Pharmaceutical Research

, 35:75 | Cite as

In Vitro’, ‘In Vivo’ and ‘In Silico’ Investigation of the Anticancer Effectiveness of Oxygen-Loaded Chitosan-Shelled Nanodroplets as Potential Drug Vector

  • Amina Khadjavi
  • Ilaria Stura
  • Mauro Prato
  • Valerio Giacomo Minero
  • Alice Panariti
  • Ilaria Rivolta
  • Giulia Rossana Gulino
  • Federica Bessone
  • Giuliana Giribaldi
  • Elena Quaglino
  • Roberta Cavalli
  • Federica Cavallo
  • Caterina Guiot
Research Paper



Chitosan-shelled/decafluoropentane-cored oxygen-loaded nanodroplets (OLN) are a new class of nanodevices to effectively deliver anti-cancer drugs to tumoral cells. This study investigated their antitumoral effects ‘per se’, using a mathematical model validated on experimental data.


OLN were prepared and characterized either in vitro or in vivo. TUBO cells, established from a lobular carcinoma of a BALB-neuT mouse, were investigated following 48 h of incubation in the absence/presence of different concentrations of OLN. OLN internalization, cell viability, necrosis, apoptosis, cell cycle and reactive oxygen species (ROS) production were checked as described in the Method section.

In vivo tumor growth was evaluated after subcutaneous transplant in BALB/c mice of TUBO cells either without treatment or after 24 h incubation with 10% v/v OLN.


OLN showed sizes of about 350 nm and a positive surface charge (45 mV). Dose-dependent TUBO cell death through ROS-triggered apoptosis following OLN internalization was detected. A mathematical model predicting the effects of OLN uptake was validated on both in vitro and in vivo results.


Due to their intrinsic toxicity OLN might be considered an adjuvant tool suitable to deliver their therapeutic cargo intracellularly and may be proposed as promising combined delivery system.


