Advanced Contrast Agents for Multimodal Biomedical Imaging Based on Nanotechnology

  • Daniel Calle
  • Paloma Ballesteros
  • Sebastián Cerdán
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1718)

Abstract

Clinical imaging modalities have reached a prominent role in medical diagnosis and patient management in the last decades. Different image methodologies as Positron Emission Tomography, Single Photon Emission Tomography, X-Rays, or Magnetic Resonance Imaging are in continuous evolution to satisfy the increasing demands of current medical diagnosis. Progress in these methodologies has been favored by the parallel development of increasingly more powerful contrast agents. These are molecules that enhance the intrinsic contrast of the images in the tissues where they accumulate, revealing noninvasively the presence of characteristic molecular targets or differential physiopathological microenvironments. The contrast agent field is currently moving to improve the performance of these molecules by incorporating the advantages that modern nanotechnology offers. These include, mainly, the possibilities to combine imaging and therapeutic capabilities over the same theranostic platform or improve the targeting efficiency in vivo by molecular engineering of the nanostructures. In this review, we provide an introduction to multimodal imaging methods in biomedicine, the sub-nanometric imaging agents previously used and the development of advanced multimodal and theranostic imaging agents based in nanotechnology. We conclude providing some illustrative examples from our own laboratories, including recent progress in theranostic formulations of magnetoliposomes containing ω-3 poly-unsaturated fatty acids to treat inflammatory diseases, or the use of stealth liposomes engineered with a pH-sensitive nanovalve to release their cargo specifically in the acidic extracellular pH microenvironment of tumors.

Key words

Imaging agents Image Guided Drug Delivery Magnetic Resonance Imaging Nanotechnology Positron Emission Tomography Single Photon Emission Tomography Theranostic agents X-Ray computed tomography 

Notes

Acknowledgements

Authors are indebted to Dr. Pilar López-Larrubia CSIC for the careful reading of the manuscript and the valuable comments provided, Mrs. Teresa Navarro CSIC for granting access to CSIC small animal MRI facilities and expert technical assistance during the MRI acquisitions, and to Mr. Javier Pérez CSIC for professional drafting of the illustrations.

Financial statement: This work was supported in part by grants SAF2014-53739-R and S2010/BMD-2349 to SC and grant CTQ2013-47669-R to PB. DC held postdoctoral contracts from Consejo Superior de Investigaciones Científicas CSIC. Funding sources were not involved in the design of the study, in the collection, analysis and interpretation of data, in the writing of the report nor in the decision to submit the article for publication.

