Different coatings on magnetic nanoparticles dictate their degradation kinetics in vivo for 15 months after intravenous administration in mice

Background The surface coating of iron oxide magnetic nanoparticle (MNPs) drives their intracellular trafficking and degradation in endolysosomes, as well as dictating other cellular outcomes. As such, we assessed whether MNP coatings might influence their biodistribution, their accumulation in certain organs and their turnover therein, processes that must be understood in vivo to optimize the design of nanoformulations for specific therapeutic/diagnostic needs. Results In this study, three different MNP coatings were analyzed, each conferring the identical 12 nm iron oxide cores with different physicochemical characteristics: 3-aminopropyl-triethoxysilane (APS), dextran (DEX), and dimercaptosuccinic acid (DMSA). When the biodistribution of these MNPs was analyzed in C57BL/6 mice, they all mainly accumulated in the spleen and liver one week after administration. The coating influenced the proportion of the MNPs in each organ, with more APS-MNPs accumulating in the spleen and more DMSA-MNPs accumulating in the liver, remaining there until they were fully degraded. The changes in the physicochemical properties of the MNPs (core size and magnetic properties) was also assessed during their intracellular degradation when internalized by two murine macrophage cell lines. The decrease in the size of the MNPs iron core was influenced by their coating and the organ in which they accumulated. Finally, MNP degradation was analyzed in the liver and spleen of C57BL/6 mice from 7 days to 15 months after the last intravenous MNP administration. Conclusions The MNPs degraded at different rates depending on the organ and their coating, the former representing the feature that was fundamental in determining the time they persisted. In the liver, the rate of degradation was similar for all three coatings, and it was faster than in the spleen. This information regarding the influence of coatings on the in vivo degradation of MNPs will help to choose the best coating for each biomedical application depending on the specific clinical requirements. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12951-022-01747-5.

and counted in a Neubauer chamber. The samples were digested in 1 ml of 37% HCl (Merck) for 1 h at 90 °C and then made up to a final volume of 10 ml with distilled water.
The amount of iron relative to the number of cells in each sample was measured using ICP-OES (Perkin Elmer-2400) at the Chemical Analysis Service of the ICMM-CSIC.

Iron quantification in blood by ICP-OES
The iron was quantified in the blood at different times after MNP administration by ICP-OES analysis, as indicated for the cell lysates (see above). The mice administered PBS alone were used as controls to determine the basal iron concentration in each sample. The amount of iron was expressed relative to the weight of the sample determined previously.

Corona formation in mouse serum
Protein corona (PC) formation was studied by incubating MNPs (APS, DEX or DMSA coated MNPs) at a concentration of 125 μg Fe/ml for different times (0, 1, 3, 5, 10, 24, 48, 72 h) in DMEM medium supplemented with 10% mouse serum (MS). As a negative control the same experimental conditions were used without MS. The hydrodynamic size was measured following the protocol described for MNP physicochemical characterization. To analyze the surface charge, the Z potential was measured at pH 7 using the NanoSizer ZS (Malvern). Figure S1 summarizes the main physicochemical characteristics of the MNPs used during this study and characterized previously(1, 2).

Evaluation of the blood circulation time of MNPs
To assess whether the particles were still circulating in the bloodstream 7 days after the last dose of MNPs was administered, two experimental approaches were followed: ICP-OES analysis, which allowed us to quantify the total amount of iron in the blood; and AC magnetic susceptibility that determined the presence of MNPs in the blood. The levels of iron detected by ICP-OES in the blood of mice treated with MNPs were lower than those of the control group not exposed to MNPs (10.57 ± 1.24 μg/mg of sample). In mice administered APS coated MNPs, the iron content detected was 7.55 (± 3.23) μg/mg of sample, while in those treated with DEX-and DMSA-MNPs the blood iron concentrations were 4.55 (± 0.41) μg/mg of sample and 3.90 (± 0.49) μg/mg of sample, respectively (Fig. S2a). Finally, a blood sample was analyzed for AC magnetic susceptibility in mice treated with APS-MNPs at 7 and 14 days, which was the type of coating where the greatest amount of iron was observed in the blood by ICP-OES analysis, and the results confirmed that no particles were found in the bloodstream after 7 days ( Fig. S2b and S2c).

