Anti-angiogenesis by dual action of R5K peptide conjugated itraconazole nanoparticles


Neovascular age-related macular degeneration (AMD) is a leading cause of central vision loss and irreversible blindness. Vascular endothelial growth factor (VEGF) plays an important role in neovascularization under the retina and macula by promoting endothelial cell proliferation, migration, and angiogenesis. Although anti-VEGF drugs have shown their efficacy in visual improvement, long-term use of these drugs leads to ocular and systemic complications due to the non-selectivity of the drug. In this study, the dual-mode anti-angiogenic drug delivery system, which potentially inhibited VEGF in two different ways, was developed. The itraconazole encapsulated nanoparticles, conjugated with R5K peptide, were fabricated to allow multivalent binding interactions with VEGF. The R5K peptide blocked VEGF binding to its receptor, while itraconazole altered the signaling pathway of VEGF stimulation. The dual action of this novel drug delivery system aimed to enhance the anti-angiogenic effects of individual drugs. R5K-ITZ-NPs demonstrated potent, cell-type specific, and dose-dependent inhibition of vascular endothelial cell proliferation, migration, and tube formation in response to VEGF stimulation. The physical stability study showed that R5K-ITZ-NPs were stable when stored at 4 °C. However, the drug remaining in R5K-ITZ-NPs when stored at 4 °C for 28 days were only 17.2%. The chemical stability test revealed that the degradation of R5K-ITZ-NPs followed second-order kinetics. The release profile showed the burst release of ITZ followed by sustained release of the drug This novel drug delivery system may be an option for neovascular AMD patients who are resistant to ITZ and may represent a novel therapy for AMD.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9


  1. 1.

    Lim LS, Mitchell P, Seddon JM, Holz FG, Wong TY. Age-related macular degeneration. Lancet. 2012;379(9827):1728–38.

    PubMed  Google Scholar 

  2. 2.

    Nowak JZ. Age-related macular degeneration (AMD): pathogenesis and therapy. Pharmacol Rep. 2006;58(3):353–63.

    CAS  PubMed  Google Scholar 

  3. 3.

    Daniel E, Toth CA, Grunwald JE, Jaffe GJ, Martin DF, Fine SL, et al. Risk of scar in the comparison of age-related macular degeneration treatments trials. Ophthalmology. 2014;121(3):656–66.

    PubMed  Google Scholar 

  4. 4.

    Ehrlich R, Harris A, Kheradiya NS, Winston DM, Ciulla TA, Wirostko B. Age-related macular degeneration and the aging eye. Clin Interv Aging. 2008;3(3):473–82.

    PubMed  PubMed Central  Google Scholar 

  5. 5.

    Tolentino M. Systemic and ocular safety of intravitreal anti-VEGF therapies for ocular neovascular disease. Surv Ophthalmol. 2011;56(2):95–113.

    PubMed  Google Scholar 

  6. 6.

    Campochiaro PA. Ocular neovascularization. J Mol Med (Berl). 2013;91(3):311–21.

    CAS  Google Scholar 

  7. 7.

    Ferrara N. VEGF and Intraocular Neovascularization: From Discovery to Therapy. Transl Vis Sci Technol. 2016;5(2):10.

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Schlingemann RO, van Hinsbergh VW. Role of vascular permeability factor/vascular endothelial growth factor in eye disease. Br J Ophthalmol. 1997;81(6):501–12.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Kvanta A, Algvere PV, Berglin L, Seregard S. Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor. Invest Ophthalmol Vis Sci. 1996;37(9):1929–34.

    CAS  PubMed  Google Scholar 

  10. 10.

    Wells JA, Murthy R, Chibber R, Nunn A, Molinatti PA, Kohner EM, et al. Levels of vascular endothelial growth factor are elevated in the vitreous of patients with subretinal neovascularisation. Br J Ophthalmol. 1996;80(4):363–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Yi X, Ogata N, Komada M, Yamamoto C, Takahashi K, Omori K, et al. Vascular endothelial growth factor expression in choroidal neovascularization in rats. Graefes Arch Clin Exp Ophthalmol. 1997;235(5):313–9.

    CAS  PubMed  Google Scholar 

  12. 12.

    Ahn JK, Moon HJ. Changes in aqueous vascular endothelial growth factor and pigment epithelium-derived factor after ranibizumab alone or combined with verteporfin for exudative age-related macular degeneration. Am J Ophthalmol. 2009;148(5):718–24 e1.

    CAS  PubMed  Google Scholar 

  13. 13.

    Funk M, Karl D, Georgopoulos M, Benesch T, Sacu S, Polak K, et al. Neovascular age-related macular degeneration: intraocular cytokines and growth factors and the influence of therapy with ranibizumab. Ophthalmology. 2009;116(12):2393–9.

