Preparation, Characterization, and In Vitro and In Vivo Evaluation of PEGylated Liposomal Doxorubicin Modified with Different cRGD Peptides

  • Mohamadreza Amin
  • Mahmoud Reza Jaafari
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


Liposomes containing cytotoxic agents and decorated with Cyclic Arg-Gly-Asp (cRGD) pentapeptides have attracted considerable attention for targeting tumor vasculature. These cRGD peptides have been used for targeting liposomes or other nano-carriers to inflamed or tumoral tissues; however, no comparative study dealing with the biological performances of liposomes decorated with different cRGDs could be found in the literature.

Herein, we prepared PEGylated Liposomal Doxorubicin (PLD) conjugated with different three cRGD peptides (RGD-PLDs). RADyC-PLD and not modified PLDs (Plain-PLD) were prepared as negative controls. Then, all the preparations were comparatively evaluated with respect to their in vitro behaviors (cell interactions and cytotoxicity) and in vivo performances (biodistribution and therapeutic efficacy) in tumored mice.

In this chapter, we present the general flowchart applied for this project as follow: (1) preparation of peptide-modified PLDs, (2) characterization of colloidal properties and stability of PLDs, (3) in vitro cell interaction and cytotoxicity, and (4) in vivo biodistribution and therapeutic efficacy in mice bearing C-26 colon carcinoma tumor model.


