The utility of asymmetric flow field-flow fractionation for preclinical characterization of nanomedicines

  • Yingwen Hu
  • Rachael M. Crist
  • Jeffrey D. ClogstonEmail author
Research Paper


Dynamic light scattering (DLS), transmission electron microscopy (TEM), and reversed phase-high performance liquid chromatography (RP-HPLC) are staples of nanoparticle characterization for size distribution, shape/morphology, and composition, respectively. These techniques are simple and provide important details on sample characteristics. However, DLS and TEM are routinely done in batch-mode, while RP-HPLC affords separation of components within the entire sample population, regardless of sample polydispersity. While batch-mode analysis is informative and should be a first-step analysis for any material, it may not be ideal for polydisperse formulations, such as many nanomedicines. Herein, we describe the utility of asymmetric flow field-flow fractionation (AF4) as a useful tool for a more thorough understanding of these inherently polydisperse materials. AF4 was coupled with in-line DLS for an enhanced separation and resolution of various size populations in a nanomaterial. Additionally, the various size populations were collected for offline analysis by TEM for an assessment of different shape populations, or RP-HPLC to provide a compositional analysis of each individual size population. This technique was also extended to assess nanoparticle stability, i.e., drug release, both in buffer and physiologically relevant matrix, as well as qualitatively evaluate the protein binding capacity of nanomedicines. Overall, AF4 is proven to be a very versatile technique and can provide a wealth of information on a material’s polydispersity and stability. Moreover, the ability to conduct analysis in physiological matrices provides an advantage that many other routine analytical techniques do not.

Graphical Abstract


Nanoparticles Size distribution Polydispersity Protein binding Stability Drug release 



The authors are grateful to Intezyne Technologies, Inc., Siva Therapeutics, and Cureport, Inc., for generously providing samples. The authors thank Joseph Meyer, Leidos Biomedical Research, Inc., for graphic illustrations.

Funding information

This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E.

Compliance with ethical standards

For plasma incubation studies, human plasma was collected from healthy volunteer donors under National Cancer Institute (NCI) at Frederick Protocol OH99-C-N046.

Conflict of interest

The authors declare that they have no competing interests.


The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.


