Long term biotransformation and toxicity of dimercaptosuccinic acid-coated magnetic nanoparticles support their use in biomedical applications

Although iron oxide magnetic nanoparticles (MNP) have been proposed for numerous biomedical applications, little is known about their biotransformation and long-term toxicity in the body. Dimercaptosuccinic acid (DMSA)-coated magnetic nanoparticles have been proven efficient for in vivo drug delivery, but these results must nonetheless be sustained by comprehensive studies of long-term distribution, degradation and toxicity. We studied DMSA-coated magnetic nanoparticle effects in vitro on NCTC 1469 non-parenchymal hepatocytes, and analyzed their biodistribution and biotransformation in vivo in C57BL/6 mice. Our results indicate that DMSA-coated magnetic nanoparticles have little effect on cell viability, oxidative stress, cell cycle or apoptosis on NCTC 1469 cells in vitro. In vivo distribution and transformation were studied by alternating current magnetic susceptibility measurements, a technique that permits distinction of MNP from other iron species. Our results show that DMSA-coated MNP accumulate in spleen, liver and lung tissues for extended periods of time, in which nanoparticles undergo a process of conversion from superparamagnetic iron oxide nanoparticles to other non-superparamagnetic iron forms, with no significant signs of toxicity. This work provides the first evidence of DMSA-coated magnetite nanoparticle biotransformation in vivo.RM holds a post-doctoral contract supported by EU-FP7 MULTIFUN project (no. 262943), LG holds a Sara Borrell post-doctoral contract (CD09/00030) from the Carlos III Health Institute, Spanish Ministry for Health, Social Services and Equality (MSSSI), and TMZ received a FPU pre-doctoral fellowship from the Spanish Ministry of Economy and Competitiveness (MINECO). This work was partially supported by grants from the MINECO (SAF-2011-23639 to DFB and MAT2011-23641 and CSD2007-00010 to MPM), the Research Network in Inflammation and Rheumatic Diseases (RIER) of the ISCIII-MSSSI Cooperative Research Thematic Network program (RD08/0075/0015 to DFB), the Madrid regional government (S009/MAT-1726 to MPM), and EU-FP7 MULTIFUN project (no. 262943 to DFB and MPM).S2009/MAT-1726/NanobiomagnetPeer Reviewe

PI3K p110δ is expressed by gp38(-)CD31(+) and gp38(+)CD31(+) spleen stromal cells and regulates their CCL19, CCL21, and LTβR mRNA levels.

The role of p110δ PI3K in lymphoid cells has been studied extensively, showing its importance in immune cell differentiation, activation and development. Altered T cell localization in p110δ-deficient mouse spleen suggested a role for p110δ in non-hematopoietic stromal cells, which maintain hematopoietic cell segregation. We tested this hypothesis using p110δ(WT/WT) mouse bone marrow to reconstitute lethally irradiated p110δ(WT/WT) or p110δ(D910A/D910A) (which express catalytically inactive p110δ) recipients, and studied localization, number and percentage of hematopoietic cell subsets in spleen and lymph nodes, in homeostatic conditions and after antigen stimulation. These analyses showed diffuse T cell areas in p110δ(D910A/D910A) and in reconstituted p110δ(D910A/D910A) mice in homeostatic conditions. In these mice, spleen CD4(+) and CD8(+) T cell numbers did not increase in response to antigen, suggesting that a p110δ(D910A/D910A) stroma defect impedes correct T cell response. FACS analysis of spleen stromal cell populations showed a decrease in the percentage of gp38(-)CD31(+) cells in p110δ(D910A/D910A) mice. qRT-PCR studies detected p110δ mRNA expression in p110δ(WT/WT) spleen gp38(-)CD31(+) and gp38(+)CD31(+) subsets, which was reduced in p110δ(D910A/D910A) spleen. Lack of p110δ activity in these cell populations correlated with lower LTβR, CCL19 and CCL21 mRNA levels; these molecules participate in T cell localization to specific spleen areas. Our results could explain the lower T cell numbers and more diffuse T cell areas found in p110δ(D910A/D910A) mouse spleen, as well as the lower T cell expansion after antigen stimulation in p110δ(D910A/D910A) compared with p110δ(WT/WT) mice

qRT-PCR analysis of homeostatic chemokines and TNF family members in spleen, LN and spleen stromal cell subsets from p110δ^WT/WT and p110δ^D910A/D910A mice.

