Skip to main content

Advertisement

SpringerLink
Log in

Riboflavin-Targeted Polymer Conjugates for Breast Tumor Delivery

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

In breast cancer, a significant decrease in riboflavin (RF) serum levels and increase in RF carrier protein occurs, indicating a potential role of RF in disease progression. To evaluate RF’s ability to serve as a targeting agent, mitomycin C (MMC)-conjugated N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesized and targeted to the RF internalization pathway in human breast cancer cells.

Methods

Competitive uptake studies were used to determine specificity of RF-targeted conjugates, and an MTT assay established the IC50 for the conjugates. Endocytic mechanisms were investigated by confocal microscopy.

Results

Studies revealed a high-affinity endocytic mechanism for RF-specific internalization of fluorescently-labeled conjugates in both MCF-7 and SKBR-3 cells, whereas folic acid-mediated endocytosis showed high specificity only in SKBR-3 cells. MMC internalization was significantly higher following nontargeted and RF-targeted MMC-conjugate administration compared to that of free MMC. Cytotoxic analysis illustrated potent IC50 values for RF-targeted MMC conjugates similar to free MMC. Maximum nuclear accumulation of MMC resulted from lysosomal release from RF-targeted and nontargeted MMC-conjugates following 6 h incubations, unlike that of free MMC seen within 10 min.

Conclusion

Targeting polymer-MMC conjugates to the RF internalization pathway in breast cancer cells enabled an increase in MMC uptake and nuclear localization, resulting in potent cytotoxic activity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

DIPEA:

Diisopropyl ethylamine

DMEM:

Dulbecco’s modified eagle medium

DMF:

Dimethylformamide

DMSO:

Dimethyl sulfoxide

DPBS:

Dulbecco’s phosphate buffered saline

EDTA:

Ethylenediaminetetraacetic acid

FA:

Folic acid

FITC:

Fluorescein isothiocyanate

HBSS:

Hank’s balanced salt solution

HPMA:

N-(2-hydroxypropyl)methacrylamide

LAMP-1:

Lysosomal-associated membrane protein 1

MMC:

Mitomycin C

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

RCP:

Riboflavin carrier protein

RF:

Riboflavin

RME:

Receptor-mediated endocytosis

TFA:

Trifluoroacetic acid

THF:

Tetrahydrofuran

References

  1. Andersson L, Davies J, Duncan R, Ferruti P, Ford J, Kneller S, et al. Poly(ethylene glycol)-poly(ester-carbonate) block copolymers carrying PEG-peptidyl-doxorubicin pendant side chains: synthesis and evaluation as anticancer conjugates. Biomacromolecules. 2005;6:914–26.

    Article  PubMed  CAS  Google Scholar 

  2. Duncan R. The dawning era of polymer therapeutics. Nat Rev Drug Discov. 2003;2:347–60.

    Article  PubMed  CAS  Google Scholar 

  3. Tanaka T, Shiramoto S, Miyashita M, Fujishima Y, Kaneo Y. Tumor targeting based on the effect of enhanced permeability and retention (EPR) and the mechanism of receptor-mediated endocytosis (RME). Int J Pharm. 2004;277:39–61.

    Article  PubMed  CAS  Google Scholar 

  4. Mamot C, Drummond DC, Hong K, Kirpotin DB, Park JW. Liposome-based approaches to overcome anticancer drug resistance. Drug Resist Updat. 2003;6:271–9.

    Article  PubMed  CAS  Google Scholar 

  5. Reddy JA, Westrick E, Vlahov I, Howard SJ, Santhapuram HK, Leamon CP. Folate receptor specific anti-tumor activity of folate-mitomycin conjugates. Cancer Chemother Pharmacol. 2006;58:229–36.

    Article  PubMed  CAS  Google Scholar 

  6. Duncan R. Designing polymer conjugates as lysosomotropic nanomedicines. Biochem Soc Trans. 2007;35:56–60.

    Article  PubMed  CAS  Google Scholar 

  7. Russell-Jones G, McTavish K, McEwan J, Rice J, Nowotnik D. Vitamin-mediated targeting as a potential mechanism to increase drug uptake by tumours. J Inorg Biochem. 2004;98:1625–33.

