Pharmaceutical Research

, Volume 29, Issue 7, pp 1843–1853 | Cite as

Fluorescence Imaging of the Lymph Node Uptake of Proteins in Mice after Subcutaneous Injection: Molecular Weight Dependence

  • Fang Wu
  • Suraj G. Bhansali
  • Wing Cheung Law
  • Earl J. Bergey
  • Paras N. Prasad
  • Marilyn E. MorrisEmail author
Research Paper



To use noninvasive fluorescence imaging to investigate the influence of molecular weight (MW) of proteins on the rate of loss from a subcutaneous (SC) injection site and subsequent uptake by the draining lymph nodes in mice.


Bevacizumab (149 kDa), bovine serum albumin (BSA, 66 kDa), ovalbumin (44.3 kDa) or VEGF-C156S (23 kDa), labeled with the near infrared dye IRDye 680, were injected SC into the front footpad of SKH-1 mice. Whole body non-invasive fluorescence imaging was performed to quantitate the fluorescence signal at the injection site and in axillary lymph nodes.


The half-life values, describing the times for 50% loss of proteins from the injection site, were 6.81 h for bevacizumab, 2.85 h for BSA, 1.57 h for ovalbumin and 0.31 h for VEGF-C156S. The corresponding axillary lymph node exposure, represented as the area of the % dose versus time curve, was 6.27, 5.13, 4.06 and 1.54% dose ∙ h, respectively.


Our results indicate that the rate of loss of proteins from a SC injection site is inversely related to MW of proteins, while lymph node exposure is proportionally related to the MW of proteins in a mouse model.


fluorescence imaging lymphatic uptake molecular weight protein subcutaneous injection 



bovine serum albumin


fraction of the dose recovered at the axillary lymph nodes


fraction of original signal remaining at the SC injection site


infrared dye


lymph node


molecular weight


region of interest




sodium dodecyl sulfate-polyacrylamide gel electrophoresis


vascular endothelial growth factor



This work is supported by a grant from the University at Buffalo Center for Protein Therapeutics to MEM. SGB was supported in part by a fellowship from Pfizer Global Research and Development. We acknowledge the valuable assistance from Dr. Rajiv Kumar, Lisa A. Vathy, Dr. Hong Ding, and Dr. Ken-Tye Yong from the Institute for Lasers, Photonics and Biophotonics, University at Buffalo.


