Pharmaceutical Research

, Volume 26, Issue 5, pp 1121–1129 | Cite as

Noninvasive Monitoring of HPMA Copolymer–RGDfK Conjugates by Magnetic Resonance Imaging

  • Bahar Zarabi
  • Mark P. Borgman
  • Jiachen Zhuo
  • Rao Gullapalli
  • Hamidreza Ghandehari
Research Paper



To evaluate the tumor targeting potential of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer–gadolinium(Gd)–RGDfK conjugates by magnetic resonance (MR) T1-mapping.


HPMA copolymers with and without RGDfK were synthesized to incorporate side chains for Gd chelation. The conjugates were characterized by their side-chain contents and r1 relaxivity. In vitro integrin-binding affinities of polymeric conjugates were assessed via competitive cell binding assays on HUVEC endothelial cells and MDA-MB-231 breast cancer cells. In vivo MR imaging was performed on MDA-MB-231 tumor-bearing SCID mice at different time points using non-targetable and targetable polymers. The specificity of αvβ3 targeting was assessed by using non-paramagnetic targetable polymer to block αvβ3 integrins followed by injection of paramagnetic targetable polymers after 2 h.


The polymer conjugates showed relaxivities higher than Gd-DOTA. Endothelial cell binding studies showed that IC50 values for the copolymer with RGDfK binding to αvβ3 integrin-positive HUVEC and MDA-MB-231 cells were similar to that of free peptide. Significantly lower T1 values were observed at the tumor site after 2 h using targetable conjugate (p < 0.012). In vivo blocking study showed significantly higher T1 values (p < 0.045) compared to targetable conjugate.


These results demonstrate the potential of this conjugate as an effective targetable MR contrast agent for tumor imaging and therapy monitoring.


contrast agents HPMA copolymers MRI targeted delivery tumor targeting 





N-(3-Aminopropyl)methacrylamide hydrochloride


N-methacryloylaminopropyl-2-(4-isothiourea-benzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid


American type culture collection


Flip angle




Dimethyl sulfoxide


1,4,7,10-tetra-azacylcododecane-N,N′,N′′,N′′′-tetraacetic acid


Ethylenediaminetetraacetic acid


Field of view


Fast protein liquid chromatography




Gadolinium chloride


High pressure liquid chromatography




Human umbilical vein endothelial cell


Inductively coupled plasma optical emission spectroscopy


Interactive data language


Kilo dalton


N-methacryloylglycylglycyl-p-nitrophenyl ester




Magnetic resonance


Magnetic resonance imaging


Molecular weight


Molecular weight cut off


Phosphate buffered saline








Longitudinal relaxivity


Region of interest


Signal intensity


Severe combined immunodeficient


Size exclusion chromatography


Longitudinal relaxation time


Echo time


Trifluoroacetic acid


Repetition time



This study received financial support from the Department of Defense Breast Cancer Research Program pre-doctoral fellowship to Bahar Zarabi (W81XWH0410341) and a grant from the National Institute of Biomedical Imaging and Bioengineering (R01-EB007171). The authors acknowledge Mrudulla Pullambhatla and Wenlian Zhu at Johns Hopkins University School of Medicine Molecular Imaging Center for their assistance with in vivo MR imaging and facilitated by a grant from the National Cancer Institute (U24 CA92871).