    Article  PubMed  CAS  Google Scholar 

  8. Bareford LM, Swaan PW. Endocytic mechanisms for targeted drug delivery. Adv Drug Deliv Rev. 2007;59:748–58.

    Article  PubMed  CAS  Google Scholar 

  9. Parker N, Turk MJ, Westrick E, Lewis JD, Low PS, Leamon CP. Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Anal Biochem. 2005;338:284–93.

    Article  PubMed  CAS  Google Scholar 

  10. Garnett MC. Targeted drug conjugates: principles and progress. Adv Drug Deliv Rev. 2001;53:171–216.

    Article  PubMed  CAS  Google Scholar 

  11. 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  PubMed  CAS  Google Scholar 

  12. Paulos CM, Reddy JA, Leamon CP, Turk MJ, Low PS. Ligand binding and kinetics of folate receptor recycling in vivo: impact on receptor-mediated drug delivery. Mol Pharmacol. 2004;66:1406–14.

    Article  PubMed  CAS  Google Scholar 

  13. Salazarand MD, Ratnam M. The folate receptor: what does it promise in tissue-targeted therapeutics? Cancer Metastasis Rev. 2007;26:141–52.

    Article  Google Scholar 

  14. Castrellonand AB, Gluck S. Chemoprevention of breast cancer. Expert Rev Anticancer Ther. 2008;8:443–52.

    Article  Google Scholar 

  15. Karande AA, Sridhar L, Gopinath KS, Adiga PR. Riboflavin carrier protein: a serum and tissue marker for breast carcinoma. Int J Cancer. 2001;95:277–81.

    Article  PubMed  CAS  Google Scholar 

  16. Vaidya SM, Kamlakar PL, Kamble SM. Molybdenum, xanthine oxidase and riboflavin levels in tamoxifen treated postmenopausal women with breast cancer. Indian J Med Sci. 1998;52:244–7.

    PubMed  CAS  Google Scholar 

  17. Mason CW, D’Souza VM, Bareford LM, Phelps MA, Ray A, Swaan PW. Recognition, co-internalization, and recycling of an avian riboflavin carrier protein in human placental trophoblasts. J Pharmacol Exp Ther. 2006;317:465–72.

    Article  PubMed  CAS  Google Scholar 

  18. Yonezawa A, Masuda S, Katsura T, Inui KI. Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1. Am J Physiol Cell Physiol. 2008;295:C632–41.

    Google Scholar 

  19. Huangand SN, Swaan PW. Involvement of a receptor-mediated component in cellular translocation of riboflavin. J Pharmacol Exp Ther. 2000;294:117–25.

    Google Scholar 

  20. Bareford LM, Phelps MA, Foraker AB, Swaan PW. Intracellular processing of riboflavin in human breast cancer cells. Mol Pharm. 2008;5:839–48.

    Article  PubMed  CAS  Google Scholar 

  21. D’Souza VM, Foraker AB, Free RB, Ray A, Shapiro PS, Swaan PW. cAMP-Coupled riboflavin trafficking in placental trophoblasts: a dynamic and ordered process. Biochemistry. 2006;45:6095–104.

    Article  PubMed  Google Scholar 

  22. Kopecek J, Kopeckova P, Minko T, Lu Z. HPMA copolymer-anticancer drug conjugates: design, activity, and mechanism of action. Eur J Pharm Biopharm. 2000;50:61–81.

    Article  PubMed  CAS  Google Scholar 

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

    Article  Google Scholar 

  24. Rihovaand B, Kubackova K. Clinical implications of N-(2-hydroxypropyl)methacrylamide copolymers. Curr Pharm Biotechnol. 2003;4:311–22.

    Article  Google Scholar 

  25. Bradner WT. Mitomycin C: a clinical update. Cancer Treat Rev. 2001;27:35–50.

    Article  PubMed  CAS  Google Scholar 

  26. Strohalmand J, Kopecek J. Poly N-(2-hydroxypropyl) methacrylamide. 4. Heterogeneous polymerization. Makromol Chem. 1978;70:109–18.