References

  1. 1.
    Suetens P (2009) Fundamentals of medical imaging, 2nd edn. Cambridge University Press, CambridgeCrossRefGoogle Scholar
  2. 2.
    Medical imaging: technology ad applications (2013). CRC PressGoogle Scholar
  3. 3.
    Textbook of contrast media (1999). CRC Press, Boca Raton, FLGoogle Scholar
  4. 4.
    Contrast agents I. Magnetic resonance imaging (2002) Topics in current chemistry, vol 221. Springer, BerlinGoogle Scholar
  5. 5.
    Contrast agents II: optical, ultrasound, x-ray and radiopharmaceutical imaging (2002) Topics in current chemistry, vol 222. Springer, BerlinGoogle Scholar
  6. 6.
    Contrast agents III: radiopharmaceuticals: from diagnosis to therapeutics (2002) Topics in current chemistry, vol 252. Springer, BerlinGoogle Scholar
  7. 7.
    Key J, Leary JF (2014) Nanoparticles for multimodal in vivo imaging in nanomedicine. Int J Nanomedicine 9:711–726. https://doi.org/10.2147/IJN.S53717 PubMedPubMedCentralGoogle Scholar
  8. 8.
    Heidt T, Nahrendorf M (2013) Multimodal iron oxide nanoparticles for hybrid biomedical imaging. NMR Biomed 26(7):756–765. https://doi.org/10.1002/nbm.2872 CrossRefPubMedGoogle Scholar
  9. 9.
    Kim J, Piao Y, Hyeon T (2009) Multifunctional nanostructured materials for multimodal imaging, and simultaneous imaging and therapy. Chem Soc Rev 38(2):372–390. https://doi.org/10.1039/b709883a CrossRefPubMedGoogle Scholar
  10. 10.
    Lee DE, Koo H, Sun IC, Ryu JH, Kim K, Kwon IC (2012) Multifunctional nanoparticles for multimodal imaging and theragnosis. Chem Soc Rev 41(7):2656–2672. https://doi.org/10.1039/c2cs15261d CrossRefPubMedGoogle Scholar
  11. 11.
    Lee N, Yoo D, Ling D, Cho MH, Hyeon T, Cheon J (2015) Iron oxide based nanoparticles for multimodal imaging and magnetoresponsive therapy. Chem Rev 115(19):10637–10689. https://doi.org/10.1021/acs.chemrev.5b00112 CrossRefPubMedGoogle Scholar
  12. 12.
    Ding H, Wu F, Nair MP (2013) Image-guided drug delivery to the brain using nanotechnology. Drug Discov Today 18(21–22):1074–1080. https://doi.org/10.1016/j.drudis.2013.06.010 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ding H, Wu F (2012) Image guided biodistribution of drugs and drug delivery. Theranostics 2(11):1037–1039. https://doi.org/10.7150/thno.5321 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Lammers T, Kiessling F, Hennink WE, Storm G (2010) Nanotheranostics and image-guided drug delivery: current concepts and future directions. Mol Pharm 7(6):1899–1912CrossRefPubMedGoogle Scholar
  15. 15.
    Yu MK, Park J, Jon S (2012) Magnetic nanoparticles and their applications in image-guided drug delivery. Drug Deliv Transl Res 2(1):3–21. https://doi.org/10.1007/s13346-011-0049-8 CrossRefPubMedGoogle Scholar
  16. 16.
    Webb S (1988) The physics of medical imaging. CRC Press, Boca Ratón, FLCrossRefGoogle Scholar
  17. 17.
    Lauterbur PC (1973) Image formation by induced local interactions: examples employing nuclear magnetic resonance. Nature 242(5394):190–191CrossRefGoogle Scholar
  18. 18.
    Farrell C, Wallace JD, Mansfield CM (1971) The use of thermography in detection of metastatic breast cancer. Am J Roentgenol 111(1):148–152CrossRefGoogle Scholar
  19. 19.
    Rontgen WC (1896) On a new kind of rays. Science 3(59):227–231. https://doi.org/10.1126/science.3.59.227 CrossRefPubMedGoogle Scholar
  20. 20.
    Brownell GL, Sweet WH (1953) Localization of brain tumors with positron emitters. Nucleonics 11(11):40–45Google Scholar
  21. 21.
    Rudin M, Weissleder R (2003) Molecular imaging in drug discovery and development. Nat Rev Drug Discov 2(2):123–131. https://doi.org/10.1038/nrd1007 CrossRefPubMedGoogle Scholar
