Advertisement

The presence of serum alters the properties of iron oxide nanoparticles and lowers their accumulation by cultured brain astrocytes

  • Mark Geppert
  • Charlotte Petters
  • Karsten Thiel
  • Ralf DringenEmail author
Research Paper

Abstract

Iron oxide nanoparticles (IONPs) are considered for various diagnostic and therapeutic applications. Such particles are able to cross the blood–brain barrier and are taken up into brain cells. To test whether serum components affect the properties of IONPs and/or their uptake into brain cells, we have incubated dimercaptosuccinate-coated magnetic IONPs without and with fetal calf serum (FCS) and have exposed cultured brain astrocytes with IONPs in the absence or presence of FCS. Incubation with FCS caused a concentration-dependent increase in the average hydrodynamic diameter of the particles and of their zeta-potential. In the presence of 10 % FCS, the diameter of the IONPs increased from 57 ± 2 to 107 ± 6 nm and the zeta-potential of the particles from −22 ± 5 to −9 ± 1 mV. FCS affected also strongly the uptake of IONPs by cultured astrocytes. The efficient time- and temperature-dependent cellular accumulation of IONPs was lowered with increasing concentration of FCS by up to 90 %. In addition, in the absence of serum, endocytosis inhibitors did not alter the IONP accumulation by astrocytes, while chlorpromazine or wortmannin lowered significantly the accumulation of IONPs in the presence of FCS, suggesting that clathrin-mediated endocytosis and macropinocytosis are involved in astrocytic IONP uptake from serum-containing medium. These data demonstrate that the presence of FCS strongly affects the properties of IONPs as well as their accumulation by cultured brain cells.

Keywords

Albumin Brain Endocytosis FCS IONPs 

Abbreviations

ANOVA

Analysis of variance

BSA

Bovine serum albumin

DMEM

Dulbecco’s modified Eagle’s medium

DMSA

Dimercaptosuccinic acid

DMSO

Dimethyl sulfoxide

EIPA

5-(N-Ethyl-N-isopropyl)amiloride

FCS

Fetal calf serum

HEPES

2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethansulfonic acid

IB

Incubation buffer

IONPs

Iron oxide nanoparticles

LDH

Lactate dehydrogenase

MTT

(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NADH

Nicotinaminde adenine dinucleotide (reduced)

PBS

Phosphate buffered saline

SD

Standard deviation

TEM

Transmission electron microscopy

Notes

Acknowledgments

M. Geppert was a recipient of a Ph.D. fellowship from the Hans-Böckler-Stiftung and a member of the graduate school “nanoToxCom.” The authors thank Dr. Malte Kleemeier (Fraunhofer IFAM, Bremen) for technical advice regarding dynamic light scattering and Dr. Jan Köser (University of Bremen) for technical advice regarding zeta-potential measurements.

