12: HRTEM monitoring of polymer-coated hollow nanostructures immersed in the lysosome-like medium for different time-lapse. The hollow nanoparticles are gradually eroded. We observe again the different crystalline petals of iron oxide that are degraded independently with different kinetics (see blue arrows).
14 D1 spleen D7 liver hepatocyte 4 µm 20 nm D7 liver hepatocyte 40 nm D7 liver 40 nm 40 nm 40 nm Figure S13: PEG-coated NHs at different time-points after IV administration in spleen and liver. We note the coexistence of gold/iron oxide intact heterostructures, resilient gold nanoparticles (white arrows) and iron rich ferritin proteins (red arrows) into the lysosomes of Kupffer cells or hepatocytes in liver and macrophages in spleen. Note the difference of contrast between iron rich ferritins and gold particles. From day 1 to day 7, the particles tend to individualize into electron dense structure or on the margin of the lysosomes.
15 Dimer spleen D14 Dimers Gold nanoparticles? 200 nm Figure S14: Polymer-coated NHs in spleen, 14 days after IV administration. We note the coexistence of gold/iron oxide dimers (blue arrow), resilient gold nanoparticles organized in chains (white arrows) and iron rich ferritin proteins (red arrows) into the lysosome. Iron rich ferritin proteins are also seen dispersed in the cytoplasm. Note the difference of contrast between iron rich ferritins and gold particles.
16 20 nm Figure S15: HR-TEM of polymer-coated NHs in spleen, 30 days after iv administration. We remark the remaining of iron oxide shell around the gold particles (gold/iron oxide interfaces), suggesting local erosion of iron oxide and excluding separation. Red arrows indicate disseminated iron-rich ferritin proteins.
17 Liver Day 90 N KC HC N HC KC 1 µm 1 µm 50 nm 50 nm 50 nm 50 nm Figure S16: Polymer-coated NHs at day 90 in liver. We note the coexistence of gold/iron oxide intact heterostructures and resilient gold nanoparticles that degrades into smaller structures forming assemblies. NHs are located with lysosomes of Kupffer cells (KC). (HC (Hepatocyte), N (Nucleus)
18 40 nm 20 nm 20 nm 40 nm 20 nm Figure S17: Polymer-coated NHs in spleen 12 months after IV administration. One still observes some isolated heterostructures (square in blue, first line middle), but principally resilient gold particles that are assembled as large aggregates (first line, right), small chains (black arrows) or that are degraded into smaller entities (blue arrows).
19 0,05 Polymer-coated PEG-coated %dose Fe injectée Polymer coated %dose Fe injectée PEG coated SP Iron (% injected dose) 0,04 0,03 0,02 0,01 0, Day post injection Figure S18: Quantification of superparamagnetic iron (SP iron) measured by ESPR in kidney as a function of time after single administration. SP iron is expressed as percentage of the injected dose. Bars are standard error of the mean for 4 to 5 mice at each time-point. A B C Figure S19: Histological sections of the liver at D14 after Pearls and Nuclear Red staining: A/ general view (mag x5) of a characteristic liver section showing no apparent pathological alterations, B and C/ magnified view of A showing scattered isolated, iron rich blue dots (mag x20 and x60).
20 Polymer-coated PEG-coated % inj. Iron / % inj. Gold Liver Spleen %Fe/%Au Polymer liver %Fe/%Au Polymer spleen e % inj. Iron / % inj. Gold Liver Spleen %Fe/%Au PEG liver %Fe/%Au PEG spleen Day post injection Day post injection Figure S20: Comparison of the SP Iron/Gold ratio in liver and spleen as function of days after injection of polymer-coated (left) and PEG-coated (right) heterostructures. The ratio is the % of SP iron injected dose over the % of gold injected dose. Blue bar indicates a ratio of 1, corresponding to the nominal SP iron/gold ratio prior to injection. We note the persistent excess of gold (compared to initial injection) in spleen for polymer coated NHs. Excess of SP iron in liver decreases with time due to iron oxide degradation. The distribution and fate of gold and SP iron depend of the organ.
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