    PubMed  Google Scholar 

  14. 14.

    Sawada O, Miyake T, Kakinoki M, Sawada T, Kawamura H, Ohji M. Aqueous vascular endothelial growth factor after intravitreal injection of pegaptanib or ranibizumab in patients with age-related macular degeneration. Retina. 2010;30(7):1034–8.

    PubMed  Google Scholar 

  15. 15.

    Comparison of Age-related Macular Degeneration Treatments Trials Research G, Martin DF, Maguire MG, Fine SL, Ying GS, Jaffe GJ, et al. Ranibizumab and bevacizumab for treatment of neovascular age-related macular degeneration: two-year results. Ophthalmology. 2012;119(7):1388–98.

    Google Scholar 

  16. 16.

    Group CR, Martin DF, Maguire MG, Ying GS, Grunwald JE, Fine SL, et al. Ranibizumab and bevacizumab for neovascular age-related macular degeneration. N Engl J Med. 2011;364(20):1897–908.

    Google Scholar 

  17. 17.

    Solomon SD, Lindsley K, Vedula SS, Krzystolik MG, Hawkins BS. Anti-vascular endothelial growth factor for neovascular age-related macular degeneration. Cochrane Database Syst Rev. 2014;8:CD005139.

    PubMed Central  Google Scholar 

  18. 18.

    Carmeliet P. Angiogenesis in life, disease and medicine. Nature. 2005;438(7070):932–6.

    CAS  PubMed  Google Scholar 

  19. 19.

    Brown DM, Kaiser PK, Michels M, Soubrane G, Heier JS, Kim RY, et al. Ranibizumab versus verteporfin for neovascular age-related macular degeneration. N Engl J Med. 2006;355(14):1432–44.

    CAS  PubMed  Google Scholar 

  20. 20.

    Brown DM, Michels M, Kaiser PK, Heier JS, Sy JP, Ianchulev T, et al. Ranibizumab versus verteporfin photodynamic therapy for neovascular age-related macular degeneration: Two-year results of the ANCHOR study. Ophthalmology. 2009;116(1):57–65 e5.

    PubMed  Google Scholar 

  21. 21.

    Gordon MS, Cunningham D. Managing patients treated with bevacizumab combination therapy. Oncology. 2005;69(Suppl 3):25–33.

    CAS  PubMed  Google Scholar 

  22. 22.

    Scappaticci FA, Skillings JR, Holden SN, Gerber HP, Miller K, Kabbinavar F, et al. Arterial thromboembolic events in patients with metastatic carcinoma treated with chemotherapy and bevacizumab. J Natl Cancer Inst. 2007;99(16):1232–9.

    PubMed  Google Scholar 

  23. 23.

    Yorston D. Anti-VEGF drugs in the prevention of blindness. Community Eye Health. 2014;27(87):44–6.

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Falavarjani KG, Nguyen QD. Adverse events and complications associated with intravitreal injection of anti-VEGF agents: a review of literature. Eye (Lond). 2013;27(7):787–94.

    Google Scholar 

  25. 25.

    Chong CR, Xu J, Lu J, Bhat S, Sullivan DJ Jr, Liu JO. Inhibition of angiogenesis by the antifungal drug itraconazole. ACS Chem Biol. 2007;2(4):263–70.

    CAS  PubMed  Google Scholar 

  26. 26.

    Head SA, Shi W, Zhao L, Gorshkov K, Pasunooti K, Chen Y, et al. Antifungal drug itraconazole targets VDAC1 to modulate the AMPK/mTOR signaling axis in endothelial cells. Proc Natl Acad Sci U S A. 2015;112(52):E7276–85.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Xu J, Dang YJ, Ren YRZ, Liu JO. Cholesterol trafficking is required for mTOR activation in endothelial cells. Proc Natl Acad Sci U S A. 2010;107(10):4764–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Nacev BA, Grassi P, Dell A, Haslam SM, Liu JO. The antifungal drug itraconazole inhibits vascular endothelial growth factor receptor 2 (VEGFR2) glycosylation, trafficking, and signaling in endothelial cells. J Biol Chem. 2011;286(51):44045–56.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Aftab BT, Dobromilskaya I, Liu JO, Rudin CM. Itraconazole inhibits angiogenesis and tumor growth in non-small cell lung cancer. Cancer Res. 2011;71(21):6764–72.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Yoo SA, Bae DG, Ryoo JW, Kim HR, Park GS, Cho CS, et al. Arginine-rich anti-vascular endothelial growth factor (anti-VEGF) hexapeptide inhibits collagen-induced arthritis and VEGF-stimulated productions of TNF-alpha and IL-6 by human monocytes. J Immunol. 2005;174(9):5846–55.