References

  1. Andersson BV, Skoglund C, Uvdal K, Solin N (2012) Preparation of amyloid-like fibrils containing magnetic iron oxide nanoparticles: effect of protein aggregation on proton relaxivity. Biochem Biophys Res Commun 419:682–686. doi: 10.1016/j.bbrc.2012.02.77 CrossRefGoogle Scholar
  2. Bajaj A, Samanta B, Yan H, Jerry DJ, Rotello VM (2009) Stability, toxicity and differential cellular uptake of protein passivated-Fe3O4 nanoparticles. J Mater Chem 19:6328–6331CrossRefGoogle Scholar
  3. Barros LF, Deitmer JW (2010) Glucose and lactate supply to the synapse. Brain Res Rev 63:149–159. doi: 10.1016/j.brainresrev.2009.10.002 CrossRefGoogle Scholar
  4. Bee A, Massart R, Neveu S (1995) Synthesis of very fine maghemite particles. J Magn Magn Mater 149:6–9. doi: 10.1016/0304-8853(95)00317-7 CrossRefGoogle Scholar
  5. Bhirde A, Xie J, Swierczewska M, Chen X (2011) Nanoparticles for cell labeling. Nanoscale 3:142–153. doi: 10.1039/c0nr00493f CrossRefGoogle Scholar
  6. Chen ZP, Zhang Y, Xu K, Xu RZ, Liu JW, Gu N (2008) Stability of hydrophilic magnetic nanoparticles under biologically relevant conditions. J Nanosci Nanotechnol 8:6260–6265. doi: 10.1166/jnn.2008.343 CrossRefGoogle Scholar
  7. Chen L, McCrate JM, Lee JC, Li H (2011) The role of surface charge on the uptake and biocompatibility of hydroxyapatite nanoparticles with osteoblast cells. Nanotechnology 22:105708. doi: 10.1088/0957-4484/22/10/105708 CrossRefGoogle Scholar
  8. Chertok B, Moffat BA, David AE, Yu FQ, Bergemann C, Ross BD, Yang VC (2008) Iron oxide nanoparticles as a drug delivery vehicle for MRI monitored magnetic targeting of brain tumors. Biomaterials 29:487–496. doi: 10.1016/j.biomaterials.2007.08.050 CrossRefGoogle Scholar
  9. Dausend J, Musyanovych A, Dass M, Walther P, Schrezenmeier H, Landfester K, Mailander V (2008) Uptake mechanism of oppositely charged fluorescent nanoparticles in HeLa cells. Macromol Biosci 8:1135–1143. doi: 10.1002/mabi.200800123 CrossRefGoogle Scholar
  10. Dringen R, Kussmaul L, Hamprecht B (1998) Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay. Brain Res Brain Res Protoc 2:223–228. doi: 10.1016/S1385-299X(97)00047-0 CrossRefGoogle Scholar
  11. Dringen R, Bishop GM, Koeppe M, Dang TN, Robinson SR (2007) The pivotal role of astrocytes in the metabolism of iron in the brain. Neurochem Res 32:1884–1890. doi: 10.1007/s11064-007-9375-0 CrossRefGoogle Scholar
  12. Eberbeck D, Kettering M, Bergemann C, Zirpel P, Hilger I, Trahms L (2010) Quantification of the aggregation of magnetic nanoparticles with different polymeric coatings in cell culture medium. J Phys D Appl Phys 43:405002–405010. doi: 10.1088/0022-3727/43/40/405002 CrossRefGoogle Scholar
  13. Fauconnier N, Pons JN, Roger J, Bee A (1997) Thiolation of maghemite nanoparticles by dimercaptosuccinic acid. J Colloid Interface Sci 194:427–433CrossRefGoogle Scholar
  14. Geppert M, Hohnholt M, Gaetjen L, Grunwald I, Bäumer M, Dringen R (2009) Accumulation of iron oxide nanoparticles by cultured brain astrocytes. J Biomed Nanotechnol 5:285–293. doi: 10.1166/jbn.2009.1033 CrossRefGoogle Scholar
  15. Geppert M, Hohnholt MC, Thiel K, Nürnberger S, Grunwald I, Rezwan K, Dringen R (2011) Uptake of dimercaptosuccinate-coated magnetic iron oxide nanoparticles by cultured brain astrocytes. Nanotechnology 22:145101–145110. doi: 10.1088/0957-4484/22/14/145101 CrossRefGoogle Scholar
  16. Geppert M, Hohnholt MC, Nürnberger S, Dringen R (2012) Ferritin upregulation and transient ROS production in cultured brain astrocytes after loading with iron oxide nanoparticles. Acta Biomater 8:3832–3839. doi: 10.1016/j.actbio.2012.06.029 Google Scholar
  17. Greulich C, Diendorf J, Simon T, Eggeler G, Epple M, Koller M (2011) Uptake and intracellular distribution of silver nanoparticles in human mesenchymal stem cells. Acta Biomater 7:347–354. doi: 10.1016/j.actbio.2010.08.003 CrossRefGoogle Scholar
