Advertisement

Pharmaceutical Research

, Volume 27, Issue 2, pp 327–339 | Cite as

Therapeutic Potential of New 4-hydroxy-tamoxifen-Loaded pH-gradient Liposomes in a Multiple Myeloma Experimental Model

  • Giorgia Urbinati
  • Davide Audisio
  • Véronique Marsaud
  • Vincent Plassat
  • Silvia Arpicco
  • Brigitte Sola
  • Elias Fattal
  • Jack-Michel Renoir
Research Paper

Abstract

Purpose

To determine the better liposomal formulation incorporating the active metabolite of tamoxifen, 4-hydroxy-tamoxifen (4HT) and the biological impact of 4HT-pH-gradient liposomes on response to in vivo treatment.

Methods

Several pegylated liposomes were formulated by varying the composition of lipids, increasing external pH from 7.4 to 9.0 and doubling the lipid concentration. Dipalmitoylphosphatidylcholine / cholesterol / distearoylphosphoethanolamine poly(ethylene)glycol liposomes (DL-9 liposomes) were chosen for their physico-chemical properties. Toxicity and release kinetics were assessed in breast cancer MCF-7 as well as in multiple myeloma (MM) cells. In vivo antitumor activity and bio-distribution were measured in the RPMI8226 MM model.

Results

Compared to conventional non-pH-gradient liposomes, 4HT-DL-9 liposomes resulted in concentration of up to 1 mM 4HT, greater stability, relative toxicity and slow 4HT release. Intravenous injections of 4HT-DL-9 liposomes at 4 mg/kg/week blocked MM tumor growth. Ki67 and CD34 labeling decreased in treated tumors, concomitantly with increase of activated caspase-3 supporting a cell proliferation arrest, a decrease of tumor vasculature and the induction of tumor cell death.

Conclusion

This antitumor effect was assumed to be the result of a modified biodistribution of 4HT once trapped in DL-9 liposomes. Such 4HT-containing pH-gradient Stealth® nanocarriers could be helpful for MM treatment.

KEY WORDS

breast cancer hydroxy-tamoxifen multiple myeloma pH-gradient Stealth® liposomes 

Abbreviations

Chol

Cholesterol

DPPC

Dipalmitoylphosphatidylcholine

DSPC

1,2-distearoyl-sn-glycero-3-phosphatidylcholine

DSPE-PEG2000

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(Polyethyleneglycol)-2000] (ammonium salt)

ePC

egg-phosphatidylcholine

ePG

egg-phosphatidylglycerol

ER

estrogen receptor

4HT

4-hydroxy-tamoxifen

Notes

Acknowledgments

We thank Besins Iscovesco for the generous gift of 4HT, J. Bignon for FACS analyses and M. Pons and P. Ballaguer for the gift of MELN cells. This work was supported by the Ligue Nationale contre le Cancer through a fellowship offered to G.U. and financial support was provided to J-M. R from the Comités du Cher, de l’Indre et des Hauts de Seine of the Ligue Nationale contre le Cancer.

