Pharmaceutical Research

, Volume 31, Issue 5, pp 1194–1209 | Cite as

Influence of Short-Chain Cell-Penetrating Peptides on Transport of Doxorubicin Encapsulating Receptor-Targeted Liposomes Across Brain Endothelial Barrier

  • Gitanjali Sharma
  • Amit Modgil
  • Tiecheng Zhong
  • Chengwen Sun
  • Jagdish Singh
Research Paper

Abstract

Purpose

To investigate the influence of different cell penetrating peptides (CPPs-TAT, Penetratin and Mastoparan), on the transport of doxorubicin encapsulating transferrin (Tf)-liposomes across brain endothelial barrier, in vitro and in vivo.

Methods

The cellular uptake of dual-functionalized, (Tf-CPP), liposomes into various tumor cells was assessed using HPLC. The transport of liposomes was also measured across a robust 3D brain tumor model constructed using chitosan-PLGA scaffolds. The growth of tumor cells was monitored using H&E staining and the fully grown tumor scaffolds were visualized using SEM. The tumor scaffolds were combined with the culture inserts carrying tightly packed brain endothelial cells. The in vitro and in vivo transport of drug (using Tf-CPP-liposomes) across the brain endothelial barrier was determined by extraction of the drug from cells and tissues followed by analysis using HPLC.

Results

The results demonstrated improved delivery of doxorubicin using dual-functionalized liposomes versus the single ligand or unmodified liposomes. Among different Tf-CPP-liposomes, the Tf-Penetratin liposomes showed efficient cellular transport of the encapsulated drug (approximately 90–98%) and maximum translocation of the drug across the brain endothelial barrier (approximately 15% across in vitro and 4% across in vivo BBB). The Tf-Penetratin and Tf-TAT liposomes demonstrated excellent cellular biocompatibility and no hemolytic activity upto 200nM phospholipid concentration.

Conclusions

The Tf-CPP liposomes showed efficient translocation of the anticancer drug across the brain endothelial barrier. In addition, an absolute and robust in vitro brain tumor model was successfully constructed to overcome the practical intricacies of developing a successful in vivo orthotopic brain tumor model.

KEY WORDS

blood brain barrier cell penetrating peptides dual-functionalized liposomes tumor 

Supplementary material

11095_2013_1242_MOESM1_ESM.docx (1.4 mb)
ESM 1(DOCX 1.37 mb)