  1. 1.
    S. Erdogan, A. Roby, and V. P. Torchilin. Enhanced tumor visualization by gamma-scintigraphy with 111In-labeled polychelating-polymer-containing immunoliposomes. Mol. Pharm. 3:525–530 (2006). doi: 10.1021/mp060055t.PubMedCrossRefGoogle Scholar
  2. 2.
    W. T. Anderson-Berg, M. Strand, T. E. Lempert, A. E. Rosenbaum, and P. M. Joseph. Nuclear magnetic resonance and gamma camera tumor imaging using gadolinium-labeled monoclonal antibodies. J. Nucl. Med. 27:829–833 (1986).PubMedGoogle Scholar
  3. 3.
    J. B. Mandeville, B. G. Jenkins, Y. C. Chen, J. K. Choi, Y. R. Kim, D. Belen, C. Liu, B. E. Kosofsky, and J. J. Marota. Exogenous contrast agent improves sensitivity of gradient-echo functional magnetic resonance imaging at 9.4 T. Magn. Reson. Med. 52:1272–1281 (2004). doi: 10.1002/mrm.20278.PubMedCrossRefGoogle Scholar
  4. 4.
    G. Niu, W. Cai, and X. Chen. Molecular imaging of human epidermal growth factor receptor 2 (HER-2) expression. Front Biosci. 13:790–805 (2008). doi: 10.2741/2720.PubMedCrossRefGoogle Scholar
  5. 5.
    H. Kim, D. E. Morgan, H. Zeng, W. E. Grizzle, J. M. Warram, C. R. Stockard, D. Wang, and K. R. Zinn. Breast tumor xenografts: diffusion-weighted MR imaging to assess early therapy with novel apoptosis-inducing anti-DR5 antibody. Radiology. 248:844–851 (2008). doi: 10.1148/radiol.2483071740.PubMedCrossRefGoogle Scholar
  6. 6.
    W. A. Weber, R. Haubner, E. Vabuliene, B. Kuhnast, H. J. Wester, and M. Schwaiger. Tumor angiogenesis targeting using imaging agents. Q. J. Nucl. Med. 45:179–182 (2001).PubMedGoogle Scholar
  7. 7.
    P. C. Brooks, R. A. Clark, and D. A. Cheresh. Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science. 264:569–571 (1994). doi: 10.1126/science.7512751.PubMedCrossRefGoogle Scholar
  8. 8.
    L. Bello, M. Francolini, P. Marthyn, J. Zhang, R. S. Carroll, D. C. Nikas, J. F. Strasser, R. Villani, D. A. Cheresh, and P. M. Black. Alpha(v)beta3 and alpha(v)beta5 integrin expression in glioma periphery. Neurosurgery. 49:380–389 (2001)discussion 390.PubMedCrossRefGoogle Scholar
  9. 9.
    D. Meitar, S. E. Crawford, A. W. Rademaker, and S. L. Cohn. Tumor angiogenesis correlates with metastatic disease, N-myc amplification, and poor outcome in human neuroblastoma. J. Clin. Oncol. 14:405–414 (1996).PubMedGoogle Scholar
  10. 10.
    G. Gasparini, P. C. Brooks, E. Biganzoli, P. B. Vermeulen, E. Bonoldi, L. Y. Dirix, G. Ranieri, R. Miceli, and D. A. Cheresh. Vascular integrin alpha(v)beta3: a new prognostic indicator in breast cancer. Clin. Cancer Res. 4:2625–2634 (1998).PubMedGoogle Scholar
  11. 11.
    S. Sengupta, N. Chattopadhyay, A. Mitra, S. Ray, S. Dasgupta, and A. Chatterjee. Role of alphavbeta3 integrin receptors in breast tumor. J. Exp. Clin. Cancer Res. 20:585–590 (2001).PubMedGoogle Scholar
  12. 12.
    A. Mitra, J. Mulholland, A. Nan, E. McNeill, H. Ghandehari, and B. R. Line. Targeting tumor angiogenic vasculature using polymer-RGD conjugates. J. Control. Release. 102:191–201 (2005). doi: 10.1016/j.jconrel.2004.09.023.PubMedCrossRefGoogle Scholar
  13. 13.
    B. R. Line, A. Mitra, A. Nan, and H. Ghandehari. Targeting tumor angiogenesis: comparison of peptide and polymer-peptide conjugates. J. Nucl. Med. 46:1552–1560 (2005).PubMedGoogle Scholar
  14. 14.
    A. Mitra, A. Nan, J. C. Papadimitriou, H. Ghandehari, and B. R. Line. Polymer-peptide conjugates for angiogenesis targeted tumor radiotherapy. Nucl. Med. Biol. 33:43–52 (2006). doi: 10.1016/j.nucmedbio.2005.09.005.PubMedCrossRefGoogle Scholar
