Small-angle X-ray scattering to quantify the incorporation and analyze the disposition of magnetic nanoparticles inside cells

Access to detailed information on cells loaded with nanoparticles with nanoscale precision is of a long-standing interest in many areas of nanomedicine. In this context, designing a single experiment able to provide statistical mean data from a large number of living unsectioned cells concerning information on the nanoparticle size and aggregation inside cell endosomes and accurate nanoparticle cell up-take is of paramount importance. Small-angle X-ray scattering (SAXS) is presented here as a tool to achieve such relevant data. Experiments were carried out in cultures of B16F0 murine melanoma and A549 human lung adenocarcinoma cell lines loaded with various iron oxide nanostructures displaying distinctive structural characteristics. Five systems of water-dispersible magnetic nanoparticles (MNP) of different size, polydispersity and morphology were analyzed, namely, nearly monodisperse MNP with 11 and 13 nm mean size coated with meso-2,3-dimercaptosuccinic acid, more polydisperse 6 nm colloids coated with citric acid and two nanoflowers (NF) systems of 24 and 27 nm in size resulting from the aggregation of 8 nm MNP. Up-take was determined for each system using B16F0 cells. Here we show that SAXS pattern provides high resolution information on nanoparticles disposition inside endosomes of the cytoplasm through the structure factor analysis, on nanoparticles size and dispersity after their incorporation by the cell and on up-take quantification from the extrapolation of the intensity in absolute scale to null scattering vector. We also report on the cell culture preparation to reach sensitivity for the observation of MNP inside cell endosomes using high brightness SAXS synchrotron source. Our results show that SAXS can become a valuable tool for analyzing MNP in cells and tissues.This work was supported by Conicet PIP 0897 and 567, UNLP x807 and UBACYT 20020130100673A and we kindly thank Brazilian Synchrotron Light Laboratory (Proposals: SAXS1-14429, SAXS2-22014, SAXS1-20160237, Campinas-Brazil, Universidad Nacional de La Plata-Argentina, and CONICET-Argentina. We thanks Maria del Puerto Morales for usefull discussion. The group of IFLP thanks Instituto de Investigaciones Bioquímicas de La Plata INIBIOLP Patología B - CONICET for allowing the use of cell culture lab, help in cell culture handling and kind suggestions on biological issues and Y-TEC S. A. for the use of TEM TALOS F200X under the supervision of A. Floridia and A. Caneiro. M. E. F. Brollo acknowledges the Brazilian agency CNPq for the grant [232947/2014-7] within the Science without Borders program A.R acknowledges financial support from the Spanish Ministry of Science and Innovation through the ‘Severo Ochoa’ Programme for Centres of Excellence in R&D (CEX2019-000917-S). M. B. Fernández van Raap, P. C. Setton-Avruj and P. Mendoza Zélis, are members of CONICET, and P. A. Soto is a fellow of CONICET, Argentina.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

Nanoclusters of crystallographically aligned nanoparticles for magnetic thermotherapy: aqueous ferrofluid, agarose phantoms and ex vivo melanoma tumour assessment

CNPEN - CENTRO NACIONAL DE PESQUISA EM ENERGIA E MATERIAISMagnetic hyperthermia is an oncological therapy where magnetic nanostructures, under a radiofrequency field, act as heat transducers increasing tumour temperature and killing cancerous cells. Nanostructure heating efficiency depends both on the field conditions and on the nanostructure properties and mobility inside the tumour. Such nanostructures are often incorrectly bench-marketed in the colloidal state and using field settings far off from the recommended therapeutic values. Here, we prepared nanoclusters composed of iron oxide magnetite nanoparticles crystallographically aligned and their specific absorption rate (SAR) values were calorimetrically determined in physiological fluids, agarose-gel-phantoms and ex vivo tumours extracted from mice challenged with B16-F0 melanoma cells. A portable, multipurpose applicator using medical field settings||100 kHz and 9.3 kA m(-1), was developed and the results were fully analysed in terms of nanoclusters' structural and magnetic properties. A careful evaluation of the nanoclusters' heating capacity in the three milieus clearly indicates that the SAR values of fluid suspensions or agarose-gel-phantoms are not adequate to predict the real tissue temperature increase or the dosage needed to heat a tumour. Our results show that besides nanostructure mobility, perfusion and local thermoregulation, the nanostructure distribution inside the tumour plays a key role in effective heating. A suppression of the magnetic material effective heating efficiency appears in tumour tissue. In fact, dosage had to be increased considerably, from the SAR values predicted from fluid or agarose, to achieve the desired temperature increase. These results represent an important contribution towards the design of more efficient nanostructures and towards the clinical translation of hyperthermia.10452126221274CNPEN - CENTRO NACIONAL DE PESQUISA EM ENERGIA E MATERIAISCNPEN - CENTRO NACIONAL DE PESQUISA EM ENERGIA E MATERIAISME-22346Agências de fomento estrangeiras apoiaram essa pesquisa, mais informações acesse artig