Liposomes Doxorubicin RGD peptides Integrins Tumor Vascular targeting 


  1. 1.
    Gottschalk KE, Kessler H (2002) The structures of Integrins and integrin-ligand complexes: implications for drug design and signal transduction. Angew Chem Int Ed 41(20):3767–3774. doi: 10.1002/1521-3773(20021018)41:20<3767::Aid-Anie3767>3.0.Co;2-TCrossRefGoogle Scholar
  2. 2.
    Chen K, Chen X (2011) Integrin targeted delivery of chemotherapeutics. Theranostics 1:189–200CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Ruoslahti E (2003) The RGD story: a personal account. Matrix Biol 22(6):459–465, S0945053X03000830 [pii]CrossRefPubMedGoogle Scholar
  4. 4.
    Pierschbacher MD, Ruoslahti E (1984) Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 309(5963):30–33CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Heckmann D, Meyer A, Marinelli L, Zahn G, Stragies R, Kessler H (2007) Probing integrin selectivity: rational design of highly active and selective ligands for the alpha 5 beta 1 and alpha v beta 3 integrin receptor. Angew Chem Int Ed 46(19):3571–3574. doi: 10.1002/anie.200700008CrossRefGoogle Scholar
  6. 6.
    Temming K, Schiffelers RM, Molema G, Kok RJ (2005) RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature. Drug Resist Updat 8(6):381–402. doi: 10.1016/j.drup.2005.10.002, S1368-7646(05)00087-7 [pii]CrossRefPubMedGoogle Scholar
  7. 7.
    Hajitou A, Pasqualini R, Arap W (2006) Vascular targeting: recent advances and therapeutic perspectives. Trends Cardiovasc Med 16(3):80–88. doi: 10.1016/j.tcm.2006.01.003CrossRefPubMedGoogle Scholar
  8. 8.
    Salvati M, Cordero FM, Pisaneschi F, Melani F, Gratteri P, Cini N, Bottoncetti A, Brandi A (2008) Synthesis, SAR and in vitro evaluation of new cyclic Arg-Gly-Asp pseudopentapeptides containing a s-cis peptide bond as integrin alphavbeta3 and alphavbeta5 ligands. Bioorg Med Chem 16(8):4262–4271. doi: 10.1016/j.bmc.2008.02.080, S0968-0896(08)00201-0 [pii]CrossRefPubMedGoogle Scholar
  9. 9.
    Cini N, Trabocchi A, Menchi G, Bottoncetti A, Raspanti S, Pupi A, Guarna A (2009) Morpholine-based RGD-cyclopentapeptides as alpha(v)beta(3)/alpha(v)beta(5) integrin ligands: role of configuration towards receptor binding affinity. Bioorg Med Chem 17(4):1542–1549. doi: 10.1016/j.bmc.2009.01.006CrossRefPubMedGoogle Scholar
  10. 10.
    Heckmann D, Kesster H (2007) Design and chemical synthesis of integrin ligands. Integrins 426:463. doi: 10.1016/S0076-6879(07)26020-3CrossRefGoogle Scholar
  11. 11.
    Mas-Moruno C, Rechenmacher F, Kessler H (2010) Cilengitide: the first anti-angiogenic small molecule drug candidate. Design, synthesis and clinical evaluation. Anticancer Agents Med Chem 10(10):753–768CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Denekamp J (1984) Vascular endothelium as the vulnerable element in tumours. Acta Radiol Oncol 23(4):217–225CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Barenholz Y (2012) Doxil(R)--the first FDA-approved nano-drug: lessons learned. J Control Release 160(2):117–134. doi: 10.1016/j.jconrel.2012.03.020, S0168-3659(12)00230-1 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Xiong XB, Huang Y, Lu WL, Zhang X, Zhang H, Nagai T, Zhang Q (2005) Enhanced intracellular delivery and improved antitumor efficacy of doxorubicin by sterically stabilized liposomes modified with a synthetic RGD mimetic. J Control Release 107(2):262–275. doi: 10.1016/j.jconrel.2005.03.030, S0168-3659(05)00121-5 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Xiong XB, Huang Y, Lu WL, Zhang X, Zhang H, Nagai T, Zhang Q (2005) Intracellular delivery of doxorubicin with RGD-modified sterically stabilized liposomes for an improved antitumor efficacy: in vitro and in vivo. J Pharm Sci 94(8):1782–1793. doi: 10.1002/jps.20397CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Schiffelers RM, Koning GA, ten Hagen TLM, Fens MHAM, Schraa AJ, Janssen ANPCA, Kok RJ, Molema G, Storm G (2003) Anti-tumor efficacy of tumor vasculature-targeted liposomal doxorubicin. J Control Release 91(1-2):115–122. doi: 10.1016/S0168-3659(03)00240-2CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Chen X, Park R, Shahinian AH, Bading JR, Conti PS (2004) Pharmacokinetics and tumor retention of 125I-labeled RGD peptide are improved by PEGylation. Nucl Med Biol 31(1):11–19, doi:S0969805103001288 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Hersel U, Dahmen C, Kessler H (2003) RGD modified polymers: biomaterials for stimulated cell adhesion and beyond. Biomaterials 24(24):4385–4415, doi:S0142961203003430 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Bibby DC, Talmadge JE, Dalal MK, Kurz SG, Chytil KM, Barry SE, Shand DG, Steiert M (2005) Pharmacokinetics and biodistribution of RGD-targeted doxorubicin-loaded nanoparticles in tumor-bearing mice. Int J Pharm 293(1-2):281–290. doi: 10.1016/j.ijpharm.2004.12.021, S0378-5173(05)00056-6 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Amin M, Badiee A, Jaafari MR (2013) Improvement of pharmacokinetic and antitumor activity of PEGylated liposomal doxorubicin by targeting with N-methylated cyclic RGD peptide in mice bearing C-26 colon carcinomas. Int J Pharm 458:324. doi: 10.1016/j.ijpharm.2013.10.018CrossRefPubMedGoogle Scholar
  21. 21.
    Zuidam NJ, Vrueh RD, Ceommelin DJA (2003) Characterization of liposomes. In: Torchilin VO, Weissig V (eds) Liposomes a practical approach, 2nd edn, Practical approach. Oxford University Press, New York, NY, pp 32–33Google Scholar
  22. 22.
    Horowitz AT, Barenholz Y, Gabizon AA (1992) In vitro cytotoxicity of liposome-encapsulated doxorubicin: dependence on liposome composition and drug release. Biochim Biophys Acta 1109(2):203–209, 0005-2736(92)90084-Y [pii]CrossRefPubMedGoogle Scholar
  23. 23.
    Huang Z, Szoka FC Jr (2008) Sterol-modified phospholipids: cholesterol and phospholipid chimeras with improved biomembrane properties. J Am Chem Soc 130(46):15702–15712. doi: 10.1021/ja8065557CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Huang Z, Jaafari MR, Szoka FC Jr (2009) Disterolphospholipids: nonexchangeable lipids and their application to liposomal drug delivery. Angew Chem Int Ed Engl 48(23):4146–4149. doi: 10.1002/anie.200900111CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Schluep T, Hwang J, Cheng J, Heidel JD, Bartlett DW, Hollister B, Davis ME (2006) Preclinical efficacy of the camptothecin-polymer conjugate IT-101 in multiple cancer models. Clin Cancer Res 12(5):1606–1614. doi: 10.1158/1078-0432.CCR-05-1566, 12/5/1606 [pii]CrossRefPubMedGoogle Scholar
  26. 26.
    Hermanson GT (2008) The chemistry of reactive groups, 2nd edn, Bioconjugate techniques. Elsevier, Houston, TX, pp 169–221Google Scholar
  27. 27.
    Vacha J (1975) Blood volume in inbred strain BALB/c, CBA/J and C57BL/10 mice determined by means of 59Fe-labelled red cells and 59Fe bound to transferrin. Physiol Bohemoslov 24(5):413–419PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Biotechnology Research Center, Nanotechnology Research Center, School of PharmacyMashhad University of Medical SciencesMashhadIran
  2. 2.Nanotechnology Research Center, School of PharmacyMashhad University of Medical SciencesMashhadIran

Personalised recommendations