  15. 15.
    A. Mitra, T. Coleman, M. Borgman, A. Nan, H. Ghandehari, and B. R. Line. Polymeric conjugates of mono- and bi-cyclic alphaVbeta3 binding peptides for tumor targeting. J. Control. Release. 114:175–183 (2006). doi: 10.1016/j.jconrel.2006.06.014.PubMedCrossRefGoogle Scholar
  16. 16.
    M. P. Borgman, T. Coleman, R. B. Kolhatkar, S. Geyser-Stoops, B. R. Line, and H. Ghandehari. Tumor-targeted HPMA copolymer-(RGDfK)-(CHX-A"-DTPA) conjugates show increased kidney accumulation. J. Control. Release. 132:193–199 (2008 ). doi: 10.1016/j.jconrel.2008.07.014.CrossRefGoogle Scholar
  17. 17.
    Y. Huang, A. Nan, G. M. Rosen, C. A. Winalski, E. Schneider, P. Tsai, and H. Ghandehari. N-(2-Hydroxypropyl)Methacrylamide (HPMA) copolymer-linked nitroxide: potential magnetic resonance contrast agent. Macromol. Biosci. 3:647–652 (2003). doi: 10.1002/mabi.200350031.CrossRefGoogle Scholar
  18. 18.
    D. Wang, S. C. Miller, M. Sima, D. Parker, H. Buswell, K. C. Goodrich, P. Kopeckova, and J. Kopecek. The arthrotropism of macromolecules in adjuvant-induced arthritis rat model: a preliminary study. Pharm. Res. 21:1741–1749 (2004). doi: 10.1023/B:PHAM.0000045232.18134.e9.PubMedCrossRefGoogle Scholar
  19. 19.
    Y. Wang, F. Ye, E. K. Jeong, Y. Sun, D. L. Parker, and Z. R. Lu. Noninvasive visualization of pharmacokinetics, biodistribution and tumor targeting of poly[N-(2-hydroxypropyl)methacrylamide] in mice using contrast enhanced MRI. Pharm. Res. 24:1208–1216 (2007). doi: 10.1007/s11095-007-9252-1.PubMedCrossRefGoogle Scholar
  20. 20.
    B. Zarabi, A. Nan, J. Zhuo, R. Gullapalli, and H. Ghandehari. Macrophage targeted N-(2-hydroxypropyl)methacrylamide conjugates for magnetic resonance imaging. Mol. Pharm. 3:550–557 (2006). doi: 10.1021/mp060072i.PubMedCrossRefGoogle Scholar
  21. 21.
    B. Zarabi, A. Nan, J. Zhuo, R. Gullapalli, and H. Ghandehari. HPMA Copolymer-doxorubicin-gadolinium conjugates: synthesis, characterization, and in vitro evaluation. Macromol. Biosci. 8:741–748 (2008). doi: 10.1002/mabi.200700290.PubMedCrossRefGoogle Scholar
  22. 22.
    J. Kopecek, P. Kopeckova, T. Minko, and Z. Lu. HPMA copolymer-anticancer drug conjugates: design, activity, and mechanism of action. Eur. J. Pharm. Biopharm. 50:61–81 (2000). doi: 10.1016/S0939-6411(00)00075-8.PubMedCrossRefGoogle Scholar
  23. 23.
    J. Strohalm, and J. Kopecek. Poly N-(2-hydroxypropyl) methacrylamide: 4. Heterogenous polymerization. Angew. Makromol. Chem. 70:109–118 (1978). doi: 10.1002/apmc.1978.050700110.CrossRefGoogle Scholar
  24. 24.
    P. Rejmanova, J. Labsky, and J. Kopecek. Aminolyses of monomeric and polymeric p-nitrophenyl esters of methacryloylated amino acids. Makromol. Chem. 178:2159–2168 (1977). doi: 10.1002/macp.1977.021780803.CrossRefGoogle Scholar
  25. 25.
    Y. Wu, X. Zhang, Z. Xiong, Z. Cheng, D. R. Fisher, S. Liu, S. S. Gambhir, and X. Chen. microPET imaging of glioma integrin {alpha}v{beta}3 expression using (64)Cu-labeled tetrameric RGD peptide. J. Nucl. Med. 46:1707–1718 (2005).PubMedGoogle Scholar
  26. 26.
    C. C. Kumar, H. Nie, C. P. Rogers, M. Malkowski, E. Maxwell, J. J. Catino, and L. Armstrong. Biochemical characterization of the binding of echistatin to integrin alphavbeta3 receptor. J. Pharmacol. Exp. Ther. 283:843–853 (1997).PubMedGoogle Scholar
  27. 27.