References

  1. 1.
    Koren E, Torchilin VP. Cell-penetrating peptides: breaking through to the other side. Trends Mol Med. 2012;18:385–93.PubMedCrossRefGoogle Scholar
  2. 2.
    Gupta B, Levchenko TS, Torchilin VP. Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides. Adv Drug Deliv Rev. 2005;57:637–51.PubMedCrossRefGoogle Scholar
  3. 3.
    Rooy IV, Mastrobattista E, Storm G, Hennink WE, Schiffelers RM. Comparison of five different targeting ligands to enhance accumulation of liposomes into the brain. J Control Release. 2011;150:30–6.PubMedCrossRefGoogle Scholar
  4. 4.
    Emerich DF, Snodgrass P, Pink M, Bloom F, Bartus RT. Central analgesic actions of loperamide following transient permeation of the blood–brain barrier with Cereport (RMP-7). Brain Res. 1998;801:259–66.PubMedCrossRefGoogle Scholar
  5. 5.
    Scherrmann JM. Drug delivery to brain via the blood–brain barrier. Vasc Pharmacol. 2002;38:349–54.CrossRefGoogle Scholar
  6. 6.
    Huwyler J, Wu D, Pardridge WM. Brain drug delivery of small molecules using immunoliposomes. Proc Natl Acad Sci U S A. 1996;93:14164–9.PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Pardridge WM, Eisenberg J, Yang J. Human blood–brain barrier insulin receptor. J Neurochem. 1985;44:1771–8.PubMedCrossRefGoogle Scholar
  8. 8.
    Oba M, Fukushima S, Kanayama N, Aoyagi K, Nishiyama N, Koyama H, et al. Cyclic RGD peptide-conjugated polyplex micelles as a targetable gene delivery system directed to cells possessing alphavbeta3 and alphavbeta5 integrins. Bioconjug Chem. 2007;18:1415–23.PubMedCrossRefGoogle Scholar
  9. 9.
    Sharma G, Modgil A, Layek B, Arora K, Sun C, Law B, et al. Cell penetrating peptide tethered bi-ligand liposomes for delivery to brain in vivo: biodistribution and transfection. J Control Release. 2013;167:1–10.PubMedCrossRefGoogle Scholar
  10. 10.
    Zhong MQ, Hongyan L, Hongzhe U, Kwokping H. Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway. Pharmacol Rev. 2002;54:561–87.CrossRefGoogle Scholar
  11. 11.
    Cheng Y, Zak O, Alsen P, Harrison SC, Watz T. Structure of the human transferrin receptor-transferrin complex. Cell. 2004;116:565–76.PubMedCrossRefGoogle Scholar
  12. 12.
    Sharma G, Modgil A, Sun C, Singh J. Grafting of cell-penetrating peptide to receptor-targeted liposomes improves their transfection efficiency and transport across blood–brain barrier model. J Pharm Sci. 2012;101:2468–78.PubMedCrossRefGoogle Scholar
  13. 13.
    Kibria G, Hatakeyama H, Ohga N, Hida K, Harashima H. Dual ligand modification of PEGylated liposomes shows better cell selectivity and efficient gene delivery. J Control Release. 2011;153:141–8.PubMedCrossRefGoogle Scholar
  14. 14.
    Varga CM, Wickham TJ, Lauffenburger DA. Receptor mediated targeting of gene delivery vectors: insights from molecular mechanisms for improved vehicle design. Biotechnol Bioeng. 2000;70:593–605.PubMedCrossRefGoogle Scholar
  15. 15.
    Bolhassani A. Potential efficacy of cell-penetrating peptides for nucleic acid and drug delivery in cancer. Biochim Biophys Acta. 1816;2011:232–46.Google Scholar
  16. 16.
    Mae M, Myrberg H, EI-Andaloussi S, Langel U. Design of a tumor homing cell penetrating peptide for drug delivery. Int J Pept Res Ther. 2009;15:11–5.CrossRefGoogle Scholar
  17. 17.
    Ziegler A, Nervi P, Durrenberger M, Seelig J. The cationic cell-penetrating peptide CPP TAT derived from the HIV-1 protein Tat is rapidly transported into living fibroblasts: optical, biophysical and metabolic evidence. Biochemistry. 2005;44:138–48.PubMedCrossRefGoogle Scholar
  18. 18.
    Meng W, Kallinteri P, Walker DA, Parker TL, Garnett MC. Evaluation of poly (Glycerol-Adipate) nanoparticle uptake in an in vitro 3-D brain tumor co-culture model. Exp Biol Med. 2007;232:1100–8.CrossRefGoogle Scholar
  19. 19.
    Murray S, Rooprai H, Selway R, Pilkington G. A novel three-dimensional “all human” in vitro brain tumor invasion model. Neuro-Oncology. 2005;7:307–8.Google Scholar
  20. 20.
    Deeken JF, Loscher W. The blood–brain barrier and cancer: transporters, treatment, and Trojan horses. Clin Cancer Res. 2007;13(6):1663–74.PubMedCrossRefGoogle Scholar
  21. 21.