Nanoclusters of crystallographically aligned nanoparticles for magnetic thermotherapy: aqueous ferrofluid, agarose phantoms and ex vivo melanoma tumour assessment

Magnetic hyperthermia is an oncological therapy where magnetic nanostructures, under a radiofrequency field, act as heat transducers increasing tumour temperature and killing cancerous cells. Nanostructure heating efficiency depends both on the field conditions and on the nanostructure properties and mobility inside the tumour. Such nanostructures are often incorrectly bench-marketed in the colloidal state and using field settings far off from the recommended therapeutic values. Here, we prepared nanoclusters composed of iron oxide magnetite nanoparticles crystallographically aligned and their specific absorption rate (SAR) values were calorimetrically determined in physiological fluids, agarose-gel-phantoms and ex vivo tumours extracted from mice challenged with B16-F0 melanoma cells. A portable, multipurpose applicator using medical field settings; 100 kHz and 9.3 kA m−1, was developed and the results were fully analysed in terms of nanoclusters’ structural and magnetic properties. A careful evaluation of the nanoclusters’ heating capacity in the three milieus clearly indicates that the SAR values of fluid suspensions or agarose-gel-phantoms are not adequate to predict the real tissue temperature increase or the dosage needed to heat a tumour. Our results show that besides nanostructure mobility, perfusion and local thermoregulation, the nanostructure distribution inside the tumour plays a key role in effective heating. A suppression of the magnetic material effective heating efficiency appears in tumour tissue. In fact, dosage had to be increased considerably, from the SAR values predicted from fluid or agarose, to achieve the desired temperature increase. These results represent an important contribution towards the design of more efficient nanostructures and towards the clinical translation of hyperthermia.Instituto de Física La PlataInstituto de Investigaciones en Electrónica, Control y Procesamiento de SeñalesInstituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

Nanoclusters of crystallographically aligned nanoparticles for magnetic thermotherapy: aqueous ferrofluid, agarose phantom and ex vivo melanoma tumour assessment

Magnetic hyperthermia is an oncologic therapy where magnetic nanostructures, under a radiofrequency field, act as heat transducers increasing tumour temperature and killing cancerous cells. Nanostructure heating efficiency depends both on the field conditions and on the nanostructure properties and mobility inside the tumour. Such nanostructures are often incorrectly bench-marketed in colloidal state and using field settings far off from the recommended therapeutic values. Here, we prepared nanoclusters composed of iron oxide magnetite nanoparticles crystallographically aligned and their specific absorption rate (SAR) values were calorimetrically determined in physiological fluids, agarose-gel-phantoms and ex vivo tumours extracted from mice challenged with B16-F0 melanoma cells. A portable, multipurpose applicator using medical field settings; 100 kHz and 9.3 kA m-1, was developed and the results were fully analysed in terms of nanoclusters structural and magnetic properties. A careful appraisal of the nanoclusters heating capacity in the three milieus clearly indicate that the SAR values of fluid suspensions or agarose-gel-phantoms are not adequate to predict real tissue temperature rise or the dosage needed to heat a tumour. Our results show that besides nanostructure mobility, perfusion and local thermoregulation, nanostructures distribution inside the tumour plays a key role in the effective heating. A suppression of magnetic material effective heating efficiency appears in tumour tissue. In fact, dosage had to be much increased, from the SAR values predicted from fluid or agarose to achieve the desired temperature increase. These results represent an important contribution towards the design of more efficient nanostructures and towards clinical translation of hyperthermia.This research was funded by CONICET (PIPs 897, 154, 524 and 567), UNLP X11/680 and X11/7884, and UBACYT 20020130100024 grants of Argentina and also partially funded by the Spanish Ministry of Economy (MAT2015-64442-R and SEV2015-0496 projects, co-funded with European Social Funds). We acknowledge, O. Moscoso-Londoño for SQUID data acquisition and M. Knobel for the use of Instituto de Física ‘Gleb Wataghin’, Universidade Estadual de Campinas (UNICAMP) magnetometry instrumentation, R. Peralta for her extreme care with cell biological sample preparation for TEM, the Brazilian Nanotechnology National Laboratory (LNNano) for the use of cryo-TEM (project: ME-22346) facilities, Y-TEC S.A. for the use of TEM TALOS F200X under the supervision of A. Floridia and A. Caneiro and F.H. Sánchez, G. Pasquevich and P. Mendoza Zélis for useful discussions during field inductor building. Monte Carlo simulation were performed in UnCaFiyQT-INIFTA-SNCAD. M.B.F.v.Raap, P. C. S. Avruj, L. Rogin, A. Veiga, E. Spinelli, V.Blank, G.P. Saracco and M.A Bab are members of CONICET, and P.A. Soto is a fellow of CONICET, Argentina.Peer reviewe

