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Leucine-Aspartic Acid-Valine Sequence as Targeting Ligand and Drug Carrier for Doxorubicin Delivery to Melanoma Cells: In Vitro Cellular Uptake and Cytotoxicity Studies

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Abstract

Purpose

To study the feasibility of Leucine-Aspartic Acid-Valine (LDV) as targeting ligand and drug carrier for targeted delivery to integrin α4β1 over-expressing cancer cells.

Methods

Poly(L,D,V) was randomly copolymerized using N-carboxyanhydrides of leucine, β-benzyl-aspartic acid, and valine. Oligo(LDV), consisting of 2-6 LDV units, were synthesized by solid phase protein synthesis (SPPS) method. Binding of Leu-Asp-Val, Val-Asp-Leu, and Leu-Asn-Val, and internalization of FITC labeled LDV by wild-type and integrin α4 knock-down A375 cells were studied. Cytotoxicity of poly(L,D,V)-Dox, oligo(LDV)-Dox, and doxorubicin (Dox) was also determined on wild-type, integrin α4 knock-down A375 cells, and normal human epithelial keratinocytes (NHEK).

Results

LDV was essential for the specific binding and internalization by cells expressing integrin α4β1. Cytotoxicity of poly(L,D,V)-Dox and oligo(LDV)-Dox was integrin α4-dependent, while free Dox did not show this differential effect. No observable cytotoxicity trend was found when increasing LDV repeating unit. Poly(L,D,V) was relatively more effective than oligo(LDV) for the delivery of Dox to A375.

Conclusion

LDV containing moieties bind specifically to integrin α4β1 expressing cancer cells. The binding, internalization, and cytotoxicity depend on the level of integrin α4β1 expression. Poly(L,D,V) and oligo(LDV) were both effective in the in vitro targeted delivery of Dox to integrin α4β1 over-expressing A375 cells.

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REFERENCES

  1. Rihova B. Receptor-mediated targeted drug or toxin delivery. Adv Drug Deliv Rev. 1998;29:273–89.

    Article  CAS  PubMed  Google Scholar 

  2. Hudson PJ, Souriau C. Engineered antibodies. Nat Med. 2003;9:129–34.

    Article  CAS  PubMed  Google Scholar 

  3. Leamon CP, Reddy JA. Folate-targeted chemotherapy. Adv Drug Deliv Rev. 2004;56:1127–41.

    Article  CAS  PubMed  Google Scholar 

  4. Chung NS, Wasan KM. Potential role of the low-density lipoprotein receptor family as mediators of cellular drug uptake. Adv Drug Deliv Rev. 2004;56:1315–34.

    Article  CAS  PubMed  Google Scholar 

  5. Fischer D, Kissel T. Histochemical characterization of primary capillary endothelial cells from porcine brains using monoclonal antibodies and fluorescein isothiocyanate-labelled lectins: implications for drug delivery. Eur J Pharm Biopharm. 2001;52:1–11.

    Article  CAS  PubMed  Google Scholar 

  6. Qian ZM, Li H, Sun H, Ho K. Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway. Pharmacol Rev. 2002;54:561–87.

    Article  CAS  PubMed  Google Scholar 

  7. Arnaout MA. Integrin structure: new twists and turns in dynamic cell adhesion. Immunol Rev. 2002;186:125–40.

    Article  CAS  PubMed  Google Scholar 

  8. Mizejewski GJ. Role of integrins in cancer: survey of expression patterns. Proc Soc Exp Biol Med. 1999;222:124–38.

    Article  CAS  PubMed  Google Scholar 

  9. Makarem R, Humphries MJ. LDV: a novel cell adhesion motif recognized by the integrin alpha 4 beta 1. Biochem Soc Trans. 1991;19:380S.

    CAS  PubMed  Google Scholar 

  10. Smith JW, Cheresh DA. Integrin (alpha v beta 3)-ligand interaction. Identification of a heterodimeric RGD binding site on the vitronectin receptor. J Biol Chem. 1990;265:2168–72.