References

  1. 1.
    Fisher B, Costantino JP, Wickerham DL, Redmond CK, Kavanah M, Cronin WM, et al. Tamoxifen for prevention of breast cancer: report of the national surgical adjuvant breast and bowel project P-1 study. J Natl Cancer Inst. 1998;90:1371–88.CrossRefPubMedGoogle Scholar
  2. 2.
    Jordan VC, Jordan VC. Tamoxifen (ICI46,474) as a targeted therapy to treat and prevent breast cancer. Br J Pharmacol. 2006;147(Suppl 1):S269–76.CrossRefPubMedGoogle Scholar
  3. 3.
    Ellis CA, Vos MD, Wickline M, Riley C, Vallecorsa T, Telford WG, et al. Tamoxifen and the farnesyl transferase inhibitor FTI-277 synergize to inhibit growth in estrogen receptor-positive breast tumor cell lines. Breast Cancer Res Treat. 2003;78:59–67.CrossRefPubMedGoogle Scholar
  4. 4.
    Obrero M, Yu DV, Shapiro DJ. Estrogen receptor-dependent and estrogen receptor-independent pathways for tamoxifen and 4-hydroxytamoxifen-induced programmed cell death. J Biol Chem. 2002;277:45695–703.CrossRefPubMedGoogle Scholar
  5. 5.
    Zheng A, Kallio A, Harkonen P. Tamoxifen-induced rapid death of MCF-7 breast cancer cells is mediated via extracellularly signal-regulated kinase signaling and can be abrogated by estrogen. Endocrinology. 2007;148:2764–77.CrossRefPubMedGoogle Scholar
  6. 6.
    Dutertreand M, Smith CL. Molecular mechanisms of selective estrogen receptor modulator (SERM) action. J Pharmacol Exp Ther. 2000;295:431–7.Google Scholar
  7. 7.
    MacGregor JI, Jordan VC. Basic guide to the mechanisms of antiestrogen action. Pharmacol Rev. 1998;50:151–96.PubMedGoogle Scholar
  8. 8.
    Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, et al. The nuclear receptor superfamily: the second decade. Cell. 1995;83:835–9.CrossRefPubMedGoogle Scholar
  9. 9.
    Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet 2005; 365:1687-717.Google Scholar
  10. 10.
    Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the women’s health initiative randomized controlled trial. JAMA. 2002;288:321–33.CrossRefPubMedGoogle Scholar
  11. 11.
    Goldenberg GJ, Froese EK. Drug and hormone sensitivity of estrogen receptor-positive and -negative human breast cancer cells in vitro. Cancer Res. 1982;42:5147–51.PubMedGoogle Scholar
  12. 12.
    Gelmann EP. Tamoxifen for the treatment of malignancies other than breast and endometrial carcinoma. Semin Oncol. 1997;24:S1-65–70.Google Scholar
  13. 13.
    de Medina P, Payre B, Boubekeur N, Bertrand-Michel J, Terce F, Silvente-Poirot S, et al. Ligands of the antiestrogen-binding site induce active cell death and autophagy in human breast cancer cells through the modulation of cholesterol metabolism. Cell Death Differ. 2009.Google Scholar
  14. 14.
    Sjak-Shie NN, Vescio RA, Berenson JR. Recent advances in multiple myeloma. Curr Opin Hematol. 2000;7:241–6.CrossRefPubMedGoogle Scholar
  15. 15.
    Treon SP, Teoh G, Urashima M, Ogata A, Chauhan D, Webb IJ, et al. Anti-estrogens induce apoptosis of multiple myeloma cells. Blood. 1998;92:1749–57.PubMedGoogle Scholar
  16. 16.
    Otsuki T, Yamada O, Kurebayashi J, Moriya T, Sakaguchi H, Kunisue H, et al. Estrogen receptors in human myeloma cells. Cancer Res. 2001;60:1434–41.Google Scholar
  17. 17.
    Gauduchon J, Gouilleux F, Maillard S, Marsaud V, Renoir JM, Sola B. The 4-hydroxytamoxifen inhibits proliferation of multiple myeloma cells in vitro and in vivo through down-regulation of c-Myc, up-regulation of p27Kip1 and modulation of Bcl-2 family members. Clin Cancer Res. 2005;11:2345–54.CrossRefPubMedGoogle Scholar
