Pharmaceutical Research

, Volume 33, Issue 5, pp 1220–1234 | Cite as

Multimodal Dispersion of Nanoparticles: A Comprehensive Evaluation of Size Distribution with 9 Size Measurement Methods

  • Fanny Varenne
  • Ali Makky
  • Mireille Gaucher-Delmas
  • Frédéric Violleau
  • Christine VauthierEmail author
Research Paper



Evaluation of particle size distribution (PSD) of multimodal dispersion of nanoparticles is a difficult task due to inherent limitations of size measurement methods. The present work reports the evaluation of PSD of a dispersion of poly(isobutylcyanoacrylate) nanoparticles decorated with dextran known as multimodal and developed as nanomedecine.


The nine methods used were classified as batch particle i.e. Static Light Scattering (SLS) and Dynamic Light Scattering (DLS), single particle i.e. Electron Microscopy (EM), Atomic Force Microscopy (AFM), Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle Tracking Analysis (NTA) and separative particle i.e. Asymmetrical Flow Field-Flow Fractionation coupled with DLS (AsFlFFF) size measurement methods.


The multimodal dispersion was identified using AFM, TRPS and NTA and results were consistent with those provided with the method based on a separation step prior to on-line size measurements. None of the light scattering batch methods could reveal the complexity of the PSD of the dispersion.


Difference between PSD obtained from all size measurement methods tested suggested that study of the PSD of multimodal dispersion required to analyze samples by at least one of the single size particle measurement method or a method that uses a separation step prior PSD measurement.


light scattering microscopy nanoparticle tracking analysis particle size distribution tunable resistive pulse sensing 



Atomic force microscopy


Asymmetrical flow field-flow fractionation


Differential centrifugal sedimentation


Dynamic light scattering


Electron microscopy




Nanoparticle tracking analysis


Photon cross-correlation spectroscopy




Particle size distribution


Particle tracking analysis


Quasi elastic light scattering


Sedimentation field-flow fractionation


Scanning electron microscopy


Static light scattering


Transmission electron microscopy


Tunable resistive pulse sensing



This work was supported by BpI France (Project NICE). The authors acknowledge the Région Ile-de-France (“Equipement mi-lourd 2012” program, DIM Malinf) and the JPK Company for their active support. The authors acknowledge all persons who performed measurement with different instruments: Camille Roesch (Izon Science Europe Ltd, Magdalen Centre, The Oxford Science Park, Oxford, UK), Pierre Peotta (Malvern, Parc club de l’Université, Orsay, France), Philippe Violle (Sympatec, Orsay, France), Serge Réteaud (Beckman Coulter, Villepinte, France), Caroline Ferré and Alain Jalocha (Cilas, Orléans, France). The present work has benefited from the facilities and expertise of the Electron Micoscopy facilities of Imagerie-Gif ( This core facility is member of the Infrastructures en Biologie Santé et Agronomie (IBiSA), and is supported by the French national Research Agency under Investments for the Future programs “France-BioImaging”, and the Labex “Saclay Plant Science” (ANR-10-INSB-04-01 and ANR-11-IDEX-0003-02, respectively).


