title: Defining the subcellular interface of nanoparticles by live-cell imaging.
abstract: Understanding of nanoparticle-bio-interactions within living cells requires knowledge about the dynamic behavior of nanomaterials during their cellular uptake, intracellular traffic and mutual reactions with cell organelles. Here, we introduce a protocol of combined kinetic imaging techniques that enables investigation of exemplary fluorochrome-labelled nanoparticles concerning their intracellular fate. By time-lapse confocal microscopy we observe fast, dynamin-dependent uptake of polystyrene and silica nanoparticles via the cell membrane within seconds. Fluorescence recovery after photobleaching (FRAP) experiments reveal fast and complete exchange of the investigated nanoparticles at mitochondria, cytoplasmic vesicles or the nuclear envelope. Nuclear translocation is observed within minutes by free diffusion and active transport. Fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS) indicate diffusion coefficients of polystyrene and silica nanoparticles in the nucleus and the cytoplasm that are consistent with particle motion in living cells based on diffusion. Determination of the apparent hydrodynamic radii by FCS and RICS shows that nanoparticles exert their cytoplasmic and nuclear effects mainly as mobile, monodisperse entities. Thus, a complete toolkit of fluorescence fluctuation microscopy is presented for the investigation of nanomaterial biophysics in subcellular microenvironments that contributes to develop a framework of intracellular nanoparticle delivery routes.
authors: Leibniz-Institute for Age Research, Fritz-Lipman-Institute, Jena, Germany. mikecz@tec-source.de
authors: Peter H
authors: PH
authors: Hemmerich
authors: Anna H
authors: AH
authors: von Mikecz
mesh_terms: D000818:Animals; D002470:Cell Survival; D004058:Diffusion; D004705:Endocytosis; D036681:Fluorescence Recovery After Photobleaching; D006367:HeLa Cells; D006801:Humans; D006835:Hydrazones / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D061848:Optical Imaging / Q000379:methods*; D010316:Particle Size; D011137:Polystyrenes / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D006835:Hydrazones; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D011137:Polystyrenes; D012822:Silicon Dioxide
references: Proc Natl Acad Sci U S A. 1987 Jul;84(14):4910-3
references: 3474634
references: Nature. 1994 Aug 25;370(6491):652-5
references: 8065451
references: Biophys J. 2004 Jun;86(6):3473-95
references: 15189848
references: Biophys J. 2008 Feb 15;94(4):1437-48
references: 17933881
references: ACS Nano. 2008 Oct 28;2(10):2085-94
references: 19206455
references: Curr Opin Biotechnol. 2005 Feb;16(1):28-34
references: 15722012
references: Dev Cell. 2006 Jun;10(6):839-50
references: 16740485
references: Nat Methods. 2006 Feb;3(2):83-9
references: 16432516
references: Methods Enzymol. 2008;438:77-93
references: 18413242
references: Proc Natl Acad Sci U S A. 2000 Nov 7;97(23):12583-8
references: 11070082
references: ACS Nano. 2008 Jan;2(1):85-96
references: 19206551
references: Nano Lett. 2007 Apr;7(4):970-7
references: 17388641
references: Science. 2003 Apr 25;300(5619):615-8
references: 12714738
references: Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6358-63
references: 11959911
references: Annu Rev Biochem. 2009;78:857-902
references: 19317650
references: Proc Natl Acad Sci U S A. 1984 Jun;81(11):3414-8
references: 6328515
references: Mol Biol Cell. 2002 Feb;13(2):425-34
references: 11854401
references: Biophys J. 1983 Dec;44(3):405-12
references: 6318842
references: Angew Chem Int Ed Engl. 2011 Feb 7;50(6):1242-58
references: 21290491
references: J Cell Biol. 1997 Jul 14;138(1):131-42
references: 9214387
references: J Cell Sci. 2008 Aug 15;121(Pt 16):2731-43
references: 18664490
references: J Cell Sci. 1997 Jan;110 ( Pt 2):105-12
references: 9044041
references: Biophys J. 2002 Jul;83(1):510-22
references: 12080138
references: Nano Lett. 2008 Sep;8(9):2779-87
references: 18687009
references: Nano Lett. 2006 Aug;6(8):1794-807
references: 16895376
references: Microsc Res Tech. 2009 Apr;72(4):323-32
references: 19067357
references: Mol Biol Cell. 2004 Jun;15(6):2819-33
references: 15064352
references: PLoS One. 2009 Aug 12;4(8):e6622
references: 19672302
references: Annu Rev Biophys. 2008;37:247-63
references: 18573081
references: J Struct Biol. 2004 Jun;146(3):281-90
references: 15099570
references: Langmuir. 2011 Sep 6;27(17):10376-85
references: 21476507
references: Methods. 2003 Jan;29(1):74-85
references: 12543073
references: Biophys J. 2005 Aug;89(2):1317-27
references: 15908582
references: Chem Soc Rev. 2012 Apr 7;41(7):2718-39
references: 22389111
references: Nano Lett. 2007 Jun;7(6):1542-50
references: 17465586
references: Cold Spring Harb Protoc. 2010 Dec 01;2010(12):pdb.top90
references: 21123431
references: ACS Nano. 2011 Mar 22;5(3):1657-69
references: 21344890
references: Exp Cell Res. 2005 Apr 15;305(1):51-62
references: 15777787
references: J Cell Biol. 2008 Feb 25;180(4):697-704
references: 18283109
references: Biomaterials. 2011 Apr;32(11):2713-24
references: 21262533
references: Nature. 2003 Mar 6;422(6927):37-44
references: 12621426
references: Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3878-82
references: 12644705
references: Clin Occup Environ Med. 2006;5(4):785-96
references: 17110292
references: Curr Opin Chem Biol. 2010 Oct;14(5):616-22
references: 20674473
references: Methods Mol Biol. 2009;464:321-41
references: 18951193
references: Biophys J. 2004 Nov;87(5):3518-24
references: 15339818
references: Nat Mater. 2009 Jul;8(7):543-57
references: 19525947
references: Biophys J. 2008 Jan 1;94(1):90-4
references: 17827216
references: Trends Neurosci. 2008 Nov;31(11):559-68
references: 18817990
references: Proc Natl Acad Sci U S A. 2007 Feb 13;104(7):2050-5
references: 17267609
references: J Cell Sci. 2001 Nov;114(Pt 21):3885-97
references: 11719555
references: Toxicol Pathol. 2011 Aug;39(5):841-9
references: 21768271
references: Biochem Mol Biol Educ. 2011 Mar-Apr;39(2):91-101
references: 21445900