    CAS  PubMed  Google Scholar 

  11. Pedchenko V, Zent R, Hudson BG. Alpha(v)beta3 and alpha(v)beta5 integrins bind both the proximal RGD site and non-RGD motifs within noncollagenous (NC1) domain of the alpha3 chain of type IV collagen: implication for the mechanism of endothelia cell adhesion. J Biol Chem. 2004;279:2772–80.

    Article  CAS  PubMed  Google Scholar 

  12. Suehiro K, Mizuguchi J, Nishiyama K, Iwanaga S, Farrell DH, Ohtaki S. Fibrinogen binds to integrin alpha(5)beta(1) via the carboxyl-terminal RGD site of the Aalpha-chain. J Biochem. 2000;128:705–10.

    CAS  PubMed  Google Scholar 

  13. MedImmune initiates phase I/II clinical study with Vitaxin. Expert Rev Anticancer Ther. 2001;1:166.

    Google Scholar 

  14. Bibby DC, Talmadge JE, Dalal MK, Kurz SG, Chytil KM, Barry SE, et al. Pharmacokinetics and biodistribution of RGD-targeted doxorubicin-loaded nanoparticles in tumor-bearing mice. Int J Pharm. 2005;293:281–90.

    Article  CAS  PubMed  Google Scholar 

  15. Maeda M, Izuno Y, Kawasaki K, Kaneda Y, Mu Y, Tsutsumi Y, et al. Amino acids and peptides. XXX. Preparation of Arg-Gly-Asp (RGD) hybrids with poly(ethylene glycol) analogs and their antimetastatic effect. Chem Pharm Bull (Tokyo). 1997;45:1788–92.

    CAS  Google Scholar 

  16. Chen R, Curran S, Curran JM, Hunt JA. The use of poly(l-lactide) and RGD modified microspheres as cell carriers in a flow intermittency bioreactor for tissue engineering cartilage. Biomaterials. 2006;27:4453–60.

    Article  CAS  PubMed  Google Scholar 

  17. Koizumi N, Mizuguchi H, Hosono T, Ishii-Watabe A, Uchida E, Utoguchi N, et al. Efficient gene transfer by fiber-mutant adenoviral vectors containing RGD peptide. Biochim Biophys Acta. 2001;1568:13–20.

    CAS  PubMed  Google Scholar 

  18. Niu JX, Zhang WJ, Ye LY, Wu LQ, Zhu GJ, Yang ZH, et al. The role of adhesion molecules, alpha v beta 3, alpha v beta 5 and their ligands in the tumor cell and endothelial cell adhesion. Eur J Cancer Prev. 2007;16:517–27.

    Article  CAS  PubMed  Google Scholar 

  19. Wayner EA, Kovach NL. Activation-dependent recognition by hematopoietic cells of the LDV sequence in the V region of fibronectin. J Cell Biol. 1992;116:489–97.

    Article  CAS  PubMed  Google Scholar 

  20. Guan JL, Hynes RO. Lymphoid cells recognize an alternatively spliced segment of fibronectin via the integrin receptor alpha 4 beta 1. Cell. 1990;60:53–61.

    Article  CAS  PubMed  Google Scholar 

  21. Chigaev A, Zwartz GJ, Buranda T, Edwards BS, Prossnitz ER, Sklar LA. Conformational regulation of alpha 4 beta 1-integrin affinity by reducing agents. :“Inside-out” signaling is independent of and additive to reduction-regulated integrin activation. J Biol Chem. 2004;279:32435–43.

    Article  CAS  PubMed  Google Scholar 

  22. Singh J, Adams S, Carter MB, Cuervo H, Lee WC, Lobb RR, et al. Rational design of potent and selective VLA-4 inhibitors and their utility in the treatment of asthma. Curr Top Med Chem. 2004;4:1497–507.

    Article  CAS  PubMed  Google Scholar 

  23. Garcia-Gila M, Cabanas C, Garcia-Pardo A. Analysis of the activation state of alpha4beta1 integrin in human B cell lines derived from myeloma, leukemia or lymphoma. FEBS Lett. 1997;418:337–40.

    Article  CAS  PubMed  Google Scholar 

  24. Zhong S, Li Y, Chan W, Jasti B, Li X. Integrin a4ß1-dependent Binding of Leucine-Aspartate-Valine (LDV) to A375 Cells: a Rationale for Targeting Drug Delivery. Vienna: Cotrolled Release Society; 2006.