    CAS  PubMed  Google Scholar 

  31. 31.

    Bae DG, Gho YS, Yoon WH, Chae CB. Arginine-rich anti-vascular endothelial growth factor peptides inhibit tumor growth and metastasis by blocking angiogenesis. J Biol Chem. 2000;275(18):13588–96.

    CAS  PubMed  Google Scholar 

  32. 32.

    Varenne F, Botton J, Merlet C, Beck-Broichsitter M, Legrand F-X. ChristineVauthier Standardization and validation of a protocol of size measurements by dynamic light scattering for monodispersed stable nanomaterial characterization. Colloids Surf A Physicochem Eng Asp. 2015;486:124–38.

    CAS  Google Scholar 

  33. 33.

    Chirila TV, Hong Y, Dalton PD, Constable IJ, Refujo MF. The use of hydrophylic polymers as artificial vitreous. Prog Polym Sci. 1998;23:475–508.

    CAS  Google Scholar 

  34. 34.

    Yang J, Wang Q, Qiao C, Lin Z, Li X, Huang Y, et al. Potent anti-angiogenesis and anti-tumor activity of a novel human anti-VEGF antibody, MIL60. Cell Mol Immunol. 2014;11(3):285–93.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Parikh SK, Dave JB, Patel CN, Ramalingan B. Stability-indicating high-performance thin-layer chromatographic method for analysis of itraconazole in bulk drug and in pharmaceutical dosage form. Pharm Methods. 2011;2(2):88–94.

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Lyu S, Sparer R, Untereker D. Analytical solutions to mathematical models of the surface and bulk erosion of solid polymers. J Polym Sci B Polym Phys. 2005;43:383–97.

    CAS  Google Scholar 

  37. 37.

    Lin SW, Huang SC, Kuo HM, Chen CH, Ma YL, Chu TH, et al. Coral-derived compound WA-25 inhibits angiogenesis by attenuating the VEGF/VEGFR2 signaling pathway. Mar Drugs. 2015;13(2):861–78.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Chu LH, Lee E, Bader JS, Popel AS. Angiogenesis interactome and time course microarray data reveal the distinct activation patterns in endothelial cells. PLoS One. 2014;9(10):e110871.

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Okuyama H, Krishnamachary B, Zhou YF, Nagasawa H, Bosch-Marce M, Semenza GL. Expression of vascular endothelial growth factor receptor 1 in bone marrow-derived mesenchymal cells is dependent on hypoxia-inducible factor 1. J Biol Chem. 2006;281(22):15554–63.

    CAS  PubMed  Google Scholar 

  40. 40.

    Hao Q, Wang L, Zhao ZJ, Tang H. Identification of Protein Kinase D2 as a Pivotal Regulator of Endothelial Cell Proliferation, Migration, and Angiogenesis. J Biol Chem. 2009;284(2):799–806.

    CAS  PubMed  Google Scholar 

  41. 41.

    Zhao Y, Liu Y, Chen Z, Korteweg C, Gu J. Immunoglobulin g (IgG) expression in human umbilical cord endothelial cells. J Histochem Cytochem. 2011;59(5):474–88.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Favot L, Keravis T, Holl V, Le Bec A, Lugnier C. VEGF-induced HUVEC migration and proliferation are decreased by PDE2 and PDE4 inhibitors. Thromb Haemost. 2003;90(2):334–43.

    CAS  PubMed  Google Scholar 

  43. 43.

    Shim JS, Li RJ, Bumpus NN, Head SA, Kumar Pasunooti K, Yang EJ, et al. Divergence of Antiangiogenic Activity and Hepatotoxicity of Different Stereoisomers of Itraconazole. Clin Cancer Res. 2016;22(11):2709–20.

    CAS  PubMed  Google Scholar 

  44. 44.

    Choi CH, Ryu JY, Cho YJ, Jeon HK, Choi JJ, Ylaya K, et al. The anti-cancer effects of itraconazole in epithelial ovarian cancer. Sci Rep. 2017;7(1):6552.

    PubMed  PubMed Central  Google Scholar 

  45. 45.

    Kim J, Tang JY, Gong RY, Kim J, Lee JJ, Clemons KV, et al. Itraconazole, a Commonly Used Antifungal that Inhibits Hedgehog Pathway Activity and Cancer Growth. Cancer Cell. 2010;17(4):388–99.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Herbst RS. Therapeutic options to target angiogenesis in human malignancies. Expert Opin Emerg Drugs. 2006;11(4):635–50.

    CAS  PubMed  Google Scholar 

  47. 47.