    Lewin M, Carlesso N, Tung CH, Tang XW, Cory D, Scadden DT, et al. Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat Biotechnol. 2000;18:410–4.PubMedCrossRefGoogle Scholar
  22. 22.
    Derossi D, Chassaing G, Prochiantz A. Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol. 1998;8:84–7.PubMedCrossRefGoogle Scholar
  23. 23.
    Pooga M, But C, Kihlmark M, Hallbrink M, Fernaeus S, Raid R, et al. Cellular translocation of proteins by transportan. J FASEB. 2001;15:1451–3.Google Scholar
  24. 24.
    Rousselle C, Smirnova M, Clair P, Lefauconnier JM, Chavanieu A, Calas B, et al. Enhanced delivery of doxorubicin into the brain via a peptide- vector-mediated strategy: saturation kinetics and specificity. JPET. 2001;296:124–31.Google Scholar
  25. 25.
    Rea JC, Barron AE, Shea LD. Peptide-mediated lipofection is governed by lipoplex physical properties and the density of surface-displayed amines. J Pharm Sci. 2008;97:4794–806.PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Miedel MC, Hulmes JD, Pan YEC. The use of fluorescamine as a detection reagent in protein microcharacterization. J Biochem Biophys Methods. 1989;18:37–52.PubMedCrossRefGoogle Scholar
  27. 27.
    Yub E, Kojima C, Sakaguchi N, Harada A, Koiwai K, Kono K. Gene delivery to dendritic cells mediated by complexes of lipoplexes and pH-sensitive fusogenic polymer-modified liposomes. J Control Release. 2008;130:77–83.CrossRefGoogle Scholar
  28. 28.
    Yamano S, Dai J, Yuvienco C, Khapli S, Moursi AM, Montclare JK. Modified Tat peptide with cationic lipids enhances gene transfection efficiency via temperature-dependent and caveolae-mediated endocytosis. J Control Release. 2011;152:278–85.PubMedCrossRefGoogle Scholar
  29. 29.
    Rejman J, Bragonzi A, Conese M. Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. Mol Ther. 2005;12:468–74.PubMedCrossRefGoogle Scholar
  30. 30.
    Vercauteren D, Vandenbroucke RE, Jones AT, Rejman J, Demeester J, De Smedt SC, et al. The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls. Mol Ther. 2010;18:561–9.PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Nam HY, Kwon SM, Chung H, Lee SY, Kwon SH, Jeon H, et al. Cellular uptake mechanism and intracellular fate of hydrophobically modified glycol chitosan nanoparticles. J Control Release. 2009;135:259–67.PubMedCrossRefGoogle Scholar
  32. 32.
    Gaillard PJ, Voorwinden HV, Nielsen JL, Ivanov A, Atsumi R, Engman H, et al. Establishment and functional characterization of an in vitro model of the blood–brain barrier comprising a co-culture of brain capillary endothelial cells and astrocytes. Eur J Pharm Sci. 2001;12:215–22.PubMedCrossRefGoogle Scholar
  33. 33.
    Fenke H, Galla HJ, Beuckmann CT. Primary cultures of brain microvessel endothelial cells: a valid and flexible model to study drug transport through the blood–brain barrier in vitro. Brain Res Brain Res Protoc. 2000;5:248–56.CrossRefGoogle Scholar
  34. 34.
    Erben M, Decker S, Franke H, Galla HJ. Electrical resistance measurements on cerebral capillary endothelial cells—a new technique to study small surface areas. J Biochem Biophys Methods. 1995;30:227–38.PubMedCrossRefGoogle Scholar
  35. 35.
    Fischer D, Li Y, Ahlemeyer B, Krieglstein J, Kissel T. In-vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials. 2003;24:1121–31.PubMedCrossRefGoogle Scholar
  36. 36.
    Lee D, Powers K, Baney R. Physicochemical properties and blood compatibility of acylated chitosan nanoparticles. Carbohydr Polym. 2004;58:371–7.CrossRefGoogle Scholar
  37. 37.
    Rodal SK, Skretting G, Garred O, Vilhardt F, Deurs BV, Sandvig K. Extraction of cholesterol with Methyl-b-Cyclodextrin perturbs formation of clathrin-coated endocytic vesicles. Mol Biol Cell. 1999;10:961–74.PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Audouy SAL, de Leiji LFMH, Hoekstra D, Molema G. In vivo characteristics of cationic liposomes as delivery vectors for gene therapy. Pharm Res. 2002;19:1599–605.PubMedCrossRefGoogle Scholar
  39. 39.
