We have efficiently produced collagen-rich microstructures in fibroblast multicellular spheroids (MCSs) as a three-dimensional in vitro tissue analog to investigate silver (Ag) nanoparticle (NP) penetration. The MCS production was examined by changing the seeding cell number (500 to 40,000 cells) and the growth period (1 to 10 days). MCSs were incubated with Ag NP suspensions with a concentration of 5 μg mL−1 for 24 h. For this study, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to visualize Ag NP localization quantitatively. Thin sections of MCSs were analyzed by LA-ICP-MS with a laser spot size of 8 μm to image distributions of 109Ag, 31P, 63Cu, 66Zn, and 79Br. A calibration using a NP suspension was applied to convert the measured Ag intensity into the number of NPs present. The determined numbers of NPs ranged from 30 to 7200 particles in an outer rim of MCS. The particle distribution was clearly correlated with the presence of 31P and 66Zn and was localized in the outer rim of proliferating cells with a width that was equal to about twice the diameter of single cells. Moreover, abundant collagens were found in the outer rim of MCSs. For only the highest seeding cell number, NPs were completely captured at the outer rim, in a natural barrier reducing particle transport, whereas Eosin (79Br) used as a probe of small molecules penetrated into the core of MCSs already after 1 min of exposure.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
Kessler R. Engineered nanoparticles in consumer products: understanding a new ingredient. Environ Health Perspect. 2011;119(3):A120–A5.
Li W-R, Xie X-B, Shi Q-S, Zeng H-Y, OU-Yang Y-S, Chen Y-B. Antibacterial activity and mechanism of silver nanoparticles on Escherichia coli. Appl Microbiol Biotechnol. 2010;85(4):1115–22.
Larguinho M, Baptista PV. Gold and silver nanoparticles for clinical diagnostics - from genomics to proteomics. J Proteome. 2012;75(10):2811–23.
Wilkinson LJ, White RJ, Chipman JK. Silver and nanoparticles of silver in wound dressings: a review of efficacy and safety. J Wound Care. 2011;20(11):543–9.
Reidy B, Haase A, Luch A, Dawson K, Lynch I. Mechanisms of silver nanoparticle release, transformation and toxicity: a critical review of current knowledge and recommendations for future studies and applications. Materials. 2013;6(6):2295.
Aaron J, Travis K, Harrison N, Sokolov K. Dynamic imaging of molecular assemblies in live cells based on nanoparticle plasmon resonance coupling. Nano Lett. 2009;9(10):3612–8.
Ando J, Fujita K, Smith NI, Kawata S. Dynamic SERS imaging of cellular transport pathways with endocytosed gold nanoparticles. Nano Lett. 2011;11(12):5344–8.
Drescher D, Kneipp J. Nanomaterials in complex biological systems: insights from Raman spectroscopy. Chem Soc Rev. 2012;41(17):5780–99.
Kneipp J, Kneipp H, Rice WL, Kneipp K. Optical probes for biological applications based on surface-enhanced Raman scattering from indocyanine green on gold nanoparticles. Anal Chem. 2005;77(8):2381–5.
Huang X, Jain PK, El-Sayed IH, El-Sayed MA. Plasmonic photothermal therapy (PPTT) using gold nanoparticles. Lasers Med Sci. 2007;23(3):217.
Nam J, Won N, Jin H, Chung H, Kim S. pH-induced aggregation of gold nanoparticles for photothermal cancer therapy. J Am Chem Soc. 2009;131(38):13639–45.
Schneider G, Guttmann P, Heim S, Rehbein S, Mueller F, Nagashima K, et al. Three-dimensional cellular ultrastructure resolved by X-ray microscopy. Nat Methods. 2010;7:985.
Guehrs E, Schneider M, Günther CM, Hessing P, Heitz K, Wittke D, et al. Quantification of silver nanoparticle uptake and distribution within individual human macrophages by FIB/SEM slice and view. J Nanobiotechnol. 2017;15(1):21.
Alkilany AM, Murphy CJ. Toxicity and cellular uptake of gold nanoparticles: what we have learned so far? J Nanopart Res. 2010;12(7):2313–33.
Krystek P, Ulrich A, Garcia CC, Manohar S, Ritsema R. Application of plasma spectrometry for the analysis of engineered nanoparticles in suspensions and products. J Anal At Spectrom. 2011;26(9):1701–21.
Laux P, Tentschert J, Riebeling C, Braeuning A, Creutzenberg O, Epp A, et al. Nanomaterials: certain aspects of application, risk assessment and risk communication. Arch Toxicol. 2018;92(1):121–41.
Sabine Becker J, Matusch A, Palm C, Salber D, Morton KA, Susanne Becker J. Bioimaging of metals in brain tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and metallomics. Metallomics. 2010;2(2):104–11.
Van Acker T, Van Malderen SJM, Van Heerden M, McDuffie JE, Cuyckens F, Vanhaecke F. High-resolution laser ablation-inductively coupled plasma-mass spectrometry imaging of cisplatin-induced nephrotoxic side effects. Anal Chim Acta. 2016;945:23–30.