    Google Scholar 

  27. Kopeček J, Rejmanová P, Strohalm J, Ulbrich K, Říhová B, Chytrý V, Lloyd JB, Duncan R. Synthetic polymeric drugs. US Patent. 5, 037, 883. Aug 6, 1991. p. 883.

  28. Rejmanova P, Labsky J, Kopecek J. Aminolyses of monomeric and polymeric p-nitrophenyl esters of methacryloylated amino acids. Makromol Chem. 1977;178:2159–68.

    Article  CAS  Google Scholar 

  29. Omelyanenko V, Kopeckova P, Gentry C, Kopecek J. Targetable HPMA copolymer-adriamycin conjugates. Recognition, internalization, and subcellular fate. J Control Release. 1998;53:25–37.

    Article  PubMed  CAS  Google Scholar 

  30. Mitra A, Nan A, Ghandehari H, McNeill E, Mulholland J, Line BR. Technetium-99m-Labeled N-(2-hydroxypropyl) methacrylamide copolymers: synthesis, characterization, and in vivo biodistribution. Pharm Res. 2004;21:1153–9.

    Article  PubMed  CAS  Google Scholar 

  31. Luo J, Smith MD, Lantrip DA, Wang S, Fuchs PL. Efficient syntheses of pyrofolic acid and pteroyl azide, reagents for the production of carboxyl-differentiated derivatives of folic acid. J Am Chem Soc. 1997;119:10004–13.

    Article  CAS  Google Scholar 

  32. Phelps MA, Foraker AB, Gao W, Dalton JT, Swaan PW. A novel rhodamine-riboflavin conjugate probe exhibits distinct fluorescence resonance energy transfer that enables riboflavin trafficking and subcellular localization studies. Mol Pharm. 2004;1:257–66.

    Article  PubMed  CAS  Google Scholar 

  33. Apodaca G, Katz LA, Mostov KE. Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes. J Cell Biol. 1994;125:67–86.

    Article  PubMed  CAS  Google Scholar 

  34. Demarre A, Soyez H, Schacht E, Shoaibi MA, Seymour LW, Rihova B. Synthesis and evaluation of macromolecular prodrugs of Mitomycin-C. J Control Release. 1995;36:87–97.

    Article  Google Scholar 

  35. Pan SS, Iracki T, Bachur NR. DNA alkylation by enzyme-activated mitomycin C. Mol Pharmacol. 1986;29:622–8.

    PubMed  CAS  Google Scholar 

  36. D’Souza VM, Bareford LM, Ray A, Swaan PW. Cytoskeletal scaffolds regulate riboflavin endocytosis and recycling in placental trophoblasts. J Nutr Biochem. 2006;17:821–9.

    Article  PubMed  Google Scholar 

  37. Foraker AB, Ray A, Da Silva TC, Bareford LM, Hillgren KM, Schmittgen TD, et al. Dynamin 2 regulates riboflavin endocytosis in human placental trophoblasts. Mol Pharmacol. 2007;72:553–62.

    Article  PubMed  CAS  Google Scholar 

  38. Guoand W, Lee RL. Receptor-targeted gene delivery via folate-conjugated polyethylenimine. AAPS Pharm Sci. 1999;1:E19.

    Google Scholar 

  39. Rachmilewitz B, Sulkes A, Rachmilewitz M, Fuks Z. Serum transcobalamin II levels in breast carcinoma patients. Isr J Med Sci. 1981;17:874–8.

    PubMed  CAS  Google Scholar 

  40. Auvinen P, Tammi R, Parkkinen J, Tammi M, Agren U, Johansson R, et al. Hyaluronan in peritumoral stroma and malignant cells associates with breast cancer spreading and predicts survival. Am J Pathol. 2000;156:529–36.

    Article  PubMed  CAS  Google Scholar 

  41. Nichifor M, Schacht EH, Seymour LW. Macromolecular prodrugs of 5-fluorouracil .2. Enzymatic degradation. J Control Release. 1996;39:79–92.