  22. 22.
    Bushberg JT, Boone JM (2011) The essential physics of medical imaging. Lippincott Williams & Wilkins, Philadelphia, PAGoogle Scholar
  23. 23.
    Weissleder R, Mahmood U (2001) Molecular imaging. Radiology 219(2):316–333. https://doi.org/10.1148/radiology.219.2.r01ma19316 CrossRefPubMedGoogle Scholar
  24. 24.
    Hounsfield GN (1973) Computerized transverse axial scanning (tomography): Part 1. Description of system. Br J Radiol 46(552):1016–1022CrossRefPubMedGoogle Scholar
  25. 25.
    Hutton BF (2011) Recent advances in iterative reconstruction for clinical SPECT/PET and CT. Acta Oncol 50(6):851–858CrossRefPubMedGoogle Scholar
  26. 26.
    Antoch G, Freudenberg LS, Beyer T, Bockisch A, Debatin JF (2004) To enhance or not to enhance? 18F-FDG and CT contrast agents in dual-modality 18F-FDG PET/CT. J Nucl Med 45(Suppl 1):56S–65SPubMedGoogle Scholar
  27. 27.
    Caravan P, Ellison JJ, McMurry TJ, Lauffer RB (1999) Gadolinium (III) chelates as MRI contrast agents: structure, dynamics, and applications. Chem Rev 99(9):2293–2352CrossRefPubMedGoogle Scholar
  28. 28.
    Pacheco-Torres J, Calle D, Lizarbe B, Negri V, Ubide C, Fayos R, Lopez Larrubia P, Ballesteros P, Cerdan S (2011) Environmentally sensitive paramagnetic and diamagnetic contrast agents for nuclear magnetic resonance imaging and spectroscopy. Curr Top Med Chem 11(1):115–130CrossRefPubMedGoogle Scholar
  29. 29.
    Elsinga PH, Dierckx RA (2014) Small molecule PET-radiopharmaceuticals. Curr Pharm Des 20:2268–2274CrossRefPubMedGoogle Scholar
  30. 30.
    Cai J, Li F (2013) Single-photon emission computed tomography tracers for predicting and monitoring cancer therapy. Curr Pharm Biotechnol 14(7):693–707CrossRefPubMedGoogle Scholar
  31. 31.
    Thomsen HS, Morcos SK (2000) Radiographic contrast media. BJU Int 86(Suppl 1):1–10PubMedGoogle Scholar
  32. 32.
    Pavel DG, Zimmer M, Patterson VN (1977) In vivo labeling of red blood cells with 99mTc: a new approach to blood pool visualization. J Nucl Med 18(3):305–308PubMedGoogle Scholar
  33. 33.
    Hahn MA, Singh AK, Sharma P, Brown SC, Moudgil BM (2011) Nanoparticles as contrast agents for in-vivo bioimaging: current status and future perspectives. Anal Bioanal Chem 399(1):3–27CrossRefPubMedGoogle Scholar
  34. 34.
    Pimlott SL, Sutherland A (2011) Molecular tracers for the PET and SPECT imaging of disease. Chem Soc Rev 40(1):149–162. https://doi.org/10.1039/b922628c CrossRefPubMedGoogle Scholar
  35. 35.
    Heinle SK, Noblin J, Goree-Best P, Mello A, Ravad G, Mull S, Mammen P, Grayburn PA (2000) Assessment of myocardial perfusion by harmonic power doppler imaging at rest and during adenosine stress comparison with 99mTc-Sestamibi SPECT imaging. Circulation 102(1):55–60CrossRefPubMedGoogle Scholar
  36. 36.
    Urtasun RC, Parliament MB, McEwan AJ, Mercer JR, Mannan RH, Wiebe LI, Morin C, Chapman JD (1996) Measurement of hypoxia in human tumours by non-invasive spect imaging of iodoazomycin arabinoside. Br J Cancer Suppl 27:S209–S212PubMedPubMedCentralGoogle Scholar
  37. 37.
    Leitha T, Glaser C, Pruckmayer M, Rasse M, Millesi W, Lang S, Nasel C, Backfrieder W, Kainberger F (1998) Technetium-99m-MIBI in primary and recurrent head and neck tumors: contribution of bone SPECT image fusion. J Nucl Med 39(7):1166–1171PubMedGoogle Scholar
  38. 38.
    Tharp K, Israel O, Hausmann J, Bettman L, Martin W, Daitzchman M, Sandler M, Delbeke D (2004) Impact of 131I-SPECT/CT images obtained with an integrated system in the follow-up of patients with thyroid carcinoma. Eur J Nucl Med Mol Imaging 31(10):1435–1442CrossRefPubMedGoogle Scholar
  39. 39.
