Quantification of the internalization patterns of superparamagnetic iron oxide nanoparticles with opposite charge

Time-resolved quantitative colocalization analysis is a method based on confocal fluorescence microscopy allowing for a sophisticated characterization of nanomaterials with respect to their intracellular trafficking. This technique was applied to relate the internalization patterns of nanoparticles i.e. superparamagnetic iron oxide nanoparticles with distinct physicochemical characteristics with their uptake mechanism, rate and intracellular fate. The physicochemical characterization of the nanoparticles showed particles of approximately the same size and shape as well as similar magnetic properties, only differing in charge due to different surface coatings. Incubation of the cells with both nanoparticles resulted in strong differences in the internalization rate and in the intracellular localization depending on the charge. Quantitative and qualitative analysis of nanoparticles-organelle colocalization experiments revealed that positively charged particles were found to enter the cells faster using different endocytotic pathways than their negative counterparts. Nevertheless, both nanoparticles species were finally enriched inside lysosomal structures and their efficiency in agarose phantom relaxometry experiments was very similar. This quantitative analysis demonstrates that charge is a key factor influencing the nanoparticle-cell interactions, specially their intracellular accumulation. Despite differences in their physicochemical properties and intracellular distribution, the efficiencies of both nanoparticles as MRI agents were not significantly different.

After 12 h of dialysis, external water was renewed in order to allow for depletive removal of the polymer.

b) Synthesis in organic solvents
SPIONs were synthesized using a published protocol by Hyeon and co-workers [2]. Briefly, 10 ml of octyl ether (Sigma) and 1.28 g of oleic acid (Sigma) were mixed and degassed in three-neck flask for 20 minutes at 60 °C. After 20 minutes temperature was increased to 100 °C. At this stage 0.28 ml of iron pentacarbonyl (Sigma) was injected and the temperature was increased up to refluxing temperature (~ 295-300 °C). The solution was kept at this temperature for 1 hour. During this time the initial yellow colour of the solution changed to black. After one hour the solution was cooled to room temperature and 0.34 g of dehydrated trimethylamine oxide (Sigma) was added. The temperature was increased to 130 °C. The solution was kept at this temperature for two hours. During this time the black colour of the solution changed into dark brown. After two hours the solution temperature was again increased to refluxing temperature in steps, each 15 °C/min. The solution was kept at refluxing temperature for another hour. During this time the solution colour again changed from dark brown to black. After one hour the reaction was stopped by removing the heating mantel. At room temperature 2-5 ml of toluene (Sigma) was added followed by 25-30 ml of methanol (Sigma). Methanol caused precipitation of the NPs, which were pelleted with centrifugation at a speed of 2800 rpm. The supernatant was removed and the precipitate was washed by using toluene and methanol. The precipitate containing the NPs was then redispersed in 10-20 ml of toluene. c) Physicochemical characterization of SPIONs.

TEM characterization and size distribution
Size and morphology of hydrophilic iron oxide cores were investigated on a JEM-3010 transmission electron microscope (Jeol Germany, Eching, Germany) at an acceleration voltage of 300 kV. Maghemite suspension droplets were placed onto carbon-coated copper grids S160-3 (Plano, Wetzlar, Germany) and allowed to dry. Core dimensions were calculated by averaging at least 200 diameters registered by ImageJ software. The TEM data give the size of the inorganic iron oxide core, which is smaller than the hydrodynamic diameter as determined via DLS.

ζ-potential
Hydrodynamic diameters and ζ-potentials of NPs after polymer functionalization were assessed by Dynamic Light Scattering (DLS) and Laser Doppler Anemometry (LDA), using a Zetasizer Nano ZS (Malvern Instruments, Herrenberg, Germany). DLS measurements were performed at 25 °C after appropriate dilution of the respective samples with ultra pure water, to avoid multiscattering events. As to LDA analysis, samples were dispersed in sodium chloride (10 mM) in order to maintain a constant ionic strength.  For the dye modified amphiphilic polymer, 1 mg amine containing dye (DY636, Dyomics Corp.) was firstly completely dissolved in 0.4 ml methanol, and then 512 µl above polymer solution was added and followed by overnight shaking incubation. The mixed solvents were exchanged to 25.  This is the diameter before the polymer-coating. They were determined by adding 2 nm (assumed 1 nm thickness shell contributed by surfactants) to the inorganic core diameter determined by TEM analysis. After the polymer coating, the γ-Fe 2 O 3 -PMA-Dy636 NPs were transferred into sodium borate buffer, and the excess polymer or dye grafted polymer were removed by gel electrophoresis. The protocol was the same as used in previously publications [8,9]. In this paper, we have used 2% agarose (UltraPure Agarose, To extract the samples from the gels, the separated sample bands were carefully cut from the gels, and then sealed in dialysis membrane tubes with a molecular weight cut off of 3500 kDa (Standard RC Dialysis Tubing, Pre-treated, Spectrum) for about 15 min gel electrophoresis running. Finally the samples were collected from the dialysis membrane tubes and concentrated by ultrafiltration with centrifuge  Table 1 in manuscript)

Quantification of intracellular iron concentration
As intensity signals were measured either on different FACS channels or in the CLSM and labeling efficiencies of the two NP systems were not homogenous, absolute comparability between γ-Fe 2 O 3 -PEI-      The polycation PEI, which is used as a surface coating for the positively charged NPs, is known as a membrane destabilizing agent [11]. In some cases green fluorescence of PEI coated NPs could be detected outside of endosomes/lysosomes within the cytosol which might indicate permeabilization of intracellular membranes due to the effect of PEI (Fig. SI-6.b.vii). Therefore, the localization of the NPs with respect to the cytosol was also studied. For this purpose, the actin cytoskeleton was stained with phalloidin-tetramethylrhodamine (shown in blue) as described before while traces of γ-Fe

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Quantitative analysis of colocalization studies A549 adenocarcinoma cells were treated as described in §Error! Reference source not found., except the fact, that they were grown on coverslips of 12 mm diameter. For the intensive quantitative colocalization studies the intracellular locations of early endosomes and lysosomes with both nanoparticle species were correlated for each of the given incubations times as shown in Table 1. Therefore either endosomes or lysosomes were stained as described in §6. For each combination at least 20 cells were imaged using a highly corrected CLSM 510 Meta (Zeiss).

Table 1 -Correlation experiments
The image material was corrected for noise by median filtering and thresholding. [12] To quantify the degree of colocalization between fluorescence signal originating from NPs and labeled endosomes (EEA1) or lysosomes (LAMP1) various correlation coefficients given in Table 2 were calculated based on confocal images. Manders' distinct colocalization coefficients M 1 and M 2 [13] were chosen for further interpretation: and (1)    [13], M 1 andM 2 : Manders' distinct colocalization coefficients [13], ICQ: Intensity correlation coefficient [15]

Results
The degree of correlation between positively charged PEI-coated NPs and early endosomes after various incubation times is shown in the left part of Figure SI-7. Manders' colocalization coefficients are close to zero for both structures (PEI-coated NPs: green, EEA1: yellow). The lack of signal overlap is also visible in Figure SI-3.b.ii exemplary. Nevertheless Pearson's correlation coefficient is slightly higher for short incubations times (blue). After 8 h a high amount of NPs can be found inside the lysosomes (right part of Figure SI-7.i, PEI-coated NPs: green, LAMP1: red) but the major fraction of lysosomal structures still does not contain any particles. For t=24 h both coefficients converge. Confocal image data underlining these observations is provided in Figure SI-