  1. 1.
    Jung H, Kittelson DB, Zachariah MR. The influence of a cerium additive on ultrafine diesel particle emissions and kinetics of oxidation. Combust Flame. 2005;142:276–88.CrossRefGoogle Scholar
  2. 2.
    Jøgensen B, Kristensen SB, Kunov-Kruse AJ, Fehrmann R, Christensen CH, Riisager A. Gas-phase oxidation of aqueous ethanol by nanoparticle vanadia/anatase catalysts. Top Catal. 2009;52:253–7.CrossRefGoogle Scholar
  3. 3.
    Wissing SA, Müller RH. Cosmetic applications for solid lipid nanoparticles (SLN). Int J Pharm. 2003;254:65–8.CrossRefPubMedGoogle Scholar
  4. 4.
    Olivier J. Drug transport to brain with targeted nanoparticles. NeuroRx. 2005;2:118–9.CrossRefGoogle Scholar
  5. 5.
    Cormode DP, Naha PC, Fayad ZA. Nanoparticle contrast agents for computed tomography: a focus on micelles. Contrast Media Mol Imaging. 2014;9(1):37–52.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Neuwelt EA, Varallyay P, Bago AG, Muldoon LL, Nesbit G, Nixon R. Imaging of iron oxide nanoparticles by MR and light microscopy in patients with malignant brain tumours. Neuropathol Appl Neurobiol. 2004;30:456–71.CrossRefPubMedGoogle Scholar
  7. 7.
    Perlman O, Weitz IS, Azhari H. Copper oxide nanoparticles as contrast agents for MRI and ultrasound dual-modality imaging. Phys Med Biol. 2015;60(15):5767–83.CrossRefPubMedGoogle Scholar
  8. 8.
    Galper MW, Saung MT, Fuster V, Roessl E, Thran A, Proksa R, et al. Effect of computed tomography scanning parameters on gold nanoparticle and iodine contrast. Invest Radiol. 2012;47(8):475–81.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Liu CJ, Wang CH, Chen ST, Chen HH, Leng WH, Chien CC, et al. Enhancement of cell radiation sensitivity by pegylated gold nanoparticles. Phys Med Biol. 2010;55(4):931–45.CrossRefPubMedGoogle Scholar
  10. 10.
    Seaton A, Tran L, Aitken R, Donaldson K. Nanoparticles, human health hazard and regulation. J R Soc Interface. 2009;7:119–29.CrossRefGoogle Scholar
  11. 11.
    Li C. Structure controlling and process scale-up in the fabrication of nanomaterials. Front Chem Eng China. 2010;4:18–25.CrossRefGoogle Scholar
  12. 12.
    Organisation for Economic Co-operation and Development (OCDE), Regulatory frameworks for nanotechnology in foods and medical products: summary results of a survey activity, DSTI/STP/NANO(2012)22/FINAL, 21 March 2013. Available from: (consulted on November 2015). Available from.
  13. 13.
    Draft guidance from FDA, Considering Whether an FDA-Regulated Product Involves the Application of Nanotechnology, 14 June 2011. Available from: (consulted on November 2015).
  14. 14.
    Reflection paper on the data requirements for intravenous liposomal products developed with reference to an innovator liposomal product, EMA/CHMP/806058/2009/Rev 02, 21 February 2013. Available from: (consulted on November 2015).
  15. 15.
    Joint MHLW/EMA reflection paper on the development of block copolymer micelle medicinal products, EMA/CHMP/13099/2013, 17 January 2013. Available from: (consulted on November 2015).
  16. 16.
    Report of the Joint Regulator -Industry Ad Hoc Working Group: Currently Available Methods for Characterization of Nanomaterials, 17 June 2011. Available from: (consulted on November 2015).
  17. 17.
    Organization for Economic Co-operation and Development (OCDE), Guidance manual for the testing of manufactured nanomaterials: OECD’s sponsorship programme; First revision ENV/JM/MONO(2009)20/REV, 2 June 2010. Available from: (consulted on November 2015).
  18. 18.
    FDA advisory committee for pharmaceutical science and clinical pharmacology meeting Topic 2 Nanotechnology - Update on FDA Activities, 9 August 2012. Available from: (consulted on November 2015).
  19. 19.
    Gaumet M, Vargas A, Gurny R, Delie F. Nanoparticles for drug delivery: the need for precision in reporting particle size parameters. Eur J Pharm Biopharm. 2008;69:1–9.CrossRefPubMedGoogle Scholar