    J. C. Bousquet, S. Saini, D. D. Stark, P. F. Hahn, M. Nigam, J. Wittenberg, and J. T. Ferrucci Jr. Gd-DOTA: characterization of a new paramagnetic complex. Radiology. 166:693–698 (1988).PubMedGoogle Scholar
  28. 28.
    T. Ke, E. K. Jeong, X. Wang, Y. Feng, D. L. Parker, and Z. R. Lu. RGD targeted poly(L-glutamic acid)-cystamine-(Gd-DO3A) conjugate for detecting angiogenesis biomarker alpha(v) beta3 integrin with MRT, mapping. Int. J. Nanomedicine. 2:191–199 (2007).PubMedGoogle Scholar
  29. 29.
    J. E. Schneider, T. Lanz, H. Barnes, D. Medway, L. A. Stork, C. A. Lygate, S. Smart, M. A. Griswold, and S. Neubauer. Ultra-fast and accurate assessment of cardiac function in rats using accelerated MRI at 9.4 Tesla. Magn. Reson. Med. 59:636–641 (2008). doi: 10.1002/mrm.21491.PubMedCrossRefGoogle Scholar
  30. 30.
    L. W. Seymour, R. Duncan, J. Strohalm, and J. Kopecek. Effect of molecular weight (Mw) of N-(2-hydroxypropyl)methacrylamide copolymers on body distribution and rate of excretion after subcutaneous, intraperitoneal, and intravenous administration to rats. J. Biomed. Mater. Res. 21:1341–1358 (1987). doi: 10.1002/jbm.820211106.PubMedCrossRefGoogle Scholar
  31. 31.
    P. Caravan, M. T. Greenfield, X. Li, and A. D. Sherry. The Gd(3+) complex of a fatty acid analogue of DOTP binds to multiple albumin sites with variable water relaxivities. Inorg. Chem. 40:6580–6587 (2001). doi: 10.1021/ic0102900.PubMedCrossRefGoogle Scholar
  32. 32.
    P. Caravan, J. J. Ellison, T. J. McMurry, and R. B. Lauffer. Gadolinium(III) chelates as MRI contrast agents: structure, dynamics, and applications. Chem. Rev. 99:2293–2352 (1999). doi: 10.1021/cr980440x.PubMedCrossRefGoogle Scholar
  33. 33.
    F. Kiessling, M. Heilmann, T. Lammers, K. Ulbrich, V. Subr, P. Peschke, B. Waengler, W. Mier, H. H. Schrenk, M. Bock, L. Schad, and W. Semmler. Synthesis and characterization of HE-24.8: a polymeric contrast agent for magnetic resonance angiography. Bioconjug. Chem. 17:42–51 (2006). doi: 10.1021/bc0501909.PubMedCrossRefGoogle Scholar
  34. 34.
    A. M. Mohs, Y. Zong, J. Guo, D. L. Parker, and Z. R. Lu. PEG-g-poly(GdDTPA-co-L-cystine): effect of PEG chain length on in vivo contrast enhancement in MRI. Biomacromolecules. 6:2305–2311 (2005). doi: 10.1021/bm050194g.PubMedCrossRefGoogle Scholar
  35. 35.
    D. R. Messroghli, S. Plein, D. M. Higgins, K. Walters, T. R. Jones, J. P. Ridgway, and M. U. Sivananthan. Human myocardium: single-breath-hold MR T1 mapping with high spatial resolution–reproducibility study. Radiology. 238:1004–1012 (2006). doi: 10.1148/radiol.2382041903.PubMedCrossRefGoogle Scholar
  36. 36.
    J. T. Yap, J. P. Carney, N. C. Hall, and D. W. Townsend. Image-guided cancer therapy using PET/CT. Cancer J. 10:221–233 (2004). doi: 10.1097/00130404-200407000-00003.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Bahar Zarabi
    • 1
    • 2
  • Mark P. Borgman
    • 1
    • 2
  • Jiachen Zhuo
    • 3
  • Rao Gullapalli
    • 2
    • 3
  • Hamidreza Ghandehari
    • 4
    • 5
  1. 1.Department of Pharmaceutical SciencesUniversity of Maryland, BaltimoreBaltimoreUSA
  2. 2.Center for Nanomedicine and Cellular DeliveryUniversity of Maryland, BaltimoreBaltimoreUSA
  3. 3.Department of RadiologyUniversity of Maryland, BaltimoreBaltimoreUSA
  4. 4.Departments of Pharmaceutics & Pharmaceutical Chemistry and BioengineeringUniversity of UtahSalt Lake CityUSA
  5. 5.Center for Nanomedicine, Nano Institute of UtahUniversity of UtahSalt Lake CityUSA

Personalised recommendations