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

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Abstract

Purpose

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

Methods

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.

Results

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.

Conclusion

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

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Abbreviations

AIBN:

Azobisisobutyronitrile

APMA:

N-(3-Aminopropyl)methacrylamide hydrochloride

APMA-DOTA:

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

ATCC:

American type culture collection

α:

Flip angle

DMF:

N,N-dimethylformamide

DMSO:

Dimethyl sulfoxide

DOTA:

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

EDTA:

Ethylenediaminetetraacetic acid

FOV:

Field of view

FPLC:

Fast protein liquid chromatography

Gd:

Gadolinium

GdCl3.6H2O:

Gadolinium chloride

HPLC:

High pressure liquid chromatography

HPMA:

N-(2-hydroxypropyl)methacrylamide

HUVEC:

Human umbilical vein endothelial cell

ICP-OES:

Inductively coupled plasma optical emission spectroscopy

IDL:

Interactive data language

kDa:

Kilo dalton

MA-GG-ONp:

N-methacryloylglycylglycyl-p-nitrophenyl ester

MA-GG-RGDfK:

N-methacryloylglycylglycyl-RGDfK

MR:

Magnetic resonance

MRI:

Magnetic resonance imaging

Mw :

Molecular weight

MwCO:

Molecular weight cut off

PBS:

Phosphate buffered saline

RGD:

Arg-Gly-Asp

RGD4C:

Lys-Ala-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys-Gly

RGDfK:

Arg-Gly-Asp-D-Phe-Lys

r1 :

Longitudinal relaxivity

ROI:

Region of interest

S:

Signal intensity

SCID:

Severe combined immunodeficient

SEC:

Size exclusion chromatography

T1:

Longitudinal relaxation time

TE:

Echo time

TEF:

Trifluoroacetic acid

TR:

Repetition time

References

  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.

    Article  PubMed  CAS  Google Scholar 

  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).

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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).

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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).

    PubMed  CAS  Google Scholar 

  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).

    PubMed  CAS  Google Scholar 

  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).

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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).

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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).

    PubMed  CAS  Google Scholar 

  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).

    PubMed  CAS  Google Scholar 

  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).

    PubMed  CAS  Google Scholar 

  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).

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

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ACKNOWLEDGMENT

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).

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Baltimore, Maryland, USA

    Bahar Zarabi & Mark P. Borgman

  2. Center for Nanomedicine and Cellular Delivery, University of Maryland, Baltimore, Baltimore, Maryland, USA

    Bahar Zarabi, Mark P. Borgman & Rao Gullapalli

  3. Department of Radiology, University of Maryland, Baltimore, Baltimore, Maryland, USA

    Jiachen Zhuo & Rao Gullapalli

  4. Departments of Pharmaceutics & Pharmaceutical Chemistry and Bioengineering, University of Utah, 383 Colorow Road, Room 343, Salt Lake City, Utah, 84108, USA

    Hamidreza Ghandehari

  5. Center for Nanomedicine, Nano Institute of Utah, University of Utah, Salt Lake City, Utah, USA

    Hamidreza Ghandehari

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  1. Bahar Zarabi
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  2. Mark P. Borgman
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  3. Jiachen Zhuo
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  4. Rao Gullapalli
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  5. Hamidreza Ghandehari
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Correspondence to Hamidreza Ghandehari.

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Zarabi, B., Borgman, M.P., Zhuo, J. et al. Noninvasive Monitoring of HPMA Copolymer–RGDfK Conjugates by Magnetic Resonance Imaging. Pharm Res 26, 1121–1129 (2009). https://doi.org/10.1007/s11095-009-9830-5

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  • DOI: https://doi.org/10.1007/s11095-009-9830-5

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