Fluorescence properties of curcumin-loaded nanoparticles for cell tracking

Bassam Felipe Mogharbel,1 Julio Cesar Francisco,1 Ana Carolina Irioda,1 Dilcele Silva Moreira Dziedzic,1 Priscila Elias Ferreira,1 Daiany de Souza,1 Carolina Maria Costa de Oliveira Souza,1 Nelson Bergonse Neto,2 Luiz Cesar Guarita-Souza,2 Celia Regina Cavichiolo Franco,3 Celso Vataru Nakamura,4 Vanessa Kaplum,4 Letícia Mazzarino,5 Elenara Lemos-Senna,6 Redouane Borsali,7 Paula A Soto,8 Patricia Setton-Avruj,8 Eltyeb Abdelwahid,9 Katherine Athayde Teixeira de Carvalho1 1Cell Therapy and Biotechnology in Regenerative Medicine Department, Pelé Pequeno Príncipe Institute, Child and Adolescent Health Research and Pequeno Príncipe Faculty, Curitiba, Paraná, Brazil; 2Institute of Biological and Health Sciences, Pontifical Catholic University of Paraná (PUCPR), Centro de Ciências Biológicas e da Saúde (CCBS), Curitiba, Brazil; 3Cell Biology Department, Federal University of Paraná, Curitiba, Paraná, Brazil; 4Department of Pharmaceutical Sciences, Universidade Estadual de Maringá, Maringá, Paraná, Brazil; 5Department of Pharmaceutical Sciences, NanoBioMat Laboratory, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil; 6Department of Pharmaceutical Sciences, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil; 7Centre de Recherches sur les Macromolécules Végétales (CERMAV), Centre National de la Recherche Scientifique (CNRS), University Grenoble Alpes, F-38000, Grenoble, France; 8Instituto de Química y Físicoquímica Biológica (IQUIFIB), Departament of Química Biológica, Facultad de Farmacia y Bíoquímica, Universidad de Buenos Aires (UBA) Consejo nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentine; 9Feinberg School of Medicine, Feinberg Cardiovascular Research Institute, Northwestern University, Chicago, Il, USA Background: Posttransplant cell tracking, via stem cell labeling, is a crucial strategy for monitoring and maximizing benefits of cell-based therapies. The structures and functionalities of polysaccharides, proteins, and lipids allow their utilization in nanotechnology systems. Materials and methods: In the present study, we analyzed the potential benefit of curcumin-loaded nanoparticles (NPC) using Vero cells (in vitro) and NPC-labeled adipose-derived mesenchymal stem cells (NPC-ADMSCs) (in vivo) in myocardial infarction and sciatic nerve crush preclinical models. Thereafter, transplantation, histological examination, real time imaging, and assessment of tissue regeneration were done. Results: Transplanted NPC-ADMSCs were clearly identified and revealed potential benefit when used in cell tracking. Conclusion: This approach may have broad applications in modeling labeled transplanted cells and in developing improved stem cell therapeutic strategies. Keywords: mesenchymal stem cells, transplantation, cell marking, myocardium infarction, sciatic nerve crus

Hemoglobin-derived Peptides as Novel Type of Bioactive Signaling Molecules

Most bioactive peptides are generated by proteolytic cleavage of large precursor proteins followed by storage in secretory vesicles from where they are released upon cell stimulation. Examples of such bioactive peptides include peptide neurotransmitters, classical neuropeptides, and peptide hormones. In the last decade, it has become apparent that the breakdown of cytosolic proteins can generate peptides that have biological activity. A case in point and the focus of this review are hemoglobin-derived peptides. In vertebrates, hemoglobin (Hb) consists of a tetramer of two α- and two β-globin chains each containing a prosthetic heme group, and is primarily involved in oxygen delivery to tissues and in redox reactions (Schechter Blood 112:3927–3938, 2008). The presence of α- and/or β-globin chain in tissues besides red blood cells including rodent and human brain and peripheral tissues (Liu et al. Proc Natl Acad Sci USA 96:6643–6647, 1999; Newton et al. J Biol Chem 281:5668–5676, 2006; Wride et al. Mol Vis 9:360–396, 2003; Setton-Avruj Exp Neurol 203:568–578, 2007; Ohyagi et al. Brain Res 635:323–327, 1994; Schelshorn et al. J Cereb Blood Flow Metab 29:585–595, 2009; Richter et al. J Comp Neurol 515:538–547, 2009) suggests that globins and/or derived peptidic fragments might play additional physiological functions in different tissues. In support of this hypothesis, a number of Hb-derived peptides have been identified and shown to have diverse functions (Ivanov et al. Biopoly 43:171–188, 1997; Karelin et al. Neurochem Res 24:1117–1124, 1999). Modern mass spectrometric analyses have helped in the identification of additional Hb peptides (Newton et al. J Biol Chem 281:5668–5676, 2006; Setton-Avruj Exp Neurol 203:568–578, 2007; Gomes et al. FASEB J 23:3020–3029, 2009); the molecular targets for these are only recently beginning to be revealed. Here, we review the status of the Hb peptide field and highlight recent reports on the identification of a molecular target for a novel set of Hb peptides, hemopressins, and the implication of these peptides to normal cell function and disease. The potential therapeutic applications for these Hb-derived hemopressin peptides will also be discussed