  18. 18.
    Gauduchon J, Seguin A, Marsaud V, Clay D, Renoir JM, Sola B. Pure antiestrogen-induced G1-arrest in myeloma cells results from the reduced kinase activity of cyclin D3/CDK6 complexes whereas apoptosis is mediated by endoplasmic reticulum-dependent caspases. Int J Cancer. 2008;122:2130–41.CrossRefPubMedGoogle Scholar
  19. 19.
    Sola B, Renoir JM. Estrogenic or antiestrogenic therapies for multiple myeloma? Mol Cancer. 2007;6:59.CrossRefPubMedGoogle Scholar
  20. 20.
    Olivier S, Close P, Castermans E, de Leval L, Tabruyn S, Chariot A, et al. Raloxifene-induced myeloma cell apoptosis: a study of nuclear factor-kappaB inhibition and gene expression signature. Mol Pharmacol. 2006;69:1615–23.CrossRefPubMedGoogle Scholar
  21. 21.
    Prall OW, Rogan EM, Sutherland RL. Estrogen regulation of cell cycle progression in breast cancer cells. J Steroid Biochem Mol Biol. 1998;65:169–74.CrossRefPubMedGoogle Scholar
  22. 22.
    Clarke R, Leonessa F, Welch JN, Skaar TC. Cellular and molecular pharmacology of antiestrogen action and resistance. Pharmacol Rev. 2001;53:25–71.PubMedGoogle Scholar
  23. 23.
    Rochefort H, Borgna JL. Differences between oestrogen receptor activation by oestrogen and antioestrogen. Nature. 1981;292:257–9.CrossRefPubMedGoogle Scholar
  24. 24.
    Katzenellenbogen BS, Montano MM, Le Goff P, Schodin DJ, Kraus WL, Bhardwaj B, et al. Antiestrogens: mechanisms and actions in target cells. J Steroid Biochem Mol Biol. 1995;53:387–93.CrossRefPubMedGoogle Scholar
  25. 25.
    Brigger I, Chaminade P, Marsaud V, Appel M, Besnard M, Gurny R, et al. Tamoxifen encapsulation within polyethylene glycol-coated nanospheres. A new antiestrogen formulation. Int J Pharm. 2001;214:37–42.CrossRefPubMedGoogle Scholar
  26. 26.
    Chawla JS, Amiji MM. Biodegradable poly(epsilon -caprolactone) nanoparticles for tumor-targeted delivery of tamoxifen. Int J Pharm. 2002;249:127–38.CrossRefPubMedGoogle Scholar
  27. 27.
    Devalapally H, Duan Z, Seiden MV, Amiji MM. Modulation of drug resistance in ovarian adenocarcinoma by enhancing intracellular ceramide using tamoxifen-loaded biodegradable polymeric nanoparticles. Clin Cancer Res. 2008;14:3193–203.CrossRefPubMedGoogle Scholar
  28. 28.
    Shenoy D, Little S, Langer R, Amiji M. Poly(ethylene oxide)-modified poly(beta-amino ester) nanoparticles as a pH-sensitive system for tumor-targeted delivery of hydrophobic drugs: part 2. In vivo distribution and tumor localization studies. Pharm Res. 2005;22:2107–14.CrossRefPubMedGoogle Scholar
  29. 29.
    Memisoglu-Bilensoy E, Vural I, Bochot A, Renoir JM, Duchene D, Hincal AA. Tamoxifen citrate loaded amphiphilic beta-cyclodextrin nanoparticles: in vitro characterization and cytotoxicity. J Control Release. 2005;104:489–96.PubMedGoogle Scholar
  30. 30.
    Hu FX, Neoh KG, Kang ET. Synthesis and in vitro anti-cancer evaluation of tamoxifen-loaded magnetite/PLLA composite nanoparticles. Biomaterials. 2006;27:5725–33.CrossRefPubMedGoogle Scholar
  31. 31.
    Renoir JM, Stella B, Ameller T, Connault E, Opolon P, Marsaud V. Improved anti-tumoral capacity of mixed and pure anti-oestrogens in breast cancer cell xenografts after their administration by entrapment in colloidal nanosystems. J Steroid Biochem Mol Biol. 2006;102:114–27.CrossRefPubMedGoogle Scholar
  32. 32.
    Ameller T, Marsaud V, Legrand P, Gref R, Barratt G, Renoir JM. Polyester-poly(ethylene glycol) nanoparticles loaded with the pure antiestrogen RU 58668: physicochemical and opsonization properties. Pharm Res. 2003;20:1063–70.CrossRefPubMedGoogle Scholar