    Article  CAS  Google Scholar 

  42. Hoste K, De Winne K, Schacht E. Polymeric prodrugs. Int J Pharm. 2004;277:119–31.

    Article  PubMed  CAS  Google Scholar 

  43. Winski SL, Swann E, Hargreaves RH, Dehn DL, Butler J, Moody CJ, et al. Relationship between NAD(P)H:quinone oxidoreductase 1 (NQO1) levels in a series of stably transfected cell lines and susceptibility to antitumor quinones. Biochem Pharmacol. 2001;61:1509–16.

    Article  PubMed  CAS  Google Scholar 

  44. Cheung RY, Rauth AM, Ronaldson PT, Bendayan R, Wu XY. In vitro toxicity to breast cancer cells of microsphere-delivered mitomycin C and its combination with doxorubicin. Eur J Pharm Biopharm. 2006;62:321–31.

    Article  PubMed  CAS  Google Scholar 

  45. Chen R, Yue Z, Eccleston ME, Williams S, Slater NK. Modulation of cell membrane disruption by pH-responsive pseudo-peptides through grafting with hydrophilic side chains. J Control Release. 2005;108:63–72.

    Article  PubMed  CAS  Google Scholar 

  46. Mukherjee S, Ghosh RN, Maxfield FR. Endocytosis. Physiol Rev. 1997;77:759–803.

    PubMed  CAS  Google Scholar 

  47. Maalmi M, Strieder W, Varma A. Ligand diffusion and receptor mediated internalization: Michaelis-Menten kinetics. Chem Eng Sci. 2001;56:5609–16.

    Article  CAS  Google Scholar 

  48. King AC, Hernaez-Davis L, Cuatrecasas P. Lysomotropic amines cause intracellular accumulation of receptors for epidermal growth factor. Proc Natl Acad Sci U S A. 1980;77:3283–7.

    Article  PubMed  CAS  Google Scholar 

  49. Watts PL, Plumb JA, Courtney JM, Scott R. Sensitivity of cell lines to mitomycin C. Br J Urol. 1996;77:363–6.

    Article  PubMed  CAS  Google Scholar 

  50. Mahoney BP, Raghunand N, Baggett B, Gillies RJ. Tumor acidity, ion trapping and chemotherapeutics. I. Acid pH affects the distribution of chemotherapeutic agents in vitro. Biochem Pharmacol. 2003;66:1207–18.

    Article  PubMed  CAS  Google Scholar 

  51. Nomura T, Saikawa A, Morita S, Sakaeda Kakutani T, Yamashita F, Honda K, et al. Pharmacokinetic characteristics and therapeutic effects of mitomycin C-dextran conjugates after intratumoural injection. J Control Release. 1998;52:239–52.

    Article  PubMed  Google Scholar 

  52. David A, Kopeckova P, Minko T, Rubinstein A, Kopecek J. Design of a multivalent galactoside ligand for selective targeting of HPMA copolymer-doxorubicin conjugates to human colon cancer cells. Eur J Cancer. 2004;40:148–57.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Hamidreza Ghandehari

    Present address: Department of Pharmaceutics & Pharmaceutical Chemistry & of Bioengineering, Utah Center for Nanomedicine, Nano Institute of Utah, University of Utah, Salt Lake City, Utah, 84108, USA

Authors and Affiliations

  1. Department of Pharmaceutical Sciences Center for Nanomedicine and Cellular Drug Delivery, University of Maryland Baltimore, 20 Penn Street, Health Sciences Facility 2, Room 543, Baltimore, Maryland, 21201, USA

    Lisa M. Bareford, Brittany R. Avaritt, Hamidreza Ghandehari, Anjan Nan & Peter W. Swaan

Authors
  1. Lisa M. Bareford
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Brittany R. Avaritt
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hamidreza Ghandehari
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anjan Nan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter W. Swaan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peter W. Swaan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bareford, L.M., Avaritt, B.R., Ghandehari, H. et al. Riboflavin-Targeted Polymer Conjugates for Breast Tumor Delivery. Pharm Res 30, 1799–1812 (2013). https://doi.org/10.1007/s11095-013-1024-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-013-1024-5

KEY WORDS

Search

Navigation