Advertisement

Pharmaceutical Research

, Volume 31, Issue 11, pp 3106–3119 | Cite as

Evaluating the Anticancer Properties of Liposomal Copper in a Nude Xenograft Mouse Model of Human Prostate Cancer: Formulation, In Vitro, In Vivo, Histology and Tissue Distribution Studies

  • Yan Wang
  • San Zeng
  • Tien-Min Lin
  • Lisa Krugner-Higby
  • Doug Lyman
  • Dana Steffen
  • May P. XiongEmail author
Research Paper

ABSTRACT

Purpose

Although Cu complexes have been investigated as anticancer agents, there has been no description of Cu itself as a cancer killing agent. A stealth liposomal Cu formulation (LpCu) was studied in vitro and in vivo.

Methods

LpCu was evaluated in prostate cancer origin PC-3 cells by a metabolic cytotoxicity assay, by monitoring ROS, and by flow cytometry. LpCu efficacy was evaluated in vivo using intratumoral and intravenous injections into mice bearing PC-3 xenograft tumors. Toxicology was assessed by performing hematological and blood biochemistry assays, and tissue histology and Cu distribution was investigated by elemental analysis.

Results

LpCu and free Cu salts displayed similar levels of cell metabolic toxicity and ROS. Flow cytometry indicated that the mechanisms of cell death were both apoptosis and necrosis. Animals injected i.t. with 3.5 mg/kg or i.v. with 3.5 and 7.0 mg/kg LpCu exhibited significant tumor growth inhibition. Kidney and eye were the main organs affected by Cu-mediated toxicities, but spleen and liver were the major organs of Cu deposition.

Conclusions

LpCu was effective at reducing tumor burden in the xenograft prostate cancer model. There was histological evidence of Cu toxicity in kidneys and eyes of animals treated at the maximum tolerated dose of LpCu 7.0 mg/kg.

KEY WORDS

liposomes copper reactive oxygen species cancer therapy toxicity 

ABBREVIATIONS

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

BCA

Bicinchoninic acid

Cr

Creatinine

Cu

Copper

DF

Dilution factor

DLS

Dynamic light scattering

DMEM

Dulbecco’s modified eagle medium

DPBS

Dulbecco’s phosphate buffered saline

EPR

Enhanced permeability and retention

FBS

Fetal bovine serum

HBSS

Hank’s balanced salt solution

HEPES

2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer

i.t.

Intratumoral

i.v.

Intravenous

LpCu

Stealth formulation of liposomal Cu

LUV

Large unilamellar vesicle

MLV

Multilamellar vesicle

MTD

Maximum tolerated dose

PBS

Phosphate buffered saline

PDI

Polydispersity index

ROS

Reactive oxygen species

TBHP

Tert-butyl hydroperoxide

WVDL

Wisconsin veterinary diagnostic laboratory

Notes

ACKNOWLEDGMENTS

This research was supported by NIH grants R00CA136970 and R01DK099596, and startup funds from the University of Wisconsin-Madison, School of Pharmacy. We are grateful to Professor Manish Patankar and Dr. Arvinder Kapur (Dept. of Ob/Gyn, University of Wisconsin-Madison), and the UWCCC Flow Cytometry Laboratory for invaluable assistance with all the flow cytometry assays. We would also like to thank the WVDL Chemistry Section for performing all the elemental tissue analyses.

Supplementary material

11095_2014_1403_MOESM1_ESM.docx (8.5 mb)
ESM 1 (DOCX 8748 kb)