    Google Scholar 

  25. Liu M, Xie C, Pan H, Pan J, Lu W. Separation of polyethylene glycols and their fluorescein-labeled compounds depending on the hydrophobic interaction by high-performance liquid chromatography. J Chromatogr A. 2006;1129:61–6.

    Article  CAS  PubMed  Google Scholar 

  26. Hyduk SJ, Chan JR, Duffy ST, Chen M, Peterson MD, Waddell TK, et al. Phospholipase C, calcium, and calmodulin are critical for alpha4beta1 integrin affinity up-regulation and monocyte arrest triggered by chemoattractants. Blood. 2007;109:176–84.

    Article  CAS  PubMed  Google Scholar 

  27. Luissint AC, Lutz PG, Calderwood DA, Couraud PO, Bourdoulous S. JAM-L-mediated leukocyte adhesion to endothelial cells is regulated in cis by alpha4beta1 integrin activation. J Cell Biol. 2008;183:1159–73.

    Article  CAS  PubMed  Google Scholar 

  28. Andersson EC, Christensen JP, Marker O, Thomsen AR. Changes in cell adhesion molecule expression on T cells associated with systemic virus infection. J Immunol. 1994;152:1237–45.

    CAS  PubMed  Google Scholar 

  29. Singh J, Van Vlijmen H, Liao Y, Lee WC, Cornebise M, Harris M, et al. Identification of potent and novel alpha4beta1 antagonists using in silico screening. J Med Chem. 2002;45:2988–93.

    Article  CAS  PubMed  Google Scholar 

  30. Stuve O, Bennett JL. Pharmacological properties, toxicology and scientific rationale for the use of natalizumab (Tysabri) in inflammatory diseases. CNS Drug Rev. 2007;13:79–95.

    Article  PubMed  Google Scholar 

  31. Jackson DY, Quan C, Artis DR, Rawson T, Blackburn B, Struble M, et al. Potent alpha 4 beta 1 peptide antagonists as potential anti-inflammatory agents. J Med Chem. 1997;40:3359–68.

    Article  CAS  PubMed  Google Scholar 

  32. Haworth D, Rees A, Alcock PJ, Wood LJ, Dutta AS, Gormley JJ, et al. Anti-inflammatory activity of c(ILDV-NH(CH2)5CO), a novel, selective, cyclic peptide inhibitor of VLA-4-mediated cell adhesion. Br J Pharmacol. 1999;126:1751–60.

    Article  CAS  PubMed  Google Scholar 

  33. Dutta AS, Gormley JJ, Coath M, Hassall L, Hayward CF, Gellert PR, et al. Potent cyclic peptide inhibitors of VLA-4 (alpha4beta1 integrin)-mediated cell adhesion. Discovery of compounds like cyclo(MePhe-Leu-Asp-Val-D-Arg-D-Arg) (ZD7349) compatible with depot formulation. J Pept Sci. 2000;6:398–412.

    Article  CAS  PubMed  Google Scholar 

  34. Dutta AS, Crowther M, Gormley JJ, Hassall L, Hayward CF, Gellert PR, et al. Potent cyclic monomeric and dimeric peptide inhibitors of VLA-4 (alpha4beta1 integrin)-mediated cell adhesion based on the Ile-Leu-Asp-Val tetrapeptide. J Pept Sci. 2000;6:321–41.

    Article  CAS  PubMed  Google Scholar 

  35. Kaneda Y, Yamamoto Y, Okada N, Tsutsuml Y, Nakagawa S, Kakiuch M, et al. Antimetastatic effect of synthetic Glu-Ile-Leu-Asp-Val peptide derivatives containing D-amino acids. Anticancer Drugs. 1997;8:702–7.

    Article  CAS  PubMed  Google Scholar 

  36. Momtaz M, Rerat V, Gharbi S, Gerard E, Pourcelle V, Marchand-Brynaert J. A graftable LDV peptidomimetic: design, synthesis and application to a blood filtration membrane. Bioorg Med Chem Lett. 2008;18:1084–90.

    Article  CAS  PubMed  Google Scholar 

  37. Nori A, Kopecek J. Intracellular targeting of polymer-bound drugs for cancer chemotherapy. Adv Drug Deliv Rev. 2005;57:609–36.