<p>Total RNA was extracted from p110δ^WT/WT and p110δ^D910A/D910A spleen, LN, and sorted spleen stromal cell subsets (<i>n</i> = 5 mice/genotype). Expression of CCL19, CCL21, LTα, LTβ and LTβR was analyzed by qRT-PCR in spleen (<b>A</b>), LN (<b>B</b>), and stromal cell subsets (<b>C</b>). Normalized quantities (mean 2^−ΔCt) of mRNA are depicted. Student's <i>t</i>-test, *p<0.05, **p<0.01, ***p<0.001.</p

Immunofluorescence analysis of immune cell distribution and white pulp area.

<p>Frozen sections of spleen and LN from p110δ^WT/WT, p110δ^D910A/D910A, and reconstituted mice were immunofluorescence-stained to detect T cells (CD3⁺, Thy1.2⁺), B cells (B220⁺), MMM (MOMA⁺) and DC (CD11c⁺). Representative images of spleen (<b>A</b>) and LN (<b>B</b>) sections for all conditions are shown (<i>n</i> = 6 mice/condition). Bar = 200 µm. (<b>C</b>) Measurement of white pulp area in hematoxylin/eosin-stained frozen spleen sections (3 sections/mouse, 6 mice/condition), quantified with ImageJ software. Mean ± SD; Kolmogorov-Smirnov test, <b>***</b>p<0.001.</p

Absolute numbers of spleen and LN total cells, CD4⁺ and CD8⁺ T cells before and after antigen stimulation.

<p>Spleens and LN were extracted from p110δ^WT/WT, p110δ^D910A/D910A, and reconstituted mice in homeostatic conditions (t = 0) and after antigen stimulation (five days post-injection of inactivated <i>C. albicans</i>, t = 5 d). Whole organ cell suspensions were counted to determine total cell number (<b>A, D</b>) and stained to determine CD4⁺ T (<b>B, E</b>) and CD8⁺ (<b>C, F</b>) cell numbers by flow cytometry (<i>n</i> = 6 mice/condition). Mean ± SD.</p

FACS analysis of stromal cell populations in spleen from p110δ^WT/WT and p110δ^D910A/D910A mice.

<p>Spleens from p110δ^WT/WT and p110δ^D910A/D910A mice were processed and stained with anti-CD45, -TER119, -CD31, and -gp38 mAb. A) Representative gating strategy for the analysis of stromal cell populations. Stromal cells were gated via the exclusion of dead, CD45-, and TER119-positive cells. B) Quantification of the percentage and absolute number of stromal cell populations in spleens of p110δ^WT/WT and p110δ^D910A/D910A mice (<i>n</i> = 3 experiments/spleen, 6 mice/group). Student's <i>t</i>-test, *p<0.05.</p

p110δ mRNA expression in spleen stromal cell populations from p110δ^WT/WT and p110δ^D910A/D910A mice.

<p>Total RNA was extracted from sorted p110δ^WT/WT and p110δ^D910A/D910A spleen stromal cell subsets (<i>n</i> = 5 mice/genotype). Lymphoid cells (CD45⁺) were sorted as control. Expression of p110δ mRNA was analyzed by qRT-PCR. Normalized quantities (mean 2^−ΔCt) of p110δ mRNA are shown.</p

Macrophage and T cell infiltration in lesions of LDLR^−/−p110γ^−/− compared to LDLR^−/−p110γ^+/− mice.

<p>Aortic sinus sections were studied in LDLR^−/−p110γ^+/− (females, <i>n</i> = 6) and LDLR^−/−p110γ^−/− mice (females, <i>n</i> = 7) after two months on a high-fat diet. (<b>A</b>) Representative photomicrographs of Mac-3⁺ cells in aortic sinus sections after immunohistochemical staining. Bar = 200 μm. Arrows indicate Mac-3⁺ area. (<b>B</b>) Percentage of Mac-3⁺-stained area relative to total lesion area, quantified with ImageJ software. (<b>C</b>) Representative photomicrographs of CD3⁺ cells in aortic sinus sections after immunohistochemical staining. Bar = 200 μm. (<b>D</b>) Percentage of CD3⁺ cells relative to total lesion area, quantified with ImageJ. (<b>E</b>) Representative photomicrographs of immunofluorescent staining for vascular smooth muscle cells (αSMA⁺) in aortic sinus sections from LDLR^−/−p110γ^+/− and LDLR^−/−p110γ^−/− mice after two months on a high-fat diet (<i>n</i> = 6 females/genotype). Bar = 100 μm. (<b>F</b>) Percentage of αSMA⁺ area relative to total lesion area, quantified with ImageJ. Mean ± SD. Student’s <i>t</i>-test.</p