AC magnetic susceptibility measurement of the PBS-treated mouse organs
AC magnetic susceptibility was measured 7 days after the last PBS dose was administered to control mice to detect any magnetic signal in these mice in the temperature range studied. These measurements were carried out under the same conditions as those used for the treated mice. As expected, no signal was detected in the temperature range where the AC magnetic susceptibility maximum for the particles appears. A small maximum in the out-of-phase magnetic susceptibility was evident around 8 K in the spleens corresponding to the ferritin signal. Ferritin in spleens was expected to occur due to the natural process of iron accumulation in this organ over the animal's life time.

Evaluation of corona formation in mouse serum
In order to evaluate the effects of different coatings on PC formation, APS-, DEX-or DMSA-MNPs were incubated in DMEM medium supplemented with 10% MS for

Western blots to assess MNP-loaded endolysosome enrichment
The vesicles loaded with each of the MNPs offered a profile of the final endolysosomal compartment and only endolysosomes containing MNPs appeared to be isolated, as witnessed by the enrichment of the lysosomal marker Lamp1 (Fig. S5a and S5b). Cell viability studies to determine the optimal concentration of MNPs to isolate

MNP-loaded endolysosomes
MNP treatment did not affect RAW 264.7 and NCTC1469 cell viability in the PrestoBlue assays (Fig. S6). The fluorescence readings from the PrestoBlue assays of the MNPtreated RAW 264.7 cells indicated a slight increase in the mitochondrial metabolism of these cells. The optimal MNP concentration selected was 125 μg/ml for both cell types.

TEM images of MNPs isolated from endolysosomes
In the Figure S7 we observed the TEM images of MNPs (APS-, DEX-or DMSA-MNPs) after being incubated for 24 h with RAW 264.7 or NCTC1469 macrophagic cells and then subcellularly located in endolysosomes. As a control of the size of the iron oxide core, the MNPs resuspended in water are observed.

Uptake of MNPs by RAW 264.7 and NCTC1469 cells
We also characterized the uptake of APS-, DEX-and DMSA-MNPs by macrophage cells.

Change of the position of the AC magnetic susceptibility maximum as evidence of nanoparticle degradation
The differences in the temperature of the MNP's magnetic susceptibility maximum may be related to a change in the interactions between particles due to less aggregation or to a decrease in the size of particles related to their degradation over time. A larger variation of the maximum temperature location was found in the livers than in the spleens, which may be associated to different particle transformation over time.  The ferritin and paramagnetic ion signal at low temperatures in the liver and spleen of PBS treated-mice was studied at different times by AC magnetic susceptibility (see Fig.   S11).

Fig. S11. Evolution of the ferritin and paramagnetic ion signals in PBS-treated mice measured by AC magnetic susceptibility at different times.
The magnetic susceptibility showed a paramagnetic signal in the in-phase magnetic susceptibility component in the liver, although no paramagnetic contribution was observed in the spleen. In addition, a ferritin signal in the out-of-phase magnetic susceptibility component that increased over time was observed in the spleen but not in the liver. The in-phase (real, χ′ -Top) and out-of-phase (imaginary, χ′′ -Bottom) component of the AC magnetic susceptibility measurements of the liver (a) and spleen (b) are shown.
In the liver tissue, a paramagnetic signal in the in-phase magnetic susceptibility component was evident at low temperatures. This signal at low temperature was studied at different times and the ferritin signal was not observed in the out-of-phase magnetic susceptibility in this organ. By contrast, a ferritin signal in the in-phase and out-of-phase magnetic susceptibility components was clearly observed in the spleen, and it increased with time. Nevertheless, no paramagnetic signal was found in the spleen tissue.