  1. 1.
    Porter CJ, Edwards GA, Charman SA. Lymphatic transport of proteins after s.c. injection: implications of animal model selection. Adv Drug Deliv Rev. 2001;50:157–71.PubMedCrossRefGoogle Scholar
  2. 2.
    Jung M, Lees P, Seewald W, King JN. Analytical determination and pharmacokinetics of robenacoxib in the dog. J Vet Pharmacol Ther. 2009;32:41–8.PubMedCrossRefGoogle Scholar
  3. 3.
    Supersaxo A, Hein WR, Steffen H. Effect of molecular weight on the lymphatic absorption of water-soluble compounds following subcutaneous administration. Pharm Res. 1990;7:167–9.PubMedCrossRefGoogle Scholar
  4. 4.
    Dreher MR, Liu W, Michelich CR, Dewhirst MW, Yuan F, Chilkoti A. Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers. J Natl Cancer Inst. 2006;98:335–44.PubMedCrossRefGoogle Scholar
  5. 5.
    Kagan L, Gershkovich P, Mendelman A, Amsili S, Ezov N, Hoffman A. The role of the lymphatic system in subcutaneous absorption of macromolecules in the rat model. Eur J Pharm Biopharm. 2007;67:759–65.PubMedCrossRefGoogle Scholar
  6. 6.
    Cubas R, Zhang S, Kwon S, Sevick-Muraca EM, Li M, Chen C, et al. Virus-like particle (VLP) lymphatic trafficking and immune response generation after immunization by different routes. J Immunother. 2009;32:118–28.PubMedCrossRefGoogle Scholar
  7. 7.
    Ding H, Yong KT, Law WC, Roy I, Hu R, Wu F, et al. Non-invasive tumor detection in small animals using novel functional Pluronic nanomicelles conjugated with anti-mesothelin antibody. Nanoscale. 2011;3:1813–22.PubMedCrossRefGoogle Scholar
  8. 8.
    Ding H, Yong KT, Roy I, Hu R, Wu F, Zhao L, et al. Bioconjugated PLGA-4-arm-PEG branched polymeric nanoparticles as novel tumor targeting carriers. Nanotechnology. 2011;22:165101.PubMedCrossRefGoogle Scholar
  9. 9.
    Yong KT, Roy I, Ding H, Bergey EJ, Prasad PN. Biocompatible near-infrared quantum dots as ultrasensitive probes for long-term in vivo imaging applications. Small. 2009;5:1997–2004.PubMedCrossRefGoogle Scholar
  10. 10.
    Yong K-T, Hu R, Roy I, Ding H, Vathy LA, Bergey EJ, et al. Tumor targeting and imaging in live animals with functionalized semiconductor quantum rods. ACS Appl Mater Interfaces. 2009;1:710–9.PubMedCrossRefGoogle Scholar
  11. 11.
    Kwon S, Sevick-Muraca EM. Noninvasive quantitative imaging of lymph function in mice. Lymphat Res Biol. 2007;5:219–31.PubMedCrossRefGoogle Scholar
  12. 12.
    Gearing AJH, Thorpe SJ, Miller K, Mangan M, Varley PG, Dudgeon T, et al. Selective cleavage of human IgG by the matrix metalloproteinases, matrilysin and stromelysin. Immunol Lett. 2002;81:41–8.PubMedCrossRefGoogle Scholar
  13. 13.
    Plasencia MD, Isailovic D, Merenbloom SI, Mechref Y, Novotny MV, Clemmer DE. Resolving and assigning N-linked glycan structural isomers from ovalbumin by IMS-MS. J Am Soc Mass Spectrom. 2008;19:1706–15.PubMedCrossRefGoogle Scholar
  14. 14.
    Bhansali SG, Balu-Iyer SV, Morris ME. Influence of route of administration and liposomal encapsulation on blood and lymph node exposure to the protein VEGF-C156S. J Pharm Sci. 2011.Google Scholar
  15. 15.
    Porter CJ, Charman SA. Lymphatic transport of proteins after subcutaneous administration. J Pharm Sci. 2000;89:297–310.PubMedCrossRefGoogle Scholar
  16. 16.
    Hong H, Sun J, Cai W. Multimodality imaging of nitric oxide and nitric oxide synthases. Free Radic Biol Med. 2009;47:684–98.PubMedCrossRefGoogle Scholar
  17. 17.
    Kim BS, Oh JM, Hyun H, Kim KS, Lee SH, Kim YH, et al. Insulin-loaded microcapsules for in vivo delivery. Mol Pharm. 2009;6:353–65.PubMedCrossRefGoogle Scholar
  18. 18.
    Kim BS, Oh JM, Kim KS, Seo KS, Cho JS, Khang G, et al. BSA-FITC-loaded microcapsules for in vivo delivery. Biomaterials. 2009;30:902–9.PubMedCrossRefGoogle Scholar
  19. 19.
    Wu F, Wuensch SA, Azadniv M, Ebrahimkhani MR, Crispe IN. Galactosylated LDL nanoparticles: a novel targeting delivery system to deliver antigen to macrophages and enhance antigen specific T cell responses. Mol Pharm. 2009;6:1506–17.PubMedCrossRefGoogle Scholar
  20. 20.
    Lu ZR. Molecular imaging of HPMA copolymers: visualizing drug delivery in cell, mouse and man. Adv Drug Deliv Rev. 2010;62:246–57.PubMedCrossRefGoogle Scholar
  21. 21.
    Erogbogbo F, Yong KT, Roy I, Hu R, Law WC, Zhao W, et al. In vivo targeted cancer imaging, sentinel lymph node mapping and multi-channel imaging with biocompatible silicon nanocrystals. ACS Nano. 2011;5:413–23.PubMedCrossRefGoogle Scholar
  22. 22.
    Ballou B, Ernst LA, Andreko S, Harper T, Fitzpatrick JA, Waggoner AS, et al. Sentinel lymph node imaging using quantum dots in mouse tumor models. Bioconjug Chem. 2007;18:389–96.PubMedCrossRefGoogle Scholar
  23. 23.
