Abstract
Nanotechnology plays an increasingly important role in the biomedical arena. Iron oxide nanoparticles (IONPs)-labelled cells is one of the most promising approaches for a fast and reliable evaluation of grafted cells in both preclinical studies and clinical trials. Current procedures to label living cells with IONPs are based on direct incubation or physical approaches based on magnetic or electrical fields, which always display very low cellular uptake efficiencies. Here we show that centrifugation-mediated internalization (CMI) promotes a high uptake of IONPs in glioblastoma tumour cells, just in a few minutes, and via clathrin-independent endocytosis pathway. CMI results in controllable cellular uptake efficiencies at least three orders of magnitude larger than current procedures. Similar trends are found in human mesenchymal stem cells, thereby demonstrating the general feasibility of the methodology, which is easily transferable to any laboratory with great potential for the development of improved biomedical applications.
Highlights
Incubation times, but with much less internalized iron oxide nanoparticles (IONPs) (c.a., 10 pg/cell)[15,16,17,18,19,20,21]
Our centrifugation-mediated internalization (CMI) method allows 100% labeling efficacy with high IONPs internalization (> 200 pg/ cell) via clathrin-independent endocytosis uptake, in short incubation times (1–20 minutes), and requiring only small initial IONPs concentrations (< 50 μ g Fe/ml), which results in cellular uptake efficiencies up to [10−6] cell−1 min−1, three orders of magnitude larger than previous ones
Consistent with previous reports showing that proteins adsorbed onto particles enhance colloidal stability instead of diminishing it, the concentration of fetal bovine serum (FBS) reduces the hydrodynamic diameter of the IONPs used in the present study from 1014 nm (0% FBS) to 357 nm (10% FBS)
Summary
U251 glioblastoma cell line was purchased from American Type Culture Collections (Manassas, VA, USA) This cell line was grown as monolayer in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with fetal bovine serum (FBS) at a final concentration of 10%, 2 mM L-glutamine, 1 μg/ml fungizone and 100 U/ml of penicillin and 100 μ g/ml streptomycin. After the internalization of IONPs in U251 cells or hMSC by DI or CMI, the cells were washed twice with PBS (AMRESCO, Ohio, USA) and fixed with 4% paraformaldehyde solution for 30 minutes at room temperature. At the end of the treatment by DI or CMI methods, the medium was removed and cells were washed twice with PBS, fixed with 4% paraformaldehyde for 30 minutes at room temperature, washed again twice with PBS and Prussian blue staining was performed as described before. Significant differences among the samples are indicated in the charts
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