  33. 33.
    Maillard S, Gauduchon J, Marsaud V, Gouilleux F, Connault E, Opolon P, et al. Improved antitumoral properties of pure antiestrogen RU 58668-loaded liposomes in multiple myeloma. J Steroid Biochem Mol Biol. 2006;100:67–78.CrossRefPubMedGoogle Scholar
  34. 34.
    Bhatia A, Kumar R, Katare OP. Tamoxifen in topical liposomes: development, characterization and in-vitro evaluation. J Pharm Pharm Sci. 2004;7:252–9.PubMedGoogle Scholar
  35. 35.
    Zeisig R, Teppke AD, Behrens D, Fichtner I. Liposomal 4-hydroxy-tamoxifen: effect on cellular uptake and resulting cytotoxicity in drug resistant breast cancer cells in vitro. Breast Cancer Res Treat. 2004;87:245–54.CrossRefPubMedGoogle Scholar
  36. 36.
    Zeisig R, Ruckerl D, Fichtner I. Reduction of tamoxifen resistance in human breast carcinomas by tamoxifen-containing liposomes in vivo. Anticancer Drugs. 2004;15:707–14.CrossRefPubMedGoogle Scholar
  37. 37.
    Daoud-Mahammed S, Couvreur P, Bouchemal K, Cheron M, Lebas G, Amiel C, et al. Cyclodextrin and Polysaccharide-Based Nanogels: Entrapment of Two Hydrophobic Molecules, Benzophenone and Tamoxifen. Biomacromolecules 2009.Google Scholar
  38. 38.
    Lasic DD, Frederik PM, Stuart MC, Barenholz Y, McIntosh TJ. Gelation of liposome interior. A novel method for drug encapsulation. FEBS Lett. 1992;312:255–8.CrossRefPubMedGoogle Scholar
  39. 39.
    Haran G, Cohen R, Bar LK, Barenholz Y. Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochim Biophys Acta. 1993;1151:201–15.CrossRefPubMedGoogle Scholar
  40. 40.
    Harrigan PR, Wong KF, Redelmeier TE, Wheeler JJ, Cullis PR. Accumulation of doxorubicin and other lipophilic amines into large unilamellar vesicles in response to transmembrane pH gradients. Biochim Biophys Acta. 1993;1149:329–38.CrossRefPubMedGoogle Scholar
  41. 41.
    Celano M, Calvagno MG, Bulotta S, Paolino D, Arturi F, Rotiroti D, et al. Cytotoxic effects of gemcitabine-loaded liposomes in human anaplastic thyroid carcinoma cells. BMC Cancer. 2004;4:63.CrossRefPubMedGoogle Scholar
  42. 42.
    Stella B, Arpicco S, Rocco F, Marsaud V, Renoir JM, Cattel L, et al. Encapsulation of gemcitabine lipophilic derivatives into polycyanoacrylate nanospheres and nanocapsules. Int J Pharm. 2007;344:71–7.CrossRefPubMedGoogle Scholar
  43. 43.
    Abraham SA, Edwards K, Karlsson G, Hudon N, Mayer LD, Bally MB. An evaluation of transmembrane ion gradient-mediated encapsulation of topotecan within liposomes. J Control Release. 2004;96:449–61.CrossRefPubMedGoogle Scholar
  44. 44.
    Abraham SA, McKenzie C, Masin D, Ng R, Harasym TO, Mayer LD, et al. In vitro and in vivo characterization of doxorubicin and vincristine coencapsulated within liposomes through use of transition metal ion complexation and pH gradient loading. Clin Cancer Res. 2004;10:728–38.CrossRefPubMedGoogle Scholar
  45. 45.
    Chemin C, Pean JM, Bourgaux C, Pabst G, Wuthrich P, Couvreur P, et al. Supramolecular organization of S12363-liposomes prepared with two different remote loading processes. Biochim Biophys Acta. 2009;1788:926–35.CrossRefPubMedGoogle Scholar
  46. 46.
    Ramsay E, Alnajim J, Anantha M, Zastre J, Yan H, Webb M, et al. A novel liposomal irinotecan formulation with significant anti-tumour activity: use of the divalent cation ionophore A23187 and copper-containing liposomes to improve drug retention. Eur J Pharm Biopharm. 2008;68:607–17.CrossRefPubMedGoogle Scholar