  18. Guarnieri D, Guaccio A, Fusco S, Netti PA (2011) Effect of serum proteins on polystyrene nanoparticle uptake and intracellular trafficking in endothelial cells. J Nanopart Res 13:4295–4309. doi: 10.1007/s11051-011-0375-2 CrossRefGoogle Scholar
  19. Hamprecht B, Löffler F (1985) Primary glial cultures as a model for studying hormone action. Methods Enzymol 109:341–345CrossRefGoogle Scholar
  20. He C, Hu Y, Yin L, Tang C, Yin C (2010) Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials 31:3657–3666. doi: 10.1016/j.biomaterials.2010.01.065 CrossRefGoogle Scholar
  21. Hirrlinger J, Dringen R (2010) The cytosolic redox state of astrocytes: maintenance, regulation and functional implications for metabolite trafficking. Brain Res Rev 63:177–188. doi: 10.1016/j.brainresrev.2009.10.003 CrossRefGoogle Scholar
  22. Hohnholt MC, Geppert M, Nürnberger S, von Byern J, Grunwald I, Dringen R (2010) Advanced biomaterials: accumulation of citrate-coated magnetic iron oxide nanoparticles by cultured brain astrocytes. Adv Eng Mater 12:B690–B694. doi: 10.1002/adem.201080055 CrossRefGoogle Scholar
  23. Huang J, Bu L, Xie J, Chen K, Cheng Z, Li X, Chen X (2010) Effects of nanoparticle size on cellular uptake and liver MRI with polyvinylpyrrolidone-coated iron oxide nanoparticles. ACS Nano 4:7151–7160. doi: 10.1021/nn101643u CrossRefGoogle Scholar
  24. Huth US, Schubert R, Peschka-Suss R (2006) Investigating the uptake and intracellular fate of pH-sensitive liposomes by flow cytometry and spectral bio-imaging. J Control Release 110:490–504. doi: 10.1016/j.jconrel.2005.10.018 CrossRefGoogle Scholar
  25. Ivanov AI (2008) Pharmocological inhibition of endocytotic pathways: is it specific enough to be useful? Methods Mol Biol 440:15–33. doi: 10.1007/978-1-59745-178-9_2 CrossRefGoogle Scholar
  26. Iversen TG, Skotland T, Sandvig K (2011) Endocytosis and intracellular transport of nanoparticles: present knowledge and need for future studies. Nano Today 6:176–185. doi: 10.1016/j.nantod.2011.02.003 CrossRefGoogle Scholar
  27. Lamkowsky M, Geppert M, Schmidt MM, Dringen R (2012) Magnetic field-induced acceleration of the accumulation of magnetic iron oxide nanoparticles by cultured brain astrocytes. J Biomed Mater Res A 100:323–334. doi: 10.1002/jbm.a.33263 Google Scholar
  28. Laurent S, Forge D, Port M, Roch A, Robic C, Elst LV, Muller RN (2008) Magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications. Chem Rev 108:2064–2110. doi: 10.1021/Cr068445e CrossRefGoogle Scholar
  29. Lesniak A, Fenaroli F, Monopoli MP, Aberg C, Dawson KA, Salvati A (2012) Effects of the presence or absence of a protein corona on silica nanoparticle uptake and impact on cells. ACS Nano 6:5845–5857. doi: 10.1021/nn300223w CrossRefGoogle Scholar
  30. Lévy M, Wilhelm C, Devaud M, Levitz P, Gazeau F (2012) How cellular processing of superparamagnetic nanoparticles affects their magnetic behavior and NMR relaxivity. Contrast Media Mol Imaging 7:373–383. doi: 10.1002/cmmi.504 CrossRefGoogle Scholar
  31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  32. Lunov O, Syrovets T, Loos C, Beil J, Delacher M, Tron K, Nienhaus GU, Musyanovych A, Mailander V, Landfester K, Simmet T (2011) Differential uptake of functionalized polystyrene nanoparticles by human macrophages and a monocytic cell line. ACS Nano 5:1657–1669. doi: 10.1021/nn2000756 CrossRefGoogle Scholar
  33. Luther EM, Koehler Y, Diendorf J, Epple M, Dringen R (2011) Accumulation of silver nanoparticles by cultured primary brain astrocytes. Nanotechnology 22:375101–375111. doi: 10.1088/0957-4484/22/37/375101 CrossRefGoogle Scholar
  34. Lynch I, Dawson KA (2008) Protein-nanoparticle interactions. Nano Today 3:40–47CrossRefGoogle Scholar
