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Evaluation of cytotoxicity profile and intracellular localisation of doxorubicin-loaded chitosan nanoparticles

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Abstract

In the emerging field of nanomedicine, targeted delivery of nanoparticle encapsulated active pharmaceutical ingredients (API) is seen as a potential significant development, promising improved pharmacokinetics and reduced side effects. In this context, understanding the cellular uptake of the nanoparticles and subsequent subcellular distribution of the API is of critical importance. Doxorubicin (DOX) was encapsulated within chitosan nanoparticles to investigate its intracellular delivery in A549 cells in vitro. Unloaded (CS-TPP) and doxorubicin-loaded (DOX-CS-TPP) chitosan nanoparticles were characterised for size (473 ± 41 nm), polydispersity index (0.3 ± 0.2), zeta potential (34 ± 4 mV), drug content (76 ± 7 μM) and encapsulation efficiency (95 ± 1 %). The cytotoxic response to DOX-CS-TPP was substantially stronger than to CS-TPP, although weaker than that of the equivalent free DOX. Fluorescence microscopy showed a dissimilar pattern of distribution of DOX within the cell, being predominantly localised in the nucleus for free form and in cytoplasm for DOX-CS-TPP. Confocal microscopy demonstrated endosomal localisation of DOX-CS-TPP. Numerical simulations, based on a rate equation model to describe the uptake and distribution of the free DOX, nanoparticles and DOX-loaded nanoparticles within the cells and the subsequent dose- and time-dependent cytotoxic responses, were used to further elucidate the API distribution processes. The study demonstrates that encapsulation of the API in nanoparticles results in a delayed release of the drug to the cell, resulting in a delayed cellular response. This work further demonstrates the potential of mathematical modelling in combination with intracellular imaging techniques to visualise and further understand the intracellular mechanisms of action of external agents, both APIs and nanoparticles in cells.

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References

  1. Moghimi SM, Hunter AC, Murray JC. Nanomedicine: current status and future prospects. FASEB J. 2005;19:311–30.

    Article  CAS  Google Scholar 

  2. Schütz CA, Juillerat-Jeanneret L, Mueller H, Lynch I, Riediker M. Therapeutic nanoparticles in clinics and under clinical evaluation. Nanomedicine-UK. 2013;8:449–67.

    Article  Google Scholar 

  3. Oberdörster G, Oberdörster E, Oberdörster J. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect. 2005;113:823–39.

    Article  Google Scholar 

  4. Octavia Y, Tocchetti CG, Gabrielson KL, Janssens S, Crijns HJ, Moens AL. Doxorubicin-induced cardiomyopathy: from molecular mechanisms to therapeutic strategies. J Mol Cell Cardiol. 2012;52:1213–25.

    Article  CAS  Google Scholar 

  5. Goldstein LJ, Galski H, Fojo A, Willingham M, Lai SL, Gazdar A, et al. Expression of multidrug resistance gene in human cancers. J Natl Cancer Inst. 1989;81:116–24.

    Article  CAS  Google Scholar 

  6. Von Hoff D, Rozencweig M, Piccart M. The cardiotoxicity of anticancer agents. Semin Oncol. 1982;9:23–33.

    Google Scholar 

  7. Primeau AJ, Rendon A, Hedley D, Lilge L, Tannock IF. The distribution of the anticancer drug doxorubicin in relation to blood vessels in solid tumors. Clin Cancer Res. 2005;11:8782–8.

    Article  CAS  Google Scholar 

  8. Hofheinz RD, Gnad-Vogt SU, Beyer U, Hochhaus A. Liposomal encapsulated anticancer drugs. Anticancer Drugs. 2005;16:691–707.

    Article  CAS  Google Scholar 

  9. Manil L, Couvreur P, Mahieu P. Acute renal toxicity of doxorubicin (adriamycin)-loaded cyanoacrylate nanoparticles. Pharm Res. 1995;12:85–7.

    Article  CAS  Google Scholar 

  10. Lorusso D, Di Stefano A, Carone V, Fagotti A, Pisconti S, Scambia G. Pegylated liposomal doxorubicin-related palmar-plantar erythrodysesthesia (‘hand-foot’ syndrome). Ann Oncol. 2007;18:1159–64.

    Article  CAS  Google Scholar 

  11. Tan ML, Choong PFM, Dass CR. Review: doxorubicin delivery systems based on chitosan for cancer therapy. J Pharm Pharmacol. 2009;61:131–42.

    Article  CAS  Google Scholar 

  12. Siqueira NM, Contri RV, Paese K, Beck RCR, Pohlmann AR, Guterres SS. Innovative sunscreen formulation based on benzophenone-3-loaded chitosan-coated polymeric nanocapsules. Skin Pharmacol Physiol. 2011;24:166–74.

    Article  CAS  Google Scholar 

  13. Nafee N, Schneider M, Schaefer UF, Lehr CM. Relevance of the colloidal stability of chitosan/PLGA nanoparticles on their cytotoxicity profile. Int J Pharm. 2009;381:130–9.