  47. 47.
    Pons M, Gagne D, Nicolas JC, Mehtali M. A new cellular model of response to estrogens: a bioluminescent test to characterize (anti) estrogen molecules. Biotechniques. 1990;9:450–9.PubMedGoogle Scholar
  48. 48.
    Balaguer P, Francois F, Comunale F, Fenet H, Boussioux AM, Pons M, et al. Reporter cell lines to study the estrogenic effects of xenoestrogens. Sci Total Environ. 1999;233:47–56.CrossRefPubMedGoogle Scholar
  49. 49.
    Maillard S, Ameller T, Gauduchon J, Gougelet A, Gouilleux F, Legrand P, et al. Innovative drug delivery nanosystems improve the anti-tumor activity in vitro and in vivo of anti-estrogens in human breast cancer and multiple myeloma. J Steroid Biochem Mol Biol. 2005;94:111–21.CrossRefPubMedGoogle Scholar
  50. 50.
    Marsaud V, Gougelet A, Maillard S, Renoir JM. Various phosphorylation pathways, depending on agonist and antagonist binding to endogenous estrogen receptor alpha (ERalpha), differentially affect ERalpha extractability, proteasome-mediated stability, and transcriptional activity in human breast cancer cells. Mol Endocrinol. 2003;17:2013–27.CrossRefPubMedGoogle Scholar
  51. 51.
    Moghimi SM, Szebeni J. Stealth liposomes and long circulating nanoparticles: critical issues in pharmacokinetics, opsonization and protein-binding properties. Prog Lipid Res. 2003;42:463–78.CrossRefPubMedGoogle Scholar
  52. 52.
    Allen TM, Cullis PR. Drug delivery systems: entering the mainstream. Science. 2004;303:1818–22.CrossRefPubMedGoogle Scholar
  53. 53.
    Watts CK, Brady A, Sarcevic B, deFazio A, Musgrove EA, Sutherland RL. Antiestrogen inhibition of cell cycle progression in breast cancer cells in associated with inhibition of cyclin-dependent kinase activity and decreased retinoblastoma protein phosphorylation. Mol Endocrinol. 1995;9:1804–13.CrossRefPubMedGoogle Scholar
  54. 54.
    Cariou S, Donovan JC, Flanagan WM, Milic A, Bhattacharya N, Slingerland JM. Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells. Proc Natl Acad Sci USA. 2000;97:9042–6.CrossRefPubMedGoogle Scholar
  55. 55.
    Otsuki T, Yamada O, Kurebayashi J, Moriya T, Sakaguchi H, Kunisue H, et al. Estrogen receptors in human myeloma cells. Cancer Res. 2000;60:1434–41.PubMedGoogle Scholar
  56. 56.
    Berman E, Adams M, Duigou-Osterndorf R, Godfrey L, Clarkson B, Andreeff M. Effect of tamoxifen on cell lines displaying the multidrug-resistant phenotype. Blood. 1991;77:818–25.PubMedGoogle Scholar
  57. 57.
    Trump DL, Smith DC, Ellis PG, Rogers MP, Schold SC, Winer EP, et al. High-dose oral tamoxifen, a potential multidrug-resistance-reversal agent: phase I trial in combination with vinblastine. J Natl Cancer Inst. 1992;84:1811–6.CrossRefPubMedGoogle Scholar
  58. 58.
    Demoy M, Gibaud S, Andreux JP, Weingarten C, Gouritin B, Couvreur P. Splenic trapping of nanoparticles: complementary approaches for in situ studies. Pharm Res. 1997;14:463–8.CrossRefPubMedGoogle Scholar
  59. 59.
    Osborne CK. Tamoxifen in the treatment of breast cancer. N Engl J Med. 1998;339:1609–18.CrossRefPubMedGoogle Scholar
  60. 60.
    Bhatia A, Bhushan S, Singh B, Katare OP. Studies on Tamoxifen Encapsulated in Lipid Vesicles: Effect on the Growth of Human Breast Cancer MCF-7 Cells. J Liposome Res. 2008;1-6.Google Scholar
  61. 61.
    Wiseman H, Quinn P, Halliwell B. Tamoxifen and related compounds decrease membrane fluidity in liposomes. Mechanism for the antioxidant action of tamoxifen and relevance to its anticancer and cardioprotective actions? FEBS Lett. 1993;330:53–6.CrossRefPubMedGoogle Scholar