  20. 20.
    Shekunov BY, Chattopadhyay P, Tong HHY, Chow AHL. Particle size analysis in pharmaceutics: principles, methods and applications. Pharm Res. 2006;24(2):203–27.CrossRefPubMedGoogle Scholar
  21. 21.
    Varenne F, Botton J, Merlet C, Beck-Broichsitter M, Legrand F-X, Vauthier C. Standardization and validation of a protocol of size measurements by dynamic light scattering for monodispersed stable nanomaterial characterization. Colloid Surf A. 2015;486:124–38.CrossRefGoogle Scholar
  22. 22.
    Braun A, Couteau O, Franks K, Kestens V, Roebben G, Lamberty A, et al. Validation of dynamic light scattering and centrifugal liquid sedimentation methods for nanoparticle characterisation. Adv Powder Technol. 2011;22:766–70.CrossRefGoogle Scholar
  23. 23.
    Woodward RC, Heeris J, St Pierre TG, Saunders M, Gilbert EP, Rutnakornpituk M, et al. A comparison of methods for the measurement of the particle-size distribution of magnetic nanoparticles. J Appl Crystallogr. 2007;40:495–500.CrossRefGoogle Scholar
  24. 24.
    Elizalde O, Leal GP, Leiza JR. Particle size distribution measurements of polymeric dispersions: a comparative study. Part Part Syst Charact. 2000;17:236–43.CrossRefGoogle Scholar
  25. 25.
    Fielding LA, Mykhaylyk OO, Armes SP, Fowler PW, Mittal V, Fitzpatrick S. Correcting for a density distribution: particle size analysis of core-shell nanocomposite particles using disk centrifuge photosedimentometry. Langmuir. 2012;28:2536–44.CrossRefPubMedGoogle Scholar
  26. 26.
    Bell NC, Minelli C, Tompkins J, Stevens MM, Shard AG. Emerging techniques for submicrometer particle sizing applied to Stoeber silica. Langmuir. 2012;28:10860–72.CrossRefPubMedGoogle Scholar
  27. 27.
    Linsinger T, Roebben G, Gilliland D, Calzolai L, Rossi F, Gibson N, et al, Requirements on measurements for the implementation of the European Commission definition of the term “nanomaterial”. JRC Reference Reports. 2012.Google Scholar
  28. 28.
    Powers KW, Brown SC, Krishna VB, Wasdo SC, Moudgil BM, Roberts SM. Research strategies for satefy evaluation of nanomaterials. Part VI. Characterization of nanoscale particles for toxicological evaluation. Toxicol Sci. 2006;90(2):296–303.CrossRefPubMedGoogle Scholar
  29. 29.
    Sowerby SJ, Broom MF, Petersen GB. Dynamically resizable nanometre-scale apertures for molecular sensing. Sensors Actuators B. 2007;123:325–30.CrossRefGoogle Scholar
  30. 30.
    Willmott GR, Vogel R, Yu SSC, Groenewegen LG, Roberts GS, Kozak D, et al. Use of tunable nanopore blockade rates to investigate colloidal dispersions. J Phys-Condens Mat. 2010;22(45):1–11.CrossRefGoogle Scholar
  31. 31.
    Vogel R, Willmott G, Kozak D, Roberts GS, Anderson W, Groenewegen L, et al. Quantitative sizing of nano/microparticles with a tunable elastomeric pore sensor. Anal Chem. 2011;83:3499–506.CrossRefPubMedGoogle Scholar
  32. 32.
    Lespes G, Gigault J. Hyphenated analytical techniques for multidimensional characterisation of submicron particles: a review. Anal Chim Acta. 2011;692:26–41.CrossRefPubMedGoogle Scholar
  33. 33.
    Anderson W, Kozak D, Coleman VA, Jämting ÅK, Trau M. A comapartive study of submicron particle sizing platforms: accuracy, precision and resolution analysis of polydisperse particle size distributions. J Colloid Interface Sci. 2013;405:322–30.CrossRefPubMedGoogle Scholar
  34. 34.
    Sokolova V, Ludwig A-K, Hornung S, Rotan O, Horn PA, Epple M, et al. Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Coilloid Surf B. 2011;87:146–50.CrossRefGoogle Scholar
  35. 35.
    Pace HE, Rogers NJ, Jarolimek C, Coleman VA, Gray EP, Higgins CP, et al. Single particle inductively coupled plasma-mass spectrometry: a performance evaluation and method comparison in the determination of nanoparticle size. Environ Sci Technol. 2012;46:12272–80.CrossRefPubMedGoogle Scholar