  1. 1.
    D’Mello SR, Cruz CN, Chen M-L, Kapoor M, Lee SL, Tyner KM. The evolving landscape of drug products containing nanomaterials in the United States. Nat Nanotechnol. 2017;12:523.CrossRefGoogle Scholar
  2. 2.
    Maguire CM, Rösslein M, Wick P, Prina-Mello A. Characterisation of particles in solution – a perspective on light scattering and comparative technologies. Sci Technol Adv Mater. 2018;19(1):732–45.CrossRefGoogle Scholar
  3. 3.
    Anderson W, Kozak D, Coleman VA, Jamting AK, Trau M. A comparative study of submicron particle sizing platforms: accuracy, precision and resolution analysis of polydisperse particle size distributions. J Colloid Interface Sci. 2013;405:322–30.CrossRefGoogle Scholar
  4. 4.
    Varenne F, Makky A, Gaucher-Delmas M, Violleau F, Vauthier C. Multimodal dispersion of nanoparticles: a comprehensive evaluation of size distribution with 9 size measurement methods. Pharm Res. 2016;33(5):1220–34.CrossRefGoogle Scholar
  5. 5.
    Caputo F, Clogston J, Calzolai L, Rosslein M, Prina-Mello A. Measuring particle size distribution of nanoparticle enabled medicinal products, the joint view of EUNCL and NCI-NCL. A step by step approach combining orthogonal measurements with increasing complexity. J Control Release. 2019;299:31–43.CrossRefGoogle Scholar
  6. 6.
    Gioria S, Caputo F, Urban P, Maguire CM, Bremer-Hoffmann S, Prina-Mello A, et al. Are existing standard methods suitable for the evaluation of nanomedicines: some case studies. Nanomedicine (London). 2018;13(5):539–54.CrossRefGoogle Scholar
  7. 7.
    Contado C. Field flow fractionation techniques to explore the “nano-world”. Anal Bioanal Chem. 2017;409(10):2501–18.CrossRefGoogle Scholar
  8. 8.
    Giddings JC. Field-flow fractionation: analysis of macromolecular, colloidal, and particulate materials. Science. 1993;260(5113):1456–65.CrossRefGoogle Scholar
  9. 9.
    Kowalkowski T, Buszewski B, Cantado C, Dondi F. Field-flow fractionation: theory, techniques, applications and the challenges. Crit Rev Anal Chem. 2006;36(2):129–35.CrossRefGoogle Scholar
  10. 10.
    Wagner M, Holzschuh S, Traeger A, Fahr A, Schubert US. Asymmetric flow field-flow fractionation in the field of nanomedicine. Anal Chem. 2014;86(11):5201–10.CrossRefGoogle Scholar
  11. 11.
    Edwards KA, Baeumner AJ. Analysis of liposomes. Talanta. 2006;68(5):1432–41.CrossRefGoogle Scholar
  12. 12.
    Moon MH, Giddings JC. Size distribution of liposomes by flow field-flow fractionation. J Pharm Biomed Anal. 1993;11(10):911–20.CrossRefGoogle Scholar
  13. 13.
    Roda B, Zattoni A, Reschiglian P, Moon MH, Mirasoli M, Michelini E, et al. Field-flow fractionation in bioanalysis: a review of recent trends. Anal Chim Acta. 2009;635(2):132–43.CrossRefGoogle Scholar
  14. 14.
    Messaud FA, Sanderson RD, Runyon JR, Otte T, Pasch H, Williams SKR. An overview on field-flow fractionation techniques and their applications in the separation and characterization of polymers. Prog Polym Sci. 2009;34(4):351–68.CrossRefGoogle Scholar
  15. 15.
    Zattoni A, Roda B, Borghi F, Marassi V, Reschiglian P. Flow field-flow fractionation for the analysis of nanoparticles used in drug delivery. J Pharmaceut Biomed. 2014;87:53–61.CrossRefGoogle Scholar
  16. 16.
    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(5):2003–18.CrossRefGoogle Scholar
  17. 17.
    Hansen M, Smith MC, Crist RM, Clogston JD, McNeil SE. Analyzing the influence of PEG molecular weight on the separation of PEGylated gold nanoparticles by asymmetric-flow field-flow fractionation. Anal Bioanal Chem. 2015;407(29):8661–72.CrossRefGoogle Scholar
  18. 18.
    Gigault J, Cho TJ, MacCuspie RI, Hackley VA. Gold nanorod separation and characterization by asymmetric-flow field flow fractionation with UV-Vis detection. Anal Bioanal Chem. 2013;405(4):1191–202.CrossRefGoogle Scholar
  19. 19.
    Nguyen TM, Gigault J, Hackley VA. PEGylated gold nanorod separation based on aspect ratio: characterization by asymmetric-flow field flow fractionation with UV-Vis detection. Anal Bioanal Chem. 2014;406(6):1651–9.CrossRefGoogle Scholar