  35. Lynch I, Cedervall T, Lundqvist M, Cabaleiro-Lago C, Linse S, Dawson KA (2007) The nanoparticle-protein complex as a biological entity; a complex fluids and surface science challenge for the 21st century. Adv Coll Interf Sci 134–135:167–174. doi: 10.1016/j.cis.2007.04.021 CrossRefGoogle Scholar
  36. Mahmoudi M, Hosseinkhani H, Hosseinkhani M, Boutry S, Simchi A, Journeay WS, Subramani K, Laurent S (2011) Magnetic resonance imaging tracking of stem cells in vivo using iron oxide nanoparticles as a tool for the advancement of clinical regenerative medicine. Chem Rev 111:253–280. doi: 10.1021/cr1001832 CrossRefGoogle Scholar
  37. Maier-Hauff K, Ulrich F, Nestler D, Niehoff H, Wust P, Thiesen B, Orawa H, Budach V, Jordan A (2011) Efficacy and safety of intratumoral thermotherapy using magnetic iron-oxide nanoparticles combined with external beam radiotherapy on patients with recurrent glioblastoma multiforme. J Neurooncol 103:317–324. doi: 10.1007/s11060-010-0389-0 CrossRefGoogle Scholar
  38. Nel AE, Madler L, Velegol D, Xia T, Hoek EM, Somasundaran P, Klaessig F, Castranova V, Thompson M (2009) Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater 8:543–557. doi: 10.1038/nmat2442 CrossRefGoogle Scholar
  39. Parpura V, Heneka MT, Montana V, Oliet SH, Schousboe A, Haydon PG, Stout RF Jr, Spray DC, Reichenbach A, Pannicke T, Pekny M, Pekna A, Zorec R, Verkhratsky A (2012) Glial cells in (patho)physiology. J Neurochem 121:4–27. doi: 10.1111/j.1471-4159.2012.07664.x CrossRefGoogle Scholar
  40. Petri-Fink A, Steitz B, Finka A, Salaklang J, Hofmann H (2008) Effect of cell media on polymer coated superparamagnetic iron oxide nanoparticles (SPIONs): colloidal stability, cytotoxicity, and cellular uptake studies. Eur J Pharm Biopharm 68:129–137. doi: 10.1016/j.ejpb.2007.02.024 CrossRefGoogle Scholar
  41. Pickard MR, Jenkins SI, Koller CJ, Furness DN, Chari DM (2011) Magnetic nanoparticle labeling of astrocytes derived for neural transplantation. Tissue Eng Part C Methods 17:89–99. doi: 10.1089/ten.TEC.2010.0170 CrossRefGoogle Scholar
  42. Rejman J, Oberle V, Zuhorn IS, Hoekstra D (2004) Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J 377:159–169. doi: 10.1042/BJ20031253 CrossRefGoogle Scholar
  43. Riemer J, Hoepken HH, Czerwinska H, Robinson SR, Dringen R (2004) Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells. Anal Biochem 331:370–375. doi: 10.1016/j.ab.2004.03.049 CrossRefGoogle Scholar
  44. Scheiber I, Dringen R (2011) Copper accelerates glycolytic flux in cultured astrocytes. Neurochem Res 36:894–903. doi: 10.1007/s11064-011-0419-0 CrossRefGoogle Scholar
  45. Scheiber I, Schmidt MM, Dringen R (2010) Zinc prevents the copper-induced damage of cultured astrocytes. Neurochem Int 57:314–322. doi: 10.1016/j.neuint.2010.06.010 CrossRefGoogle Scholar
  46. Smith PJ, Giroud M, Wiggins HL, Gower F, Thorley JA, Stolpe B, Mazzolini J, Dyson RJ, Rappoport JZ (2012) Cellular entry of nanoparticles via serum sensitive clathrin-mediated endocytosis, and plasma membrane permeabilization. Int J Nanomedicine 7:2045–2055. doi: 10.2147/IJN.S29334ijn-7-2045 Google Scholar
  47. Sofroniew MV, Vinters HV (2010) Astrocytes: biology and pathology. Acta Neuropathol 119:7–35. doi: 10.1007/s00401-009-0619-8 CrossRefGoogle Scholar
  48. Tedja R, Lim M, Amal R, Marquis C (2012) Effects of serum adsorption on cellular uptake profile and consequent impact of titanium dioxide nanoparticles on human lung cell lines. ACS Nano 6:4083–4093. doi: 10.1021/nn3004845 CrossRefGoogle Scholar
  49. Tenzer S, Docter D, Rosfa S, Wlodarski A, Kuharev J, Rekik A, Knauer SK, Bantz C, Nawroth T, Bier C, Sirirattanapan J, Mann W, Treuel L, Zellner R, Maskos M, Schild H, Stauber RH (2011) Nanoparticle size is a critical physicochemical determinant of the human blood plasma corona: a comprehensive quantitative proteomic analysis. ACS Nano 5:7155–7167. doi: 10.1021/nn201950e CrossRefGoogle Scholar