    Kim JW, Kim TD, Hong BS, Kim OY, Yoon WH, Chae CB, et al. A serum-stable branched dimeric anti-VEGF peptide blocks tumor growth via anti-angiogenic activity. Exp Mol Med. 2010;42(7):514–23.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Alhowyan AA, Altamimi MA, Kalam MA, Khan AA, Badran M, Binkhathlan Z, et al. Antifungal efficacy of Itraconazole loaded PLGA-nanoparticles stabilized by vitamin-E TPGS: In vitro and ex vivo studies. J Microbiol Methods. 2019;161:87–95.

    CAS  PubMed  Google Scholar 

  49. 49.

    Zhang N, Chittasupho C, Duangrat C, Siahaan TJ, Berkland C. PLGA nanoparticle--peptide conjugate effectively targets intercellular cell-adhesion molecule-1. Bioconjug Chem. 2008;19(1):145–52.

    CAS  PubMed  Google Scholar 

  50. 50.

    Chittasupho C, Posritong P, Ariyawong P. Stability, Cytotoxicity, and Retinal Pigment Epithelial Cell Binding of Hyaluronic Acid-Coated PLGA Nanoparticles Encapsulating Lutein. AAPS PharmSciTech. 2018;20(1):4.

    PubMed  Google Scholar 

  51. 51.

    Izak-Nau E, Huk A, Reidy B, Uggerud H, Vadset M, Eiden S, et al. Impact of storage conditions and storage time on silver nanoparticles' physicochemical properties and implications for their biological effects. RSC Adv. 2015;5(102):84172–85.

    CAS  Google Scholar 

  52. 52.

    Parikh T, Sandhu HK, Talele TT, Serajuddin AT. Characterization of Solid Dispersion of Itraconazole Prepared by Solubilization in Concentrated Aqueous Solutions of Weak Organic Acids and Drying. Pharm Res. 2016;33(6):1456–71.

    CAS  PubMed  Google Scholar 

  53. 53.

    Liu CW, Lin WJ. Polymeric nanoparticles conjugate a novel heptapeptide as an epidermal growth factor receptor-active targeting ligand for doxorubicin. Int J Nanomedicine. 2012;7:4749–67.

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Khoee S, Rahmatolahzadeh R. Synthesis and characterization of pH-responsive and folated nanoparticles based on self-assembled brush-like PLGA/PEG/AEMA copolymer with targeted cancer therapy properties: A comprehensive kinetic study. Eur J Med Chem. 2012;50:416–27.

    CAS  PubMed  Google Scholar 

  55. 55.

    Vey E, Rodger C, Meehan L, Booth J, Claybourn M, Miller AF, et al. The impact of chemical composition on the degradation kinetics of poly(lactic-co-glycolic) acid copolymers cast films in phosphate buffer solution. Polym Degrad Stab. 2012;97(3):358–65.

    CAS  Google Scholar 

  56. 56.

    Jahan ST, Haddadi A. Investigation and optimization of formulation parameters on preparation of targeted anti-CD205 tailored PLGA nanoparticles. Int J Nanomedicine. 2015;10:7371–84.

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Dinarvand R, Sepehri N, Manoochehri S, Rouhani H, Atyabi F. Polylactide-co-glycolide nanoparticles for controlled delivery of anticancer agents. Int J Nanomedicine. 2011;6:877–95.

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Makadia HK, Siegel SJ. Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Polymers (Basel). 2011;3(3):1377–97.

    CAS  Google Scholar 

  59. 59.

    Maiz-Fernandez S, Perez-Alvarez L, Ruiz-Rubio L, Perez Gonzalez R, Saez-Martinez V, Ruiz Perez J, et al. Synthesis and Characterization of Covalently Crosslinked pH-Responsive Hyaluronic Acid Nanogels: Effect of Synthesis Parameters. Polymers (Basel). 2019;11(4).

  60. 60.

    Almalik A, Benabdelkamel H, Masood A, Alanazi IO, Alradwan I, Majrashi MA, et al. Hyaluronic Acid Coated Chitosan Nanoparticles Reduced the Immunogenicity of the Formed Protein Corona. Sci Rep. 2017;7(1):10542.

    PubMed  PubMed Central  Google Scholar 

Download references


The authors gratefully acknowledge the use of the facility of the Research Center for Drug Discovery and Development, Srinakharinwirot University.


This work was supported by the Office of the Higher Education Commission and the Thailand Research Fund (MRG6180064).

Author information



Corresponding author

Correspondence to Chuda Chittasupho.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chittasupho, C., Kengtrong, K., Chalermnithiwong, S. et al. Anti-angiogenesis by dual action of R5K peptide conjugated itraconazole nanoparticles. AAPS PharmSciTech 21, 74 (2020).

Download citation


  • angiogenesis
  • R5K
  • itraconazole
  • PLGA