    Koyama Y, Talanov VS, Bernardo M, Hama Y, Regino CA, Brechbiel MW, et al. A dendrimer-based nanosized contrast agent dual-labeled for magnetic resonance and optical fluorescence imaging to localize the sentinel lymph node in mice. J Magn Reson Imaging. 2007;25:866–71.PubMedCrossRefGoogle Scholar
  24. 24.
    Kim D, Lee ES, Park K, Kwon IC, Bae YH. Doxorubicin loaded pH-sensitive micelle: antitumoral efficacy against ovarian A2780/DOXR tumor. Pharm Res. 2008;25:2074–82.PubMedCrossRefGoogle Scholar
  25. 25.
    Zou P, Xu S, Povoski SP, Wang A, Johnson MA, Martin Jr EW, et al. Near-infrared fluorescence labeled anti-TAG-72 monoclonal antibodies for tumor imaging in colorectal cancer xenograft mice. Mol Pharm. 2009;6:428–40.PubMedCrossRefGoogle Scholar
  26. 26.
    Hu R, Yong KT, Roy I, Ding H, Law WC, Cai H, et al. Functionalized near-infrared quantum dots for in vivo tumor vasculature imaging. Nanotechnology. 2010;21:145105.PubMedCrossRefGoogle Scholar
  27. 27.
    Diagaradjane P, Orenstein-Cardona JM, Colon-Casasnovas NE, Deorukhkar A, Shentu S, Kuno N, et al. Imaging epidermal growth factor receptor expression in vivo: pharmacokinetic and biodistribution characterization of a bioconjugated quantum dot nanoprobe. Clin Cancer Res. 2008;14:731–41.PubMedCrossRefGoogle Scholar
  28. 28.
    Fox GB, Chin CL, Luo F, Day M, Cox BF. Translational neuroimaging of the CNS: novel pathways to drug development. Mol Interv. 2009;9:302–13.PubMedCrossRefGoogle Scholar
  29. 29.
    Prajapati SI, Martinez CO, Bahadur AN, Wu IQ, Zheng W, Lechleiter JD, et al. Near-infrared imaging of injured tissue in living subjects using IR-820. Mol Imaging. 2009;8:45–54.PubMedGoogle Scholar
  30. 30.
    Koning GA, Krijger GC. Targeted multifunctional lipid-based nanocarriers for image-guided drug delivery. Anticancer Agents Med Chem. 2007;7:425–40.PubMedCrossRefGoogle Scholar
  31. 31.
    Kumar R, Roy I, Ohulchanskky TY, Vathy LA, Bergey EJ, Sajjad M, et al. In vivo biodistribution and clearance studies using multimodal organically modified silica nanoparticles. ACS Nano. 2010;4:699–708.PubMedCrossRefGoogle Scholar
  32. 32.
    Barrett T, Choyke PL, Kobayashi H. Imaging of the lymphatic system: new horizons. Contrast Media Mol Imaging. 2006;1:230–45.PubMedCrossRefGoogle Scholar
  33. 33.
    Weissleder R. Scaling down imaging: molecular mapping of cancer in mice. Nat Rev Cancer. 2002;2:11–8.PubMedCrossRefGoogle Scholar
  34. 34.
    Wang H, Chen K, Niu G, Chen X. Site-specifically biotinylated VEGF(121) for near-infrared fluorescence imaging of tumor angiogenesis. Mol Pharm. 2009;6:285–94.PubMedCrossRefGoogle Scholar
  35. 35.
    Zou P, Xu S, Wang A, Povoski S, Johnson M, Martin E, et al. Near-infrared fluorescence labeled anti-TAG-72 monoclonal antibodies for tumor imaging in colorectal cancer xenograft mice. Mol Pharm. 2009.Google Scholar
  36. 36.
    Engeset A. The route of peripheral lymph to the blood stream; an x-ray study of the barrier theory. J Anat. 1959;93:96–100.PubMedGoogle Scholar
  37. 37.
    Kubik S. Anatomy of the lymphatic system. In: Foldi M, Foldi E, Kubik S, editors. Textbook of lymphology. San Francisco: Elsevier GmbH; 2003. p. 1–166.Google Scholar
  38. 38.
    Hildebrandt P. Subcutaneous absorption of insulin in insulin-dependent diabetic patients. Influence of species, physico-chemical properties of insulin and physiological factors. Dan Med Bull. 1991;38:337–46.PubMedGoogle Scholar
  39. 39.
    Kang S, Brange J, Burch A, Volund A, Owens DR. Subcutaneous insulin absorption explained by insulin’s physicochemical properties. Evidence from absorption studies of soluble human insulin and insulin analogues in humans. Diabetes Care. 1991;14:942–8.PubMedCrossRefGoogle Scholar
  40. 40.
    Charman SA, McLennan DN, Edwards GA, Porter CJ. Lymphatic absorption is a significant contributor to the subcutaneous bioavailability of insulin in a sheep model. Pharm Res. 2001;18:1620–6.PubMedCrossRefGoogle Scholar
  41. 41.
    Harvey AJ, Kaestner SA, Sutter DE, Harvey NG, Mikszta JA, Pettis RJ. Microneedle-based intradermal delivery enables rapid lymphatic uptake and distribution of protein drugs. Pharm Res. 2011;28:107–16.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Fang Wu
    • 1
  • Suraj G. Bhansali
    • 1
    • 2
  • Wing Cheung Law
    • 3
  • Earl J. Bergey
    • 3
  • Paras N. Prasad
    • 3
  • Marilyn E. Morris
    • 1
    Email author
  1. 1.Department of Pharmaceutical Sciences School of Pharmacy and Pharmaceutical SciencesUniversity at Buffalo, State University of New YorkAmherstUSA
  2. 2.Novartis Pharmaceuticals Corporation, Clinical PKPDEast HanoverUSA
  3. 3.Institute for Lasers, Photonics and BiophotonicsUniversity at Buffalo, State University of New YorkAmherstUSA

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