Giesen C, Waentig L, Mairinger T, Drescher D, Kneipp J, Roos PH, et al. Iodine as an elemental marker for imaging of single cells and tissue sections by laser ablation inductively coupled plasma mass spectrometry. J Anal At Spectrom. 2011;26(11):2160–5.
Drescher D, Giesen C, Traub H, Panne U, Kneipp J, Jakubowski N. Quantitative imaging of gold and silver nanoparticles in single eukaryotic cells by laser ablation ICP-MS. Anal Chem. 2012;84(22):9684–8.
Scharlach C, Müller L, Wagner S, Kobayashi Y, Kratz H, Ebert M, et al. LA-ICP-MS allows quantitative microscopy of europium-doped iron oxide nanoparticles and is a possible alternative to ambiguous Prussian blue iron staining. J Biomed Nanotechnol. 2016;12(5):1001–10.
Büchner T, Drescher D, Traub H, Schrade P, Bachmann S, Jakubowski N, et al. Relating surface-enhanced Raman scattering signals of cells to gold nanoparticle aggregation as determined by LA-ICP-MS micromapping. Anal Bioanal Chem. 2014;406(27):7003–14.
Drescher D, Zeise I, Traub H, Guttmann P, Seifert S, Büchner T, et al. In situ characterization of SiO2 nanoparticle biointeractions using BrightSilica. Adv Funct Mater. 2014;24(24):3765–75.
Theiner S, Schreiber-Brynzak E, Jakupec MA, Galanski M, Koellensperger G, Keppler BK. LA-ICP-MS imaging in multicellular tumor spheroids - a novel tool in the preclinical development of metal-based anticancer drugs. Metallomics. 2016;8(4):398–402.
Theiner S, Van Malderen SJM, Van Acker T, Legin A, Keppler BK, Vanhaecke F, et al. Fast high-resolution laser ablation-inductively coupled plasma mass spectrometry imaging of the distribution of platinum-based anticancer compounds in multicellular tumor spheroids. Anal Chem. 2017;89(23):12641–5.
Furukawa KS, Ushida T, Sakai Y, Kunii K, Suzuki M, Tanaka J, et al. Tissue-engineered skin using aggregates of normal human skin fibroblasts and biodegradable material. J Artif Organs. 2001;4(4):353–6.
Priwitaningrum DL, Blondé J-BG, Sridhar A, van Baarlen J, Hennink WE, Storm G, et al. Tumor stroma-containing 3D spheroid arrays: a tool to study nanoparticle penetration. J Control Release. 2016;244:257–68.
Jorgenson AJ, Choi KM, Sicard D, Smith KMJ, Hiemer SE, Varelas X, et al. TAZ activation drives fibroblast spheroid growth, expression of profibrotic paracrine signals, and context-dependent ECM gene expression. Am J Phys Cell Phys. 2017;312(3):C277–C85.
Sapudom J, Pompe T. Biomimetic tumor microenvironments based on collagen matrices. Biomater Sci. 2018;6(8):2009–24.
Emon B, Bauer J, Jain Y, Jung B, Saif T. Biophysics of tumor microenvironment and cancer metastasis - a mini review. Comput Struct Biotechnol J. 2018;16:279–87.
Suzuki T, Sakata S, Makino Y, Obayashi H, Ohara S, Hattori K, et al. iQuant2: software for rapid and quantitative imaging using laser ablation-ICP mass spectrometry. Mass Spectrom. 2018;7(1):A0065-A.
Schneider CA, Rasband WS, Eliceiri KW. NIH image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671.
Curcio E, Salerno S, Barbieri G, De Bartolo L, Drioli E, Bader A. Mass transfer and metabolic reactions in hepatocyte spheroids cultured in rotating wall gas-permeable membrane system. Biomaterials. 2007;28(36):5487–97.
Drescher D. Spektro-Mikroskopische Charakterisierung von Nano-Bio-Wechselwirkungen in Zellen. PhD thesis. Humboldt University Berlin; 2016.
Drescher D, Guttmann P, Buchner T, Werner S, Laube G, Hornemann A, et al. Specific biomolecule corona is associated with ring-shaped organization of silver nanoparticles in cells. Nanoscale. 2013;5(19):9193–8.
We thank Konrad Löhr (Bundesanstalt für Materialforschung und -prüfung) for the support and training for the non-contact piezo-driven array spotter and Akvile Häckel (Charité Universitätsmedizin Berlin) for providing access to and support with using the cryomicrotome.
Conflict of interest
The authors declare that they have no conflicts of interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Arakawa, A., Jakubowski, N., Flemig, S. et al. High-resolution laser ablation inductively coupled plasma mass spectrometry used to study transport of metallic nanoparticles through collagen-rich microstructures in fibroblast multicellular spheroids. Anal Bioanal Chem 411, 3497–3506 (2019). https://doi.org/10.1007/s00216-019-01827-w
- Laser ablation inductively coupled plasma mass spectrometry
- Silver nanoparticles
- Fibroblast cells
- Multicellular spheroids