  20. 20.
    Chuan YP, Fan YY, Lua L, Middelberg AP. Quantitative analysis of virus-like particle size and distribution by field-flow fractionation. Biotechnol Bioeng. 2008;99(6):1425–33.CrossRefGoogle Scholar
  21. 21.
    Mathaes R, Winter G, Engert J, Besheer A. Application of different analytical methods for the characterization of non-spherical micro- and nanoparticles. Int J Pharm. 2013;453(2):620–9.CrossRefGoogle Scholar
  22. 22.
    Burchard W, Schmidt M, Stockmayer WH. Information on polydispersity and branching from combined quasi-elastic and integrated scattering. Macromolecules. 1980;13:1265–72.CrossRefGoogle Scholar
  23. 23.
    Tande BM, Wagner NJ, Mackay ME, Hawker CJ, Jeong M. Viscosimetric, hydrodynamic, and conformational properties of dendrimers and dendrons. Macromolecules. 2001;34(24):8580–5.CrossRefGoogle Scholar
  24. 24.
    Carie A, Rios-Doria J, Costich T, Burke B, Slama R, Skaff H, et al. IT-141, a polymer micelle encapsulating SN-38, induces tumor regression in multiple colorectal cancer models. J Drug Deliv. 2011;2011:869027.CrossRefGoogle Scholar
  25. 25.
    Costich TL, Carie A, Semple JE, Sullican B, Vojkovsky T, Ellis T, et al. IT-143, a polymer micelle nanoparticle, widens therapeutic window of daunorubicin. Pharm Nanotechnol. 2016;4(1):3–15.CrossRefGoogle Scholar
  26. 26.
    Clogston JD, Crist RM, McNeil SE. Physicochemical characterization of polymer nanoparticles: challenges and present limitations. In: Vauthier C, Ponchel G, editors. Polymer nanoparticles for nanomedicines: a guide for their design, preparation and development. Cham: Springer International Publishing; 2016. p. 187–203.CrossRefGoogle Scholar
  27. 27.
    Podzimek S. Asymmetric flow field flow fractionation. In: Light scattering, size exclusion chromatography and asymmetric flow field flow fractionation: powerful tools for the characterization of polymers, proteins and nanoparticles: John Wiley & Sons, Inc; 2011. p. 259–305.Google Scholar
  28. 28.
    Clogston JD, Patri AK. Importance of physicochemical characterization prior to immunological studies. Handbook of Immunological Properties of Engineered Nanomaterials; 2013. 1:25-52.Google Scholar
  29. 29.
    Dos Santos N, Allen C, Doppen AM, Anantha M, Cox KA, Gallagher RC, et al. Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: relating plasma circulation lifetimes to protein binding. Biochim Biophys Acta. 2007;1768(6):1367–77.CrossRefGoogle Scholar
  30. 30.
    Digiacomo L, Pozzi D, Amenitsch H, Caracciolo G. Impact of the biomolecular corona on the structure of PEGylated liposomes. Biomater Sci-Uk. 2017;5(9):1884–8.CrossRefGoogle Scholar
  31. 31.
    Bartucci R, Pantusa M, Marsh D, Sportelli L. Interaction of human serum albumin with membranes containing polymer-grafted lipids: spin-label ESR studies in the mushroom and brush regimes. Biochim Biophys Acta. 2002;1564(1):237–42.CrossRefGoogle Scholar
  32. 32.
  33. 33.
  34. 34.
    AmBisome prescribing information.
  35. 35.
    Beckett R, Giddings JC. Entropic contribution to the retention of nonspherical particles in field-flow fractionation. J Colloid Interface Sci. 1997;186(1):53–9.CrossRefGoogle Scholar
  36. 36.
    Dubascoux S, Le Hecho I, Hasselloev M, Von de Krammer F, Gautier MP, Lespes G. Anal At Spectrom. 2010;25:613–23.CrossRefGoogle Scholar
  37. 37.
    Makan AC, Sinha P, Ngaza N, van Aswegen W, Pasch H. Asymmetrical flow field-flow fractionation as a novel technique for the analysis of PS-b-PI copolymers. Anal Bioanal Chem. 2013;405(28):9041–7.CrossRefGoogle Scholar
  38. 38.
    Dobrovolskaia MA, Neun BW, Man S, Ye X, Hansen M, Patri AK, et al. Protein corona composition does not accurately predict hematocompatibility of colloidal gold nanoparticles. Nanomed Nanotechnol Biol Med. 2014;10(7):1453–63.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Yingwen Hu
    • 1
  • Rachael M. Crist
    • 1
  • Jeffrey D. Clogston
    • 1
    Email author
  1. 1.Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc.Frederick National Laboratory for Cancer ResearchFrederickUSA

Personalised recommendations