Quantitative histochemistry for macrophage biodistribution on mice liver and spleen after the administration of a pharmacological-relevant dose of polyacrylic acid-coated iron oxide nanoparticles

Abstract

Understanding in vivo biodistribution of iron oxide nanoparticles (IONs), and the involvement of the phagocyte system in this process, is crucial for the assessment of their potential health risk. In the present study, the histochemical expression of iron in liver and spleen sections of CD-1 mice (aged 8 weeks) was quantified, 24 h after intravenous administration of polyacrylic acid-coated IONs (PAA-coated IONs) (8, 20, 50 mg/kg). Organ sections were stained with Perls’ Prussian blue for iron detection, followed by the quantification of iron deposition with ImageJ software. Our study revealed the existence of a linear dose-dependent increase of iron deposition in macrophages of both organs. Exposed animals showed hepatic iron deposition in all zones, although most marked in periportal region. In the spleen, no iron was detected in the white splenic pulp of both control and treated animals. When compared with control mice, a positive correlation between histochemical detection of iron and PAA-coated ION doses was observed in splenic red pulp of animals. The results confirmed our assumption that liver and spleen are involved in the clearance pathways of PAA-coated IONs from the blood. Excess iron was cytotoxic at the highest dose of PAA-coated IONs tested, but no significant morphologic alterations were observed for the lower doses. Clusters of early necrotic hepatocytes were observed in the hepatic periportal region of mice injected with the higher dose (50 mg/kg) of PAA-coated IONs. Further studies are necessary to determine if liver and spleen macrophages will degrade these IONs, or eventually exocytose both the degraded and intact ones.

Keywords:

• Iron oxide nanoparticles
• histopathology
• Kupffer cells
• splenic macrophage
• Perls’ reaction

Acknowledgements

This work received financial support from National funds [Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência (FCT/MEC)] and European Union funds [Fundo Europeu de Desenvolvimento Regional (FEDER)] under the program PT2020 (PT2020 UID/MULTI/04378/2013 – POCI/01/0145/FEDER/007728), the QOPNA research Unit (FCT UID/QUI/00062/2013), the framework of QREN (NORTE-01-0145-FEDER-000024), and Programa Operacional Competitividade e Internacionalização (COMPETE) (PTDC/QEQ-QAN/1742/2014 – POCI-01-0145-FEDER-016530). Vera Marisa Costa also acknowledges the FCT financial support for the Pos-doc grant SFRH/BPD/110001/2015 within “POPH – QREN – Tipologia 4.1 – Formação Avançada” co-sponsored by FSE and national funds of MCTES.

Compliance with ethical standards

All animal experiments were done according to a protocol approved by the Ethical Committee of the Faculty of Pharmacy, University of Porto (opinion n∘ 9/07/2014).

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência (FCT/MEC)] and European Union funds [Fundo Europeu de Desenvolvimento Regional (FEDER)] under the program PT2020 (PT2020 UID/MULTI/04378/2013 – POCI/01/0145/FEDER/007728), the QOPNA research Unit (FCT UID/QUI/00062/2013), the framework of QREN (NORTE-01-0145-FEDER-000024), and Programa Operacional Competitividade e Internacionalização (COMPETE) (PTDC/QEQ-QAN/1742/2014 – POCI-01-0145-FEDER-016530).