    Jones SW, Christison R, Bundell K, Voyce CJ, Brockbank SMV, Newham P, et al. Characterization of cell-penetrating peptide-mediated peptide delivery. Br J Pharmacol. 2005;145:1093–102.PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Iden DL, Allen TM. In vitro and in vivo comparison of immunoliposomes made by conventional coupling techniques with those made by a new post-insertion approach. Biochim Biophys Acta. 2001;1513:207–16.PubMedCrossRefGoogle Scholar
  41. 41.
    Moreira JN, Ishida T, Gaspar R, Allen TM. Use of the post-insertion technique to insert peptide ligands into preformed stealth liposomes with retention of binding activity and cytotoxicity. Pharm Res. 2002;19:265–9.PubMedCrossRefGoogle Scholar
  42. 42.
    Chen Y, Liu L. Modern methods for delivery of drugs across the blood–brain barrier. Adv Drug Deliv Rev. 2012;64:640–65.PubMedCrossRefGoogle Scholar
  43. 43.
    Roney CC, Kulkarni P, Arora V, Antich P, Bonte F, Wu A, et al. Targeted nanoparticles for drug delivery through the blood–brain barrier for Alzheimer’s disease. J Control Release. 2005;108:193–214.PubMedCrossRefGoogle Scholar
  44. 44.
    Krex D, Klink B, Hartmann C, Deimling AV, Pietsch T, Simon M, et al. German Glioma Network, long-term survival with glioblastoma multiforme. Brain. 2007;130:2596–606.PubMedCrossRefGoogle Scholar
  45. 45.
    McNeil DE, Cote TR, Clegg L, Rorke BL. Incidence and trends in pediatric malignancies medulloblastoma/primitive neuroectodermal tumor: a SEER update: surveillance epidemiology and end results. Med Pediatr Oncol. 2002;39:190–4.PubMedCrossRefGoogle Scholar
  46. 46.
    Saar K, Lindgren M, Hansen M, Eiriksdottir E, Jiang Y, Rosenthal-Aizman K, et al. Cell-penetrating peptides: a comparative membrane toxicity study. Anal Biochem. 2005;345:55–65.PubMedCrossRefGoogle Scholar
  47. 47.
    Kim JB. Three-dimensional tissue culture models in cancer biology. Semin Cancer Biol. 2005;15:365–77.PubMedCrossRefGoogle Scholar
  48. 48.
    Sourla A, Doillon C, Koutsilieris M. Three-dimensional type I collagen gel system containing MG-63 osteoblasts-like cells as model for studying local bone reaction caused by metastatic cancer cells. Anticancer Res. 1996;16:2773–80.PubMedGoogle Scholar
  49. 49.
    Bell E. Strategy for the selection of scaffolds for tissue engineering. Tissue Eng. 1995;1:163–79.PubMedCrossRefGoogle Scholar
  50. 50.
    Provenzale JM, Mukundan S, Dewhirst M. The role of blood–brain barrier permeability in brain tumor imaging and therapeutics. Am J Roentgenol. 2005;185:763–7.CrossRefGoogle Scholar
  51. 51.
    Ishihara H, Kubota H, Lindberg RL, Leppert D, Gloor SM, Errede M, et al. Endothelial cell barrier impairment induced by glioblastomas and transforming growth factor beta2 involves matrix metalloproteinases and tight junction proteins. J Neuropathol Exp Neurol. 2008;67:435–48.PubMedCrossRefGoogle Scholar
  52. 52.
    Janzer RC, Raff MC. Astrocytes induce blood–brain barrier properties in endothelial cells. Nature. 1987;325:253–7.PubMedCrossRefGoogle Scholar
  53. 53.
    Antohi S, Brumfeld V. Polycation-cell surface interactions and plasma membrane compartments in mammals. Interference of oligocation with polycationic condensation. Z Naturforsch C. 1984;39:767–75.PubMedGoogle Scholar
  54. 54.
    Cardozo AK, Buchillier V, Mathieu M, Chen J, Ortis F, Ladrire L, et al. Cell-permeable peptides induce dose- and length-dependent cytotoxic effects. Biochim Biophys Acta Biomembr. 2007;1768:2222–34.CrossRefGoogle Scholar
  55. 55.
    Deaglio S, Capobianco A, Cali A, Bellora F, Alberti F, Righi L, et al. Structural, functional, and tissue distribution analysis of human transferrin receptor-2 by murine monoclonal antibodies and a polyclonal antiserum. Blood. 2002;100:3782–9.PubMedCrossRefGoogle Scholar
  56. 56.
    Jefferies WA, Brandon MR, Hunt SV, Williams AF, Gatter KC, Mason DY. Transferrin receptor on endothelium of brain capillaries. Nature. 1984;312:162–3.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Gitanjali Sharma
    • 1
  • Amit Modgil
    • 1
  • Tiecheng Zhong
    • 1
  • Chengwen Sun
    • 1
  • Jagdish Singh
    • 1
    • 2
  1. 1.Department of Pharmaceutical Sciences College of Pharmacy Nursing and Allied SciencesNorth Dakota State UniversityFargoUSA
  2. 2.Department of Pharmaceutical Sciences College of Pharmacy, Nursing & Allied SciencesNorth Dakota State UniversityFargoUSA

Personalised recommendations