  36. 36.
    van der Pol E, Coumans FAW, Grootemaat AE, Gardiner C, Sargent IL, Harrison P, et al. Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J Thromb Haemost. 2014;12:1182–92.CrossRefPubMedGoogle Scholar
  37. 37.
    Cascio C, Gilliland D, Rossi F, Calzolai L, Contado C. Critical experimental evaluation of Key methods to detect, size and quantify nanoparticulate silver. Anal Chem. 2014;86:12143–51.CrossRefPubMedGoogle Scholar
  38. 38.
    Calzolai L, Gilliland D, Garcìa CP, Rossi F. Separation and characterization of gold nanoparticle mixtures by flow-field-flow fractionation. J Chromatogr A. 2011;1218:4234–9.CrossRefPubMedGoogle Scholar
  39. 39.
    Ingebrigtsen L, Brandl M. Determination of the size distribution of liposomes by SEC fractionation, and PCS analysis and enzymatic assay of lipid content. AAPS Pharm Sci Tech. 2002;3(2):9–15.CrossRefGoogle Scholar
  40. 40.
    Sitar S, Kejžar A, Pahovnik D, Kogej K, Tušek-Žnidarič M, Lenassi M, et al. Size characterization and quantification of exosomes by asymmetrical-flow field-flow fractionation. Anal Chem. 2015;87:9225–33.CrossRefPubMedGoogle Scholar
  41. 41.
    Gun’ko VM, Klyueva AV, Levchuk YN, Leboda R. Photon correlation spectroscopy investigations of proteins. Adv Colloid Interface. 2003;105:201–328.CrossRefGoogle Scholar
  42. 42.
    ISO/TS 10797:2012: Nanotechnologies - Characterization of single-wall carbon nanotubes using transmission electron microscopy.Google Scholar
  43. 43.
    ISO 13322-1:2004 Particle size analysis - Image analysis methods - Part 1: Static image analysis, methods.Google Scholar
  44. 44.
    Vauthier C, Persson B, Lindner P, Cabane B. Protein adsorption and complement activation for di-block copolymer nanoparticles. Biomaterials. 2011;32:1646–56.CrossRefPubMedGoogle Scholar
  45. 45.
    Rasband W. ImageJ (Computer Program), National Institute of Health, 2013.Google Scholar
  46. 46.
    Cybernetics M. Image-Pro Plus (Computer Program), Roper Industries, 2013.Google Scholar
  47. 47.
    Binnig G, Quate CF, Gerber C. Atomic force microscope. Phys Rev Lett. 1986;56:930–3.CrossRefPubMedGoogle Scholar
  48. 48.
    Meyer G, Amer NM. Novel optical approach to atomic force microscopy. Appl Phys Lett. 1988;53(12):1045–7.CrossRefGoogle Scholar
  49. 49.
    Couteau O, Roebben G. Measurement of the size of spherical nanoparticles by means of atomic force microscopy. Meas Sci Technol. 2001;22(6):65101–8.CrossRefGoogle Scholar
  50. 50.
    Filipe V, Hawe A, Jiskoot W. Critical evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates. Pharm Res. 2010;27:796–810.CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    ISO 22 412:2008(E): Particle size analysis - dynamic light scattering (DLS).Google Scholar
  52. 52.
    Cho TJ, Hackley VA. Fractionation and characterization of gold nanoparticles in aqueous solution: asymmetric-flow field flow fractionation with MALS, DLS, and UV–vis detection. Anal Bioanal Chem. 2010;398:2003–18.CrossRefPubMedGoogle Scholar
  53. 53.
    Rbii K, Violleau F, Guedj S, Surel O. Analysis of aged gelatin by AFlFFF-MALS: Identification of high molar mass components and their influence on solubility. Food Hydrocoll. 2009;23:1024–30.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Fanny Varenne
    • 1
  • Ali Makky
    • 1
  • Mireille Gaucher-Delmas
    • 2
  • Frédéric Violleau
    • 3
    • 4
  • Christine Vauthier
    • 1
    Email author
  1. 1.Institut Galien Paris-Sud, CNRS, Univ. Paris-SudUniversity Paris-SaclayChâtenay-MalabryFrance
  2. 2.INP - Ecole d’Ingénieurs de PURPAN, Département Sciences Agronomiques & AgroalimentairesUniversité de ToulouseToulouseFrance
  3. 3.INP - Ecole d’Ingénieurs de PURPAN, Laboratoire de Chimie Agro-IndustrielleUniversité de ToulouseToulouseFrance
  4. 4.INRA, UMR 1010 CAIToulouseFrance

Personalised recommendations