  62. 62.
    Chauhan D, Catley L, Hideshima T, Li G, Leblanc R, Gupta D, et al. 2-Methoxyestradiol overcomes drug resistance in multiple myeloma cells. Blood. 2002;100:2187–94.CrossRefPubMedGoogle Scholar
  63. 63.
    Cuendet M, Christov K, Lantvit DD, Deng Y, Hedayat S, Helson L, et al. Multiple myeloma regression mediated by bruceantin. Clin Cancer Res. 2004;10:1170–9.CrossRefPubMedGoogle Scholar
  64. 64.
    Hideshima T, Anderson KC. Molecular mechanisms of novel therapeutic approaches for multiple myeloma. Nat Rev Cancer. 2002;2:927–37.CrossRefPubMedGoogle Scholar
  65. 65.
    Garvin S, Dabrosin C. Tamoxifen inhibits secretion of vascular endothelial growth factor in breast cancer in vivo. Cancer Res. 2003;63:8742–8.PubMedGoogle Scholar
  66. 66.
    Bouclier C, Marsaud V, Bawa O, Nicolas V, Moine L, Opolon P, et al. Coadministration of nanosystems of short silencing RNAs targeting oestrogen receptor alpha and anti-oestrogen synergistically induces tumour growth inhibition in human breast cancer xenografts. Breast Cancer Res Treat 2009.Google Scholar
  67. 67.
    Podar K, Anderson KC. The pathophysiologic role of VEGF in hematologic malignancies: therapeutic implications. Blood. 2005;105:1383–95.CrossRefPubMedGoogle Scholar
  68. 68.
    Barlogie B, Shaughnessy J, Tricot G, Jacobson J, Zangari M, Anaissie E, et al. Treatment of multiple myeloma. Blood. 2004;103:20–32.CrossRefPubMedGoogle Scholar
  69. 69.
    Obeng EA, Carlson LM, Gutman DM, Harrington WJ Jr, Lee KP, Boise LH. Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells. Blood. 2006;107:4907–16.CrossRefPubMedGoogle Scholar
  70. 70.
    Fribley A, Wang CY. Proteasome inhibitor induces apoptosis through induction of endoplasmic reticulum stress. Cancer Biol Ther. 2006;5:745–8.PubMedGoogle Scholar
  71. 71.
    Doisneau-Sixou SF, Sergio CM, Carroll JS, Hui R, Musgrove EA, Sutherland RL. Estrogen and antiestrogen regulation of cell cycle progression in breast cancer cells. Endocr Relat Cancer. 2003;10:179–86.CrossRefPubMedGoogle Scholar
  72. 72.
    Caldon CE, Daly RJ, Sutherland RL, Musgrove EA. Cell cycle control in breast cancer cells. J Cell Biochem. 2006;97:261–74.CrossRefPubMedGoogle Scholar
  73. 73.
    Bursch W, Ellinger A, Kienzl H, Torok L, Pandey S, Sikorska M, et al. Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy. Carcinogenesis. 1996;17:1595–607.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Giorgia Urbinati
    • 1
    • 2
    • 3
  • Davide Audisio
    • 2
    • 3
    • 4
  • Véronique Marsaud
    • 1
    • 2
    • 3
  • Vincent Plassat
    • 1
    • 2
    • 3
  • Silvia Arpicco
    • 5
  • Brigitte Sola
    • 6
  • Elias Fattal
    • 1
    • 2
    • 3
    • 7
  • Jack-Michel Renoir
    • 1
    • 2
    • 3
  1. 1.CNRS, UMR 8612, Physico-Chimie, Pharmacotechnie, Biopharmacie, Laboratoire Pharmacologie Cellulaire et Moléculaire des Anticancéreux, Faculté de PharmacieChâtenay-MalabryFrance
  2. 2.University Paris-SudOrsayFrance
  3. 3.IFR 141Châtenay-MalabryFrance
  4. 4.CNRS, BIOCIS-UMR 8076Laboratoire de Chimie ThérapeutiqueChâtenay-MalabryFrance
  5. 5.Facoltà di FarmaciaUniversità degli studi di TorinoTorinoItaly
  6. 6.Biologie Moléculaire et Cellulaire de la Signalisation, EA 3919, IFR186Université de Caen Basse NormandieCaenFrance
  7. 7.CNRS, UMR 8612Laboratoire de Vectorisation Pharmaceutique de Molécules fragilesChâtenay-MalabryFrance

Personalised recommendations