    Article  CAS  Google Scholar 

  14. Janes KA, Fresneau MP, Marazuela A, Fabra A, Alonso MJ. Chitosan nanoparticles as delivery systems for doxorubicin. J Control Release. 2001;73:255–67.

    Article  CAS  Google Scholar 

  15. Sureshkumar MK, Das D, Mallia MB, Gupta PC. Adsorption of uranium from aqueous solution using chitosan-tripolyphosphate (CTPP) beads. J Hazard Mater. 2010;184:65–72.

    Article  CAS  Google Scholar 

  16. Bugnicourt L, Alcouffe P, Ladavière C. Elaboration of chitosan nanoparticles: favorable impact of a mild thermal treatment to obtain finely divided, spherical, and colloidally stable objects. Colloids Surf A Physicochem Eng Asp. 2014;457:476–86.

    Article  CAS  Google Scholar 

  17. European Commission. Definition of nanoparticle. In: Scientific Committees Toolbox. Scientific Committee on Emergent and Newly Identified Health Risks. 2007. http://ec.europa.eu/health/scientific_committees/opinions_layman/glossary/mno/nanoparticle.htm. Accessed 19 Apr 2016.

  18. International Organisation for Standardization. In: Technical Committee 229 Nanotechnologies (ISO/TC 229). 2005. http://www.iso.org/iso/iso_technical_committee?commid=381983. Accessed 19 Apr 2016.

  19. Keating M, Byrne HJ. Raman spectroscopy in nanomedicine: current status and future perspectives. Nanomedicine. 2013;8:1–17.

    Article  Google Scholar 

  20. European Parliament and the Council of the European Union. Directive 2010/63/EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes. OJEU 2010, L 276/33.

  21. Huang Y-W, C-H W, Aronstam RS. Toxicity of transition metal oxide nanoparticles: recent insights from in vitro studies. Materials. 2010;3:4842–59.

    Article  CAS  Google Scholar 

  22. Mukherjee SP, Davoren M, Byrne HJ. In vitro mammalian cytotoxicological study of PAMAM dendrimers—towards quantitative structure activity relationships. Toxicol In Vitro. 2010;24:169–77.

    Article  CAS  Google Scholar 

  23. Maher MA, Naha PC, Mukerjee SP, Byrne HJ. Numerical simulations of in vitro nanoparticle toxicity—the case of poly(amido amine) dendrimers. Toxicol In Vitro. 2014;28:1449–60.

    Article  CAS  Google Scholar 

  24. Nawaz H, Bonnier F, Knief P, Howe O, Lyng FM, Meade AD, et al. Evaluation of the potential of Raman microspectroscopy for prediction of chemotherapeutic response to cisplatin in lung adenocarcinoma. Analyst. 2010;135:3070–6.

    Article  CAS  Google Scholar 

  25. Nawaz H, Bonnier F, Meade AD, Lyng FM, Byrne HJ. Comparison of subcellular responses for the evaluation and prediction of the chemotherapeutic response to cisplatin in lung adenocarcinoma using Raman spectroscopy. Analyst. 2011;136:2450–63.

    Article  CAS  Google Scholar 

  26. Nawaz H, Garcia A, Meade AD, Lyng FM, Byrne HJ. Raman micro spectroscopy study of the interaction of vincristine with A549 cells supported by expression analysis of bcl-2 protein. Analyst. 2013;138:6177–84.

    Article  CAS  Google Scholar 

  27. Poletto FS, Jager E, Cruz L, Pohlmann AR, Guterres SS. The effect of polymeric wall on the permeability of drug-loaded nanocapsules. Mater Sci Eng C. 2008;28:472–8.

    Article  CAS  Google Scholar 

  28. Ravanello A, Dadalt G, Torres BGS, Hurtado FK, Marcolino AIP, Rolim CMB. Development and validation of an UV-spectrophotometric method for the dissolution studies of sitagliptin tablets. Lat Am J Pharm. 2010;29:962–7.

    CAS  Google Scholar 

  29. Atkinson KE. An introduction to numerical analysis. 2nd ed. New York: Wiley; 1989.

    Google Scholar 

  30. Salvati A, Aberg C, Dos Santos T. Experimental and theoretical comparison of intracellular import of polymeric nanoparticles and small molecules: toward models of uptake kinetics. Nanomedicine: NBM. 2011;7:818–26.

    CAS  Google Scholar 

  31. Mukherjee SP, Byrne HJ. Polyamidoamine dendrimer nanoparticle cytotoxicity, oxidative stress, caspase activation and inflammatory response: experimental observation and numerical simulation. Nanomedicine: NBM. 2013;9:202–11.