    Fukuyama H, Ouchi Y, Matsuzaki S, Nagahama Y, Yamauchi H, Ogawa M, Kimura J, Shibasaki H (1997) Brain functional activity during gait in normal subjects: a SPECT study. Neurosci Lett 228(3):183–186CrossRefPubMedGoogle Scholar
  40. 40.
    Strauss LG (1996) Fluorine-18 deoxyglucose and false-positive results: a major problem in the diagnostics of oncological patients. Eur J Nucl Med 23(10):1409–1415CrossRefPubMedGoogle Scholar
  41. 41.
    Aime S, Barge A, Delli Castelli D, Fedeli F, Mortillaro A, Nielsen FU, Terreno E (2002) Paramagnetic lanthanide (III) complexes as pH-sensitive chemical exchange saturation transfer (CEST) contrast agents for MRI applications. Magn Reson Med 47(4):639–648CrossRefPubMedGoogle Scholar
  42. 42.
    Hancu I, Dixon WT, Woods M, Vinogradov E, Sherry AD, Lenkinski RE (2010) CEST and PARACEST MR contrast agents. Acta Radiol 51(8):910–923. https://doi.org/10.3109/02841851.2010.502126 CrossRefPubMedGoogle Scholar
  43. 43.
    Sun PZ, Sorensen AG (2008) Imaging pH using the chemical exchange saturation transfer (CEST) MRI: correction of concomitant RF irradiation effects to quantify CEST MRI for chemical exchange rate and pH. Magn Reson Med 60(2):390–397CrossRefPubMedGoogle Scholar
  44. 44.
    McRae R, Bagchi P, Sumalekshmy S, Fahrni CJ (2009) In situ imaging of metals in cells and tissues. Chem Rev 109(10):4780–4827. https://doi.org/10.1021/cr900223a CrossRefPubMedGoogle Scholar
  45. 45.
    Zhang S, Malloy CR, Sherry AD (2005) MRI thermometry based on PARACEST agents. J Am Chem Soc 127(50):17572–17573. https://doi.org/10.1021/ja053799t CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Portman MA, Lassen NA, Cooper TG, Sills AM, Potchen EJ (1991) Intra- and extracellular pH of the brain in vivo studied by 31P-NMR during hyper- and hypocapnia. J Appl Physiol 71(6):2168–2172CrossRefPubMedGoogle Scholar
  47. 47.
    Fisher MJ, Dillon PF (1987) Phenylphosphonate: a 31P-NMR indicator of extracellular pH and volume in the isolated perfused rabbit bladder. Circ Res 60(4):472–477CrossRefPubMedGoogle Scholar
  48. 48.
    Garcia-Martin ML, Herigault G, Remy C, Farion R, Ballesteros P, Coles JA, Cerdan S, Ziegler A (2001) Mapping extracellular pH in rat brain gliomas in vivo by 1H magnetic resonance spectroscopic imaging: comparison with maps of metabolites. Cancer Res 61(17):6524–6531PubMedGoogle Scholar
  49. 49.
    Provent P, Benito M, Hiba B, Farion R, Lopez-Larrubia P, Ballesteros P, Remy C, Segebarth C, Cerdan S, Coles JA, Garcia-Martin ML (2007) Serial in vivo spectroscopic nuclear magnetic resonance imaging of lactate and extracellular pH in rat gliomas shows redistribution of protons away from sites of glycolysis. Cancer Res 67(16):7638–7645. https://doi.org/10.1158/0008-5472.CAN-06-3459 CrossRefPubMedGoogle Scholar
  50. 50.
    Gallagher FA, Kettunen MI, Day SE, DE H, Ardenkjaer-Larsen JH, Zandt R, Jensen PR, Karlsson M, Golman K, Lerche MH, Brindle KM (2008) Magnetic resonance imaging of pH in vivo using hyperpolarized 13C-labelled bicarbonate. Nature 453(7197):940–943. https://doi.org/10.1038/nature07017 CrossRefPubMedGoogle Scholar
  51. 51.
    Pacheco-Torres J, Calle D, Lizarbe B, Negri V, Ubide C, Fayos R, Lopez Larrubia P, Ballesteros P, Cerdan S (2011) Environmentally sensitive paramagnetic and diamagnetic contrast agents for nuclear magnetic resonance imaging and spectroscopy. Curr Top Med Chem 11(1):115–130CrossRefPubMedGoogle Scholar
  52. 52.
    Frangioni JV (2003) In vivo near-infrared fluorescence imaging. Curr Opin Chem Biol 7(5):626–634CrossRefPubMedGoogle Scholar
  53. 53.