  50. Thiesen B, Jordan A (2008) Clinical applications of magnetic nanoparticles for hyperthermia. Int J Hyperther 24:467–474. doi: 10.1080/02656730802104757 CrossRefGoogle Scholar
  51. Valois CR, Braz JM, Nunes ES, Vinolo MA, Lima EC, Curi R, Kuebler WM, Azevedo RB (2010) The effect of DMSA-functionalized magnetic nanoparticles on transendothelial migration of monocytes in the murine lung via a beta2 integrin-dependent pathway. Biomaterials 31:366–374. doi: 10.1016/j.biomaterials.2009.09.053 CrossRefGoogle Scholar
  52. Walczyk D, Baldelli Bombelli F, Monopoli MP, Lynch I, Dawson KA (2010) What the cell “sees” in bionanoscience. J Am Chem Soc 132:5761–5768. doi: 10.1021/ja910675v CrossRefGoogle Scholar
  53. Wang J, Chen Y, Chen B, Ding J, Xia G, Gao C, Cheng J, Jin N, Zhou Y, Li X, Tang M, Wang XM (2010) Pharmacokinetic parameters and tissue distribution of magnetic Fe3O4 nanoparticles in mice. Int J Nanomedicine 5:861–866. doi: 10.2147/IJN.S13662 Google Scholar
  54. Weinstein JS, Varallyay CG, Dosa E, Gahramanov S, Hamilton B, Rooney WD, Muldoon LL, Neuwelt EA (2010) Superparamagnetic iron oxide nanoparticles: diagnostic magnetic resonance imaging and potential therapeutic applications in neurooncology and central nervous system inflammatory pathologies, a review. J Cereb Blood Flow Metab 30:15–35. doi: 10.1038/jcbfm.2009.192 CrossRefGoogle Scholar
  55. Wilhelm C, Billotey C, Roger J, Pons JN, Bacri JC, Gazeau F (2003) Intracellular uptake of anionic superparamagnetic nanoparticles as a function of their surface coating. Biomaterials 24:1001–1011CrossRefGoogle Scholar
  56. Winer JL, Kim PE, Law M, Liu CY, Apuzzo ML (2011) Visualizing the future: enhancing neuroimaging with nanotechnology. World Neurosurg 75:626–637. doi: 10.1016/j.wneu.2011.02.016 CrossRefGoogle Scholar
  57. Wiogo HTR, Lim M, Bulmus V, Yun J, Amal R (2011) Stabilization of magnetic iron oxide nanoparticles in biological media by fetal bovine serum (FBS). Langmuir 27:843–850. doi: 10.1021/La104278m CrossRefGoogle Scholar
  58. Yan GP, Robinson L, Hogg P (2007) Magnetic resonance imaging contrast agents: overview and perspectives. Radiography 13:e5–e19. doi: 10.1016/j.radi.2006.07.005 CrossRefGoogle Scholar
  59. Yang QQ, Liang JG, Han HY (2009) Probing the interaction of magnetic iron oxide nanoparticles with bovine serum albumin by spectroscopic techniques. J Phys Chem B 113:10454–10458. doi: 10.1021/Jp904004w CrossRefGoogle Scholar
  60. Yang X, Hong H, Grailer JJ, Rowland IJ, Javadi A, Hurley SA, Xiao Y, Yang Y, Zhang Y, Nickles RJ, Cai W, Steeber DA, Gong S (2011) cRGD-functionalized, DOX-conjugated, and (64)Cu-labeled superparamagnetic iron oxide nanoparticles for targeted anticancer drug delivery and PET/MR imaging. Biomaterials 32:4151–4160. doi: 10.1016/j.biomaterials.2011.02.006 CrossRefGoogle Scholar
  61. Yue ZG, Wei W, Lv PP, Yue H, Wang LY, Su ZG, Ma GH (2011) Surface charge affects cellular uptake and intracellular trafficking of chitosan-based nanoparticles. Biomacromolecules 12:2440–2446. doi: 10.1021/bm101482r CrossRefGoogle Scholar
  62. Zhu Y, Li WX, Li QN, Li YG, Li YF, Zhang XY, Huang Q (2009) Effects of serum proteins on intracellular uptake and cytotoxicity of carbon nanoparticles. Carbon 47:1351–1358. doi: 10.1016/j.carbon.2009.01.026 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Mark Geppert
    • 1
    • 2
  • Charlotte Petters
    • 1
    • 2
  • Karsten Thiel
    • 3
  • Ralf Dringen
    • 1
    • 2
    Email author
  1. 1.Centre for Biomolecular Interactions BremenUniversity of BremenBremenGermany
  2. 2.Centre for Environmental Research and Sustainable TechnologyUniversity of BremenBremenGermany
  3. 3.Fraunhofer Institute for Manufacturing Technology and Advanced MaterialsBremenGermany

Personalised recommendations