    Article  CAS  PubMed  Google Scholar 

  38. Nowak G. Methods and data analyses in radioligand-receptor binding: unnecessary transformations may cause biased results. Pol J Pharmacol. 1998;50:101–5.

    CAS  PubMed  Google Scholar 

  39. Steinitz M, Baraz L. A rapid method for estimating the binding of ligands to ELISA microwells. J Immunol Methods. 2000;238:143–50.

    Article  CAS  PubMed  Google Scholar 

  40. Dardik BN, Schwartzkopf CD, Stevens DE, Chatelain RE. A quantitative assay for the non-covalent association between apolipoprotein[a] and apolipoprotein B: an alternative measure of Lp[a] assembly. J Lipid Res. 2000;41:1013–9.

    CAS  PubMed  Google Scholar 

  41. Sriramarao P, Mendler M, Bourdon MA. Endothelial cell attachment and spreading on human tenascin is mediated by alpha 2 beta 1 and alpha v beta 3 integrins. J Cell Sci. 1993;105(Pt 4):1001–12.

    CAS  PubMed  Google Scholar 

  42. Schraa AJ, Kok RJ, Berendsen AD, Moorlag HE, Bos EJ, Meijer DK, et al. Endothelial cells internalize and degrade RGD-modified proteins developed for tumor vasculature targeting. J Control Release. 2002;83:241–51.

    Article  CAS  PubMed  Google Scholar 

  43. Mould AP, Komoriya A, Yamada KM, Humphries MJ. The CS5 peptide is a second site in the IIICS region of fibronectin recognized by the integrin alpha 4 beta 1. Inhibition of alpha 4 beta 1 function by RGD peptide homologues. J Biol Chem. 1991;266:3579–85.

    CAS  PubMed  Google Scholar 

  44. Murata J, Saiki I, Ogawa R, Nishi N, Tokura S, Azuma I. Molecular properties of poly(RGD) and its binding capacities to metastatic melanoma cells. Int J Pept Protein Res. 1991;38:212–7.

    CAS  PubMed  Google Scholar 

  45. Saiki I, Murata J, Matsuno K, Ogawa R, Nishi N, Tokura S, et al. Anti-metastatic and anti-invasive effects of polymeric Arg-Gly-Asp (RGD) peptide, poly(RGD), and its analogues. Jpn J Cancer Res. 1990;81:660–7.

    CAS  PubMed  Google Scholar 

  46. Saiki I, Iida J, Murata J, Ogawa R, Nishi N, Sugimura K, et al. Inhibition of the metastasis of murine malignant melanoma by synthetic polymeric peptides containing core sequences of cell-adhesive molecules. Cancer Res. 1989;49:3815–22.

    CAS  PubMed  Google Scholar 

  47. Tressler RJ, Belloni PN, Nicolson GL. Correlation of inhibition of adhesion of large cell lymphoma and hepatic sinusoidal endothelial cells by RGD-containing peptide polymers with metastatic potential: role of integrin-dependent and -independent adhesion mechanisms. Cancer Commun. 1989;1:55–63.

    CAS  PubMed  Google Scholar 

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ACKNOWLEDGEMENTS

Authors would like to thank Dr. Jianhua Ren and Xiaoning Zhao for providing us with magnetic beads, and Dr. Der Thor for his help on the siRNA knock-down technique. This study is supported by NIH 1R15CA100182-01A2.

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Authors and Affiliations

  1. Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA, 95211, USA

    Sha Zhong, Shiladitya Bhattacharya, William Chan, Bhaskara Jasti & Xiaoling Li

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  1. Sha Zhong
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Correspondence to Xiaoling Li.

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Zhong, S., Bhattacharya, S., Chan, W. et al. Leucine-Aspartic Acid-Valine Sequence as Targeting Ligand and Drug Carrier for Doxorubicin Delivery to Melanoma Cells: In Vitro Cellular Uptake and Cytotoxicity Studies. Pharm Res 26, 2578–2587 (2009). https://doi.org/10.1007/s11095-009-9971-6

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  • DOI: https://doi.org/10.1007/s11095-009-9971-6

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