    Weissleder R, Tung CH, Mahmood U, Bogdanov A Jr (1999) In vivo imaging of tumors with protease-activated near-infrared fluorescent probes. Nat Biotechnol 17(4):375–378. https://doi.org/10.1038/7933 CrossRefPubMedGoogle Scholar
  54. 54.
    Chan WC, Maxwell DJ, Gao X, Bailey RE, Han M, Nie S (2002) Luminescent quantum dots for multiplexed biological detection and imaging. Curr Opin Biotechnol 13(1):40–46CrossRefPubMedGoogle Scholar
  55. 55.
    Yang M, Baranov E, Li XM, Wang JW, Jiang P, Li L, Moossa AR, Penman S, Hoffman RM (2001) Whole-body and intravital optical imaging of angiogenesis in orthotopically implanted tumors. Proc Natl Acad Sci U S A 98(5):2616–2621. https://doi.org/10.1073/pnas.051626698 CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Sedghi S, Fields JZ, Klamut M, Urban G, Durkin M, Winship D, Fretland D, Olyaee M, Keshavarzian A (1993) Increased production of luminol enhanced chemiluminescence by the inflamed colonic mucosa in patients with ulcerative colitis. Gut 34(9):1191–1197CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Barichello JM, Morishita M, Takayama K, Nagai T (1999) Encapsulation of hydrophilic and lipophilic drugs in PLGA nanoparticles by the nanoprecipitation method. Drug Dev Ind Pharm 25(4):471–476. https://doi.org/10.1081/DDC-100102197 CrossRefPubMedGoogle Scholar
  58. 58.
    Na HB, Song IC, Hyeon T (2009) Inorganic nanoparticles for MRI contrast agents. Adv Mater 21(21):2133–2148CrossRefGoogle Scholar
  59. 59.
    Bonnemain B (1998) Superparamagnetic agents in magnetic resonance imaging: physicochemical characteristics and clinical applications. A review. J Drug Target 6(3):167–174. https://doi.org/10.3109/10611869808997890 CrossRefPubMedGoogle Scholar
  60. 60.
    Liu F, Laurent S, Fattahi H, Vander Elst L, Muller RN (2011) Superparamagnetic nanosystems based on iron oxide nanoparticles for biomedical imaging. Nanomedicine 6(3):519–528. https://doi.org/10.2217/nnm.11.16 CrossRefPubMedGoogle Scholar
  61. 61.
    Grant CW, Karlik S, Florio E (1989) A liposomal MRI contrast agent: phosphatid ylethanolamine-DTPA. Magn Reson Med 11(2):236–243CrossRefPubMedGoogle Scholar
  62. 62.
    Mukundan S Jr, Ghaghada KB, Badea CT, Kao CY, Hedlund LW, Provenzale JM, Johnson GA, Chen E, Bellamkonda RV, Annapragada A (2006) A liposomal nanoscale contrast agent for preclinical CT in mice. AJR Am J Roentgenol 186(2):300–307. https://doi.org/10.2214/AJR.05.0523 CrossRefPubMedGoogle Scholar
  63. 63.
    Torchilin VP (2002) PEG-based micelles as carriers of contrast agents for different imaging modalities. Adv Drug Deliv Rev 54(2):235–252CrossRefPubMedGoogle Scholar
  64. 64.
    Wegner KD, Hildebrandt N (2015) Quantum dots: bright and versatile in vitro and in vivo fluorescence imaging biosensors. Chem Soc Rev 44(14):4792–4834. https://doi.org/10.1039/c4cs00532e CrossRefPubMedGoogle Scholar
  65. 65.
    Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S (2005) Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307(5709):538–544. https://doi.org/10.1126/science.1104274 CrossRefPubMedPubMedCentralGoogle Scholar
  66. 66.
    Wang L, Zhu X, Tang X, Wu C, Zhou Z, Sun C, Deng SL, Ai H, Gao J (2015) A multiple gadolinium complex decorated fullerene as a highly sensitive T(1) contrast agent. Chem Commun 51(21):4390–4393. https://doi.org/10.1039/c5cc00285k CrossRefGoogle Scholar
  67. 67.
    Braun K, Dunsch L, Pipkorn R, Bock M, Baeuerle T, Yang S, Waldeck W, Wiessler M (2010) Gain of a 500-fold sensitivity on an intravital MR contrast agent based on an endohedral gadolinium-cluster-fullerene-conjugate: a new chance in cancer diagnostics. Int J Med Sci 7(3):136–146CrossRefPubMedPubMedCentralGoogle Scholar
  68. 68.