    CAS  Google Scholar 

  32. Kaufmann SH, Earnshaw WC. Induction of apoptosis by cancer chemotherapy. Exp Cell Res. 2000;256:42–9.

    Article  CAS  Google Scholar 

  33. Black JW, Leff P. Operational models of pharmacological agonism. Proc R Soc Lond [Biol]. 1983;220:141–62.

    Article  CAS  Google Scholar 

  34. Gewirtz DA. A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem Pharmacol. 1999;57:727–41.

    Article  CAS  Google Scholar 

  35. Nel A, Xia T, Mädler L, Li N. Toxic potential of materials at the nanolevel. Science. 2006;311:622–7.

    Article  CAS  Google Scholar 

  36. International Conference on Harmonisation (ICH). Validation of analytical procedures: text and methodology Q2(R1). 2005. http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf. Accessed 15 Jul 2015.

  37. 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.

    Article  Google Scholar 

  38. De Fries R, Mitsuhashi M. Quantification of mitogen induced human lymphocyte proliferation: comparison of alamarbluetm assay to 3h-thymidine incorporation assay. J Clin Lab Anal. 1995;9:89–95.

    Article  Google Scholar 

  39. Poornima P, Kumar VB, Weng CF, Padma VV. Doxorubicin induced apoptosis was potentiated by neferine in human lung adenocarcinoma, A549 cells. Food Chem Toxicol. 2014;68:87–98.

    Article  CAS  Google Scholar 

  40. Lv S, Tang Z, Li M, Lin J, Song W, Liu H, et al. Co-delivery of doxorubicin and paclitaxel by PEG-polypeptide nanovehicle for the treatment of non-small cell lung cancer. Biomaterials. 2014;35:6118–29.

    Article  CAS  Google Scholar 

  41. Farhane Z, Bonnier F, Casey A, Byrne HJ. Raman microspectroscopy for in vitro drug screening: subcellular localization and interactions of doxorubicin. Analyst. 2015;140:4212–23.

    Article  CAS  Google Scholar 

  42. Prabaharan M, Grailer JJ, Pilla S, Steeber DA, Gong SQ. Amphiphilic multi-arm-block copolymer conjugated with doxorubicin via pH-sensitive hydrazone bond for tumor-targeted drug delivery. Biomaterials. 2009;30:5757–66.

    Article  CAS  Google Scholar 

  43. Li Q, Lv S, Tang Z, Liu M, Zhang D, Yang Y, et al. A co-delivery system based on paclitaxel grafted mPEG-b-PLG loaded with doxorubicin: preparation, in vitro and in vivo evaluation. Int J Pharm. 2014;471:412–20.

    Article  CAS  Google Scholar 

  44. Chittasupho C, Lirdprapamongkol K, Kewsuwan P, Sarisuta N. Targeted delivery of doxorubicin to A549 lung cancer cells by CXCR4 antagonist conjugated PLGA nanoparticles. Eur J Pharm Biopharm. 2014;88:529–38.

    Article  CAS  Google Scholar 

  45. Wang X-B, Zhou H-Y. Molecularly targeted gemcitabine-loaded nanoparticulate system towards the treatment of EGFR overexpressing lung cancer. Biomed Pharmacother. 2015;70:123–8.

    Article  CAS  Google Scholar 

  46. Uhrich KE, Cannizzaro SM, Langer RS, Shakesheff KM. Polymeric systems for controlled drug release. Chem Rev. 1999;99:3181–98.

    Article  CAS  Google Scholar 

  47. Siddique MI, Katas H, Amin MCIM, Ng SF, Zulfakar MH, Buang F, et al. Minimization of local and systemic adverse effects of topical glucocorticoids by nanoencapsulation: in vivo safety of hydrocortisone–hydroxytyrosol loaded chitosan nanoparticles. J Pharm Sci. 2015;104:4276–86.

    Article  CAS  Google Scholar 

  48. Yoshitomita T, Ozakia Y, Thangavela S, Nagasakia Y. Redox nanoparticle therapeutics to cancer—increase in therapeutic effect of doxorubicin, suppressing its adverse effect. J Control Release. 2013;172:137–43.

    Article  Google Scholar 

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Acknowledgments

This research was carried out under funding by the Brazilian National Council for Scientific and Technological Development (CNPq), through the Science without Borders Program grant no. 236817/2013-2, awarded to Gabriele Dadalt Souto. Zeineb Farhane, Alan Casey, Esen Efeoglu, Jennifer McIntyre and Hugh James Byrne are supported by Science Foundation Ireland Principle Investigator Award 11/PI/1108.

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  1. Gabriele Dadalt Souto

    Present address: Faculdade de Farmácia, Universidade do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, 90610-000, Brazil

Authors and Affiliations

  1. FOCAS Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland

    Gabriele Dadalt Souto, Zeineb Farhane, Alan Casey, Esen Efeoglu, Jennifer McIntyre & Hugh James Byrne

  2. School of Physics, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland

    Zeineb Farhane & Esen Efeoglu

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  1. Gabriele Dadalt Souto
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Correspondence to Gabriele Dadalt Souto.

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Souto, G.D., Farhane, Z., Casey, A. et al. Evaluation of cytotoxicity profile and intracellular localisation of doxorubicin-loaded chitosan nanoparticles. Anal Bioanal Chem 408, 5443–5455 (2016). https://doi.org/10.1007/s00216-016-9641-6

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