REFERENCES

  1. 1.
    D’Autreauxand B, Toledano MB. ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat Rev Mol Cell Biol. 2007;8:813–24.CrossRefGoogle Scholar
  2. 2.
    Krause KH. Aging: a revisited theory based on free radicals generated by NOX family NADPH oxidases. Exp Gerontol. 2007;42:256–62.PubMedCrossRefGoogle Scholar
  3. 3.
    Alfaddaand AA, Sallam RM. Reactive oxygen species in health and disease. J Biomed Biotechnol. 2012;2012:936486.Google Scholar
  4. 4.
    Halliwelland B, Gutteridge JM. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol. 1990;186:1–85.CrossRefGoogle Scholar
  5. 5.
    Imlay JA, Chin SM, Linn S. Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science. 1988;240:640–2.PubMedCrossRefGoogle Scholar
  6. 6.
    Lu W, Ogasawara MA, Huang P. Models of reactive oxygen species in cancer. Drug Disc Today Dis Model. 2007;4:67–73.CrossRefGoogle Scholar
  7. 7.
    Wang Y, Hodgkinson V, Zhu S, Weisman GA, Petris MJ. Advances in the understanding of mammalian copper transporters. Adv Nutr. 2011;2:129–37.PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Allenand TM, Cullis PR. Drug delivery systems: entering the mainstream. Science. 2004;303:1818–22.CrossRefGoogle Scholar
  9. 9.
    Fanciullinoand R, Ciccolini J. Liposome-encapsulated anticancer drugs: still waiting for the magic bullet? Curr Med Chem. 2009;16:4361–71.CrossRefGoogle Scholar
  10. 10.
    Kawahara K, Sekiguchi A, Kiyoki E, Ueda T, Shimamura K, Kurosaki Y, et al. Effect of TRX-liposomes size on their prolonged circulation in rats. Chem Pharm Bull. 2003;51:336–8.PubMedCrossRefGoogle Scholar
  11. 11.
    Wisse E, Jacobs F, Topal B, Frederik P, De Geest B. The size of endothelial fenestrae in human liver sinusoids: implications for hepatocyte-directed gene transfer. Gene Ther. 2008;15:1193–9.PubMedCrossRefGoogle Scholar
  12. 12.
    Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release. 2000;65:271–84.PubMedCrossRefGoogle Scholar
  13. 13.
    Wangand T, Guo Z. Copper in medicine: homeostasis, chelation therapy and antitumor drug design. Curr Med Chem. 2006;13:525–37.CrossRefGoogle Scholar
  14. 14.
    Marzano C, Pellei M, Tisato F, Santini C. Copper complexes as anticancer agents. Anticancer Agents Med Chem. 2009;9:185–211.PubMedCrossRefGoogle Scholar
  15. 15.
    Barve V, Ahmed F, Adsule S, Banerjee S, Kulkarni S, Katiyar P, et al. Synthesis, molecular characterization, and biological activity of novel synthetic derivatives of chromen-4-one in human cancer cells. J Med Chem. 2006;49:3800–8.PubMedCrossRefGoogle Scholar
  16. 16.
    Filomeni G, Cerchiaro G, Da Costa Ferreira AM, De Martino A, Pedersen JZ, Rotilio G, et al. Pro-apoptotic activity of novel Isatin-Schiff base copper(II) complexes depends on oxidative stress induction and organelle-selective damage. J Biol Chem. 2007;282:12010–21.PubMedCrossRefGoogle Scholar
  17. 17.
    Awasthi VD, Garcia D, Goins BA, Phillips WT. Circulation and biodistribution profiles of long-circulating PEG-liposomes of various sizes in rabbits. Int J Pharm. 2003;253:121–32.PubMedCrossRefGoogle Scholar
  18. 18.
    Brennerand AJ, Harris ED. A quantitative test for copper using bicinchoninic acid. Anal Biochem. 1995;226:80–4.CrossRefGoogle Scholar
  19. 19.
    O’Brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem. 2000;267:5421–6.PubMedCrossRefGoogle Scholar
  20. 20.
    Wangand H, Joseph JA. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med. 1999;27:612–6.CrossRefGoogle Scholar
  21. 21.
    Perkins WR, Minchey SR, Ahl PL, Janoff AS. The determination of liposome captured volume. Chem Phys Lipids. 1993;64:197–217.PubMedCrossRefGoogle Scholar
  22. 22.