    Yang H, Lu C, Liu Z, Jin H, Che Y, Olmstead MM, Balch AL (2008) Detection of a family of gadolinium-containing endohedral fullerenes and the isolation and crystallographic characterization of one member as a metal-carbide encapsulated inside a large fullerene cage. J Am Chem Soc 130(51):17296–17300. https://doi.org/10.1021/ja8078303 CrossRefPubMedGoogle Scholar
  69. 69.
    Avti PK, Talukdar Y, Sirotkin MV, Shroyer KR, Sitharaman B (2013) Toward single-walled carbon nanotube-gadolinium complex as advanced MRI contrast agents: pharmacodynamics and global genomic response in small animals. J Biomed Mater Res B Appl Biomater 101(6):1039–1049. https://doi.org/10.1002/jbm.b.32914 CrossRefPubMedGoogle Scholar
  70. 70.
    Negri V, Cerpa A, Lopez-Larrubia P, Nieto-Charques L, Cerdan S, Ballesteros P (2010) Nanotubular paramagnetic probes as contrast agents for magnetic resonance imaging based on the diffusion tensor. Angew Chem 49(10):1813–1815. https://doi.org/10.1002/anie.200906415 CrossRefGoogle Scholar
  71. 71.
    Lian T, Ho RJ (2001) Trends and developments in liposome drug delivery systems. J Pharm Sci 90(6):667–680CrossRefPubMedGoogle Scholar
  72. 72.
    Torchilin VP (2005) Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov 4(2):145–160CrossRefPubMedGoogle Scholar
  73. 73.
    Hilgenbrink AR, Low PS (2005) Folate receptor-mediated drug targeting: from therapeutics to diagnostics. J Pharm Sci 94(10):2135–2146CrossRefPubMedGoogle Scholar
  74. 74.
    Danhier F, Feron O, Preat V (2010) To exploit the tumor microenvironment: passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J Control Release 148(2):135–146CrossRefPubMedGoogle Scholar
  75. 75.
    Calle D, Negri V, Ballesteros P, Cerdan S (2015) Magnetoliposomes loaded with poly-unsaturated fatty acids as novel theranostic anti-inflammatory formulations. Theranostics 5(5):489–503. https://doi.org/10.7150/thno.10069 CrossRefPubMedPubMedCentralGoogle Scholar
  76. 76.
    Calle D (2014) Novel contrast agents for multimodal biomedical imaging based in nanotechnology. Dissertation, Universidad Autónoma de MadridGoogle Scholar
  77. 77.
    Robey IF, Baggett BK, Kirkpatrick ND, Roe DJ, Dosescu J, Sloane BF, Hashim AI, Morse DL, Raghunand N, Gatenby RA, Gillies RJ (2009) Bicarbonate increases tumor pH and inhibits spontaneous metastases. Cancer Res 69(6):2260–2268. https://doi.org/10.1158/0008-5472.CAN-07-5575 CrossRefPubMedPubMedCentralGoogle Scholar
  78. 78.
    Raghunand N, Mahoney BP, Gillies RJ (2003) Tumor acidity, ion trapping and chemotherapeutics. II. pH-dependent partition coefficients predict importance of ion trapping on pharmacokinetics of weakly basic chemotherapeutic agents. Biochem Pharmacol 66(7):1219–1229CrossRefPubMedGoogle Scholar
  79. 79.
    Mahoney BP, Raghunand N, Baggett B, Gillies RJ (2003) Tumor acidity, ion trapping and chemotherapeutics. I. Acid pH affects the distribution of chemotherapeutic agents in vitro. Biochem Pharmacol 66(7):1207–1218CrossRefPubMedGoogle Scholar
  80. 80.
    Pacheco-Torres J, Mukherjee N, Walko M, Lopez-Larrubia P, Ballesteros P, Cerdan S, Kocer A (2015) Image guided drug release from pH-sensitive Ion channel-functionalized stealth liposomes into an in vivo glioblastoma model. Nanomedicine 11(6):1345–1354. https://doi.org/10.1016/j.nano.2015.03.014 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Daniel Calle
    • 1
  • Paloma Ballesteros
    • 2
  • Sebastián Cerdán
    • 1
  1. 1.Instituto de Investigaciones Biomédicas “Alberto Sols”CSIC/UAMMadridSpain
  2. 2.Facultad de CienciasUniversidad Nacional de Educación a Distancia UNEDMadridSpain

Personalised recommendations