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Title

Analysis of the intra-lysosomal degradative interactome of magnetic nanoparticles.

AuthorsPortilla, Y.; Sanz-Ortega, L.; Pérez-Yagüe, S.; Paradela, A.; Morales, M. P. ; Barber, D. F.
Issue Date2017
CitationNanomaterials Applied to Life Sciences (2016)
AbstractSuperparamagnetic iron oxide nanoparticles (SPIONs) have a great potential in the field of biomedicine due to its size and physical properties. Some examples would be the targeted drug release, the therapeutic hyperthermia and magnetic resonance imaging. The achieved success in lab assays using SPIONs led us to think that a fast transition to the clinic practice would occur, however this progress was slowed down by the lack of information regarding the molecular and cellular mechanisms that regulate the degradation of the nanoparticles, since it could affect the therapeutic efficiency of the SPIONs or its accumulation and toxicity on the long term. Once the SPIONs enter the body intravenously, they accumulate mostly in the liver and the spleen, where they are captured by macrophages of the reticuloendothelial system (1,2). SPIONs are all superparamagnetic but as time passes they gradually lose this magnetism, which suggests that the lysosome degrades SPIONs (2). Several studies, including those from our research group, have shown that after a period of time, these SPIONs are completely degraded. Such disposal processes point to a specific iron transformation metabolism in the lysosomes, which might be part of the cell¿s physiological iron degradation pathway or might imply the existence of another degenerative process in the cell interactome. To analyze this process and the proteins of the lysosome involved is the main goal of our ongoing research. The first step to achieve this objective is to isolate the organelles where the degradative process takes place: obtain the intra-lysosomal degradative interactome of the SPIONs. To achieve this, a magnetic isolation method was used. To corroborate that the obtained cellular fraction was rich on SPION loaded lysosomes, several techniques were performed: Western blot, microscopy (confocal, fluorescence and TEM) and mass spectrometry analysis. Western blot experiments showed that the sample was rich in LAMP 1 protein, which is typically found in the lysosome membrane, which confirmed that lysosomes were clearly present. Once obtained, we tested its integrity via microscopy. In fluorescence and confocal microscopy, the areas in which SPION appeared were surrounded by lysosomes. In a subsequent analysis by TEM, we observed full lysosomes. This cell fraction was then used for proteomic analysis. It allowed us to obtain a first approximation for the description of the cellular interactome induced by treatment with SPIONs. In this experiment we validate the enrichment of the lysosomal fraction and identify possible proteins involved in the degradation of the nanoparticles. So far, a SPION-rich lysosome fraction has been isolated, which allow us to further study the process of degradation over time of the SPIONs and the proteins involved in it. This knowledge will contribute to make the use of the SPIONs on the clinical practice a reality and to develop a more efficient second generation of nanoparticles.
DescriptionPoster presented at the Nanomaterials Applied to Life Sciences (NALS), held in Gijón (Spain) on December 13-15th, 2017.
URIhttp://hdl.handle.net/10261/187708
Appears in Collections:(CNB) Comunicaciones congresos
(ICMM) Comunicaciones congresos
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