    Litzingerand DC, Huang L. Amphipathic poly(ethylene glycol) 5000-stabilized dioleoylphosphatidylethanolamine liposomes accumulate in spleen. Biochim et Biophys Acta (BBA) Lipids Lipid Metab. 1992;1127:249–54.CrossRefGoogle Scholar
  23. 23.
    Gabizon A, Goren D, Horowitz AT, Tzemach D, Lossos A, Siegal T. Long-circulating liposomes for drug delivery in cancer therapy: a review of biodistribution studies in tumor-bearing animals. Adv Drug Deliv Rev. 1997;24:337–44.CrossRefGoogle Scholar
  24. 24.
    Allen TM, Hansen C, Rutledge J. Liposomes with prolonged circulation times: factors affecting uptake by reticuloendothelial and other tissues. Biochim Biophys Acta. 1989;981:27–35.PubMedCrossRefGoogle Scholar
  25. 25.
    Chugh KS, Sharma BK, Singhal PC, Das KC, Datta BN. Acute renal failure following copper sulphate intoxication. Postgrad Med J. 1977;53:18–23.PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Oldenquistand G, Salem M. Parenteral copper sulfate poisoning causing acute renal failure. Nephrol Dial Transpl. 1999;14:441–3.CrossRefGoogle Scholar
  27. 27.
    Dayan MR, Cottrell DG, Mitchell KW. Reversible retinal toxicity associated with retained intravitreal copper foreign body in the absence of intraocular inflammation. Acta Ophthalmol Scand. 1999;77:597–8.PubMedCrossRefGoogle Scholar
  28. 28.
    Gaetkeand LM, Chow CK. Copper toxicity, oxidative stress, and antioxidant nutrients. Toxicology. 2003;189:147–63.CrossRefGoogle Scholar
  29. 29.
    Galhardi CM, Diniz YS, Faine LA, Rodrigues HG, Burneiko RC, Ribas BO, et al. Toxicity of copper intake: lipid profile, oxidative stress and susceptibility to renal dysfunction. Food Chem Toxicol. 2004;42:2053–60.PubMedCrossRefGoogle Scholar
  30. 30.
    Dos Santos N, Allen C, Doppen AM, Anantha M, Cox KA, Gallagher RC, et al. Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: relating plasma circulation lifetimes to protein binding. Biochim Biophys Acta. 2007;1768:1367–77.PubMedCrossRefGoogle Scholar
  31. 31.
    Tseng LP, Liang HJ, Chung TW, Huang YY, Liu DZ. Liposomes incorporated with cholesterol for drug release triggered by magnetic field. J Med Biol Eng. 2007;27:29–34.Google Scholar
  32. 32.
    Düzgüneşand N, Nir S. Mechanisms and kinetics of liposome–cell interactions. Adv Drug Deliv Rev. 1999;40:3–18.CrossRefGoogle Scholar
  33. 33.
    Wang YF, Hodgkinson V, Zhu S, Weisman GA, Petris MJ. Advances in the understanding of mammalian copper transporters. Adv Nutr. 2011;2:129–37.PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Petris MJ, Smith K, Lee J, Thiele DJ. Copper-stimulated endocytosis and degradation of the human copper transporter, hCtr1. J Biol Chem. 2003;278:9639–46.PubMedCrossRefGoogle Scholar
  35. 35.
    White C, Kambe T, Fulcher YG, Sachdev SW, Bush AI, Fritsche K, et al. Copper transport into the secretory pathway is regulated by oxygen in macrophages. J Cell Sci. 2009;122:1315–21.PubMedCrossRefPubMedCentralGoogle Scholar
  36. 36.
    Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Itty Ipe B, et al. Renal clearance of quantum dots. Nat Biotechnol. 2007;25:1165–70.PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    Andrews NC. Disorders of iron metabolism. N Engl J Med. 1999;341:1986–95.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Yan Wang
    • 1
  • San Zeng
    • 1
  • Tien-Min Lin
    • 1
  • Lisa Krugner-Higby
    • 2
  • Doug Lyman
    • 3
  • Dana Steffen
    • 1
  • May P. Xiong
    • 1
    Email author
  1. 1.School of PharmacyUniversity of Wisconsin – MadisonMadisonUnited State of America
  2. 2.Research Animal Resources CenterUniversity of Wisconsin-MadisonMadisonUnited State of America
  3. 3.Wisconsin Veterinary Diagnostic LaboratoryMadisonUnited State of America

Personalised recommendations