Analytical and Bioanalytical Chemistry

, Volume 404, Issue 9, pp 2749–2763

In vitro dose–response effects of poly(amidoamine) dendrimers [amino-terminated and surface-modified with N-(2-hydroxydodecyl) groups] and quantitative determination by a liquid chromatography–hybrid quadrupole/time-of-flight mass spectrometry based method

  • M. D. Hernando
  • P. Rosenkranz
  • M. M. Ulaszewska
  • M. L. Fernández-Cruz
  • A. R. Fernández-Alba
  • J. M. Navas
Original Paper
  • 267 Downloads

Abstract

This article presents a dose–response study of the effects of two types of third-generation (G3) and fourth-generation poly(amidoamine) (PAMAM) dendrimers on two cell lines (RTG-2 and H4IIE) by in vitro cytotoxicity assays with 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red uptake (NRU), and lactate dehydrogenase (LDH) assays. We particularly investigated the potential cytotoxic effect of positive surface charge, which a cationic amino-terminated PAMAM dendrimer can display, on the marked ability of PAMAM dendrimers to cross the cell membrane compared with PAMAM dendrimers functionalized with chains of N-(2-hydroxydodecyl). Quantification of dose–response effects was performed by use of mass spectrometry analysis. The analytical method using liquid chromatography–hybrid quadrupole/time-of-flight mass spectrometry that we developed allowed characterization of defective dendrimers instead of “ideal structures.” Identification was based on accurate mass measurement, assignment of elemental composition, and the fully resolved 13 C/12 C isotopic clusters of the multiply charged ions of PAMAM dendrimers. Validation of the liquid chromatography–mass spectrometry method made possible reliable and reproducible quantification of the extracellular and intracellular concentration of dendrimers at a micromolar level (limits of detection from 0.14 to 1.34 μM and from 0.43 to 1.82 μM in standard and culture medium, respectively). A higher cytotoxicity was found with the H4IIE cell line for surface-modified PAMAM dendrimers. The LDH assay was significantly more sensitive than the MTT and NRU assays, with half-maximal inhibitory concentrations (IC50) of 12.96 and 38.31 μg mL-1 for surface-modified G3 and G4 dendrimers, respectively. No cytotoxic effects, in terms of IC50, of amino-terminated PAMAM dendrimers were observed on both H4IIE and RTG-2 cells when the concentration was below 500 μg mL-1 for G3 and G4 dendrimers.

Figure

Liquid chromatography-electrospray ionization-quadrupole/time-of-flight mass spectrometry (LC-ESI-QTOFMS) based method for quantitative determination of PAMAM dendrimers in cytotoxicity assays

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Keywords

Amino-terminated poly(amidoamine) dendrimer Surface-modified poly(amidoamine) dendrimer Cytotoxicity Dose–response assessment Quantification Liquid chromatography–electrospray ionization quadrupole/time-of-flight mass spectrometry 

References

  1. 1.
    Project on Emerging Nanotechnologies (2006) Nanotechnology consumer products inventory. http://www.nanotechproject.org/inventories/consumer/. Accessed Apr 2012
  2. 2.
    Menjoge AR, Kannan RM, Tomalia DA (2010) Drug Discov Today 15:171–185CrossRefGoogle Scholar
  3. 3.
    Klajnert B, Bryszewska M (2007) Dendrimers in medicine. Nova, New YorkGoogle Scholar
  4. 4.
    Ulaszewska MM, Hernando MD, Uclés A, Rosal R, Rodríguez A, García E, Fernández-Alba AR (2012) In: Barceló D, Farré M (eds) Analysis and risk of nanomaterials in environmental and food samples, 1st edn. Elsevier, AmsterdamGoogle Scholar
  5. 5.
    Yellepeddi VK, Kumar A, Palakurthi S (2009) Expert Opin Drug Deliv 6:835–850CrossRefGoogle Scholar
  6. 6.
    Naha PC, Davoren M, Casey A, Byrne HJ (2009) Environ Sci Technol 43:6864–6869CrossRefGoogle Scholar
  7. 7.
    Mukherjee SP, Davoren M, Byrne HJ (2010) Toxicol In Vitro 24:169–177CrossRefGoogle Scholar
  8. 8.
    Giri J, Diallo MS, Goddard WA, Dalleska NF, Fang X, Tang Y (2009) Environ Sci Technol 43:5123–5129CrossRefGoogle Scholar
  9. 9.
    Mullen DG, Borgmeier EL, Desai AM (2010) Chemistry 16:10675–10678CrossRefGoogle Scholar
  10. 10.
    Cason CA, Fabré TA, Buhrlage A, Haik KL, Bullen HA (2012) Int J Anal Chem. doi:10.1155/2012/341260
  11. 11.
    Caminade AM, Laurent R, Majoral JP (2005) Adv Drug Deliv Rev 57:2130–2146CrossRefGoogle Scholar
  12. 12.
    Giordanengo R, Mazarin M, Wu J, Peng L, Charles L (2007) Int J Mass Spectrom 266:62–75CrossRefGoogle Scholar
  13. 13.
    Schwartz BL, Rockwood AL, Smith RD, Tomalia DA, Spindler R (1995) Rapid Commun Mass Spectrom 9:1552–1555CrossRefGoogle Scholar
  14. 14.
    Blasco C, Picó Y (2011) Trends Anal Chem 30:84–99CrossRefGoogle Scholar
  15. 15.
    Jain K, Kesharwani P, Gupta U, Jain NK (2010) Int J Pharm 394:122–142CrossRefGoogle Scholar
  16. 16.
    Mosmann T (1983) J Immunol Methods 65:55–63CrossRefGoogle Scholar
  17. 17.
    Borenfreund E, Puerner JA (1985) Toxicol Lett 24:119–124CrossRefGoogle Scholar
  18. 18.
    Brown DM, Wilson MR, Macnee W, Stone V, Donaldson K (2001) Toxicol Appl Pharmacol 175:191–199CrossRefGoogle Scholar
  19. 19.
    Segner H (2004) Altern Lab Anim 32:375–382Google Scholar
  20. 20.
    Hong S, Bielinska AU, Mecke A, Keszler B, Beals JL, Shi X, Balogh L, Orr BG, Baker JR, Banaszak Holl MM (2004) Bioconjug Chem 15:774–782CrossRefGoogle Scholar
  21. 21.
    Metullio L, Ferrone M, Coslanich A, Fuchs S, Fermeglia M, Paneni MS, Pricl S (2004) Biomacromolecules 5:1371–1378CrossRefGoogle Scholar
  22. 22.
    Zhou L, Russell DH, Zhao M, Crooks RM (2001) Macromolecules 34:3567–3573CrossRefGoogle Scholar
  23. 23.
    Kallos GJ, Tomalia DA, Hedstrand DM, Lewis S, Zhou J (1991) Rapid Commun Mass Spectrom 5:383–386CrossRefGoogle Scholar
  24. 24.
    Hernando MD, Agüera A, Fernández-Alba AR (2007) Anal Bioanal Chem 387:1269–1285CrossRefGoogle Scholar
  25. 25.
    Janaszewska A, Mączyńska K, Matuszko G, Appelhans D, Voit B, Klajnert B, Bryszewska M (2012) New J Chem 36:428–437CrossRefGoogle Scholar
  26. 26.
    Parimi S, Barnes TJ, Callen DF, Prestidge CA (2010) Biomacromolecules 11:382–389CrossRefGoogle Scholar
  27. 27.
    Saovapakhiran A, D’Emanuele A, Attwood D, Penny J (2009) Bioconjug Chem 20:693–701CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • M. D. Hernando
    • 1
  • P. Rosenkranz
    • 1
  • M. M. Ulaszewska
    • 2
  • M. L. Fernández-Cruz
    • 1
  • A. R. Fernández-Alba
    • 3
    • 2
  • J. M. Navas
    • 1
  1. 1.Spanish National Institute for Agricultural and Food Research and Technology - INIAMadridSpain
  2. 2.IMDEA-Water (Instituto Madrileño De Estudios Avanzados- Agua), Parque Científico TecnológicoUniversity of AlcaláMadridSpain
  3. 3.Pesticide Residue Research Group, Department of Hydrogeology and Analytical ChemistryUniversity of AlmeríaAlmeríaSpain

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