antitumor nanodevice breast cancer chitosan nanodroplet nanocarrier oxygen 



Diamidino-2-phenylindole dihydrochloride


Dulbecco’s modified eagle medium


Fluorescein isothiocyanate


Lactate dehydrogenase


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


Oxygen-loaded Nanodroplets


Oxygen Reactive Species


  1. 1.
    Talevi A, Gantner ME, Ruiz ME. Applications of nanosystems to anticancer drug therapy (Part I. Nanogels, nanospheres, nanocapsules). Recent Pat Anticancer Drug Discov. 2014;9(1):83–98.CrossRefPubMedGoogle Scholar
  2. 2.
    Ferrari M. Nanovector therapeutics. Curr Opin Chem Biol. 2005;9(4):343–6.CrossRefPubMedGoogle Scholar
  3. 3.
    Gao H, Shi W, Freund LB. Mechanics of receptor-mediated endocytosis. Proc Natl Acad Sci U S A. 2005;102(27):9469–74.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Mora-Huertas CE, Fessi H, Elaissari A. Polymer-based nanocapsules for drug delivery. Int J Pharm. 2010;385(1–2):113–42.CrossRefPubMedGoogle Scholar
  5. 5.
    Barrera G, Daga M, Ferrara B, Dianzani C, Pizzimenti S, Argenziano M, et al. Drug delivery nanoparticles in treating Chemoresistant tumor cells. Curr Med Chem. 2017;24(42):4800–15.PubMedGoogle Scholar
  6. 6.
    Lin Q, Bao C, Yang Y, Liang Q, Zhang D, Cheng S, et al. Highly discriminating photorelease of anticancer drugs based on hypoxia activatable phototrigger conjugated chitosan nanoparticles. Adv Mater. 2013;25(14):1981–6.CrossRefPubMedGoogle Scholar
  7. 7.
    Magnetto C, Prato M, Khadjavi A, Giribaldi G, Fenoglio I, Jose J, et al. Ultrasound-activated decafluoropentane-cored and chitosan-shelled nanodroplets for oxygen delivery to hypoxic cutaneous tissues. RSC Adv. 2014;4(72):38433–41.CrossRefGoogle Scholar
  8. 8.
    Prato M, Magnetto C, Jose J, Khadjavi A, Cavallo F, Quaglino E, et al. 2H,3H-decafluoropentane-based nanodroplets: new perspectives for oxygen delivery to hypoxic cutaneous tissues. PLoS One. 2015;10(3):e0119769.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Cavalli R, Bisazza A, Giustetto P, Civra A, Lembo D, Trotta G, et al. Preparation and characterization of dextran nanobubbles for oxygen delivery. Int J Pharm. 2009;381(2):160–5.CrossRefPubMedGoogle Scholar
  10. 10.
    Cavalli R, Bisazza A, Rolfo A, Balbis S, Madonnaripa D, Caniggia I, et al. Ultrasound-mediated oxygen delivery from chitosan nanobubbles. Int J Pharm. 2009;378(1–2):215–7.CrossRefPubMedGoogle Scholar
  11. 11.
    Banche G, Prato M, Magnetto C, Allizond V, Giribaldi G, Argenziano M, et al. Antimicrobial chitosan nanodroplets: new insights for ultrasound-mediated adjuvant treatment of skin infection. Future Microbiol. 2015;10(6):929–39.CrossRefPubMedGoogle Scholar
  12. 12.
    Khadjavi A, Magnetto C, Panariti A, Argenziano M, Gulino GR, Rivolta I, et al. Chitosan-shelled oxygen-loaded nanodroplets abrogate hypoxia dysregulation of human keratinocyte gelatinases and inhibitors: new insights for chronic wound healing. Toxicol Appl Pharmacol. 2015;286(3):198–206.CrossRefPubMedGoogle Scholar
  13. 13.
    Rovero S, Amici A, Carlo ED, Bei R, Nanni P, Quaglino E, et al. DNA vaccination against rat her-2/Neu p185 more effectively inhibits carcinogenesis than transplantable carcinomas in transgenic BALB/c mice. J Immunol. 2000;165(9):5133–42.CrossRefPubMedGoogle Scholar
  14. 14.
    Minero VG, Khadjavi A, Costelli P, Baccino FM, Bonelli G. JNK activation is required for TNFalpha-induced apoptosis in human hepatocarcinoma cells. Int Immunopharmacol. 2013;17(1):92–8.CrossRefPubMedGoogle Scholar
  15. 15.
    Salvati A, Åberg C, dos Santos T, Varela J, Pinto P, Lynch I, et al. Experimental and theoretical comparison of intracellular import of polymeric nanoparticles and small molecules: toward models of uptake kinetics. Nanomedicine. 2011;7(6):818–26.CrossRefPubMedGoogle Scholar
  16. 16.
    Maher MA, Naha PC, Mukherjee SP, Byrne HJ. Numerical simulations of in vitro nanoparticle toxicity - the case of poly(amido amine) dendrimers. Toxicol in Vitro. 2014;28(8):1449–60.CrossRefPubMedGoogle Scholar
  17. 17.
    Souto GD, Farhane Z, Casey A, Efeoglu E, McIntyre J, Byrne HJ. Evaluation of cytotoxicity profile and intracellular localisation of doxorubicin-loaded chitosan nanoparticles. Anal Bioanal Chem. 2016;408(20):5443–55.CrossRefPubMedGoogle Scholar
  18. 18.
    Kanapathipillai M, Brock A, Ingber DE. Nanoparticle targeting of anti-cancer drugs that alter intracellular signaling or influence the tumor microenvironment. Adv Drug Deliv Rev. 2014;79-80:107–18.CrossRefPubMedGoogle Scholar
  19. 19.
    Duchene D, Cavalli R, Gref R. Cyclodextrin-based polymeric nanoparticles as efficient carriers for anticancer drugs. Curr Pharm Biotechnol. 2016;17(3):248–55.CrossRefPubMedGoogle Scholar
  20. 20.
    Li Q, Dunn ET, Grandmaison EW, Goosen MFA. Applications and properties of chitosan. J Bioact Compat Polym. 1992;7(4):370–97.CrossRefGoogle Scholar
  21. 21.
    Kumar MNVR, Muzzarelli RAA, Muzzarelli C, Sashiwa H, Domb AJ. Chitosan chemistry and pharmaceutical perspectives. Chem Rev. 2004;104(12):6017–84.CrossRefPubMedGoogle Scholar
  22. 22.
    Qi LF, Xu ZR, Li Y, Jiang X, Han XY. In Vitro effects of chitosan nanoparticles on proliferation of human gastric carcinoma cell line MGC803 cells. World J Gastroenterol. 2005;11(33):5136–41.PubMedPubMedCentralGoogle Scholar
  23. 23.
    Qi LF, Xu ZR. In vivo antitumor activity of chitosan nanoparticles. Bioorg Med Chem Lett. 2006;16(16):4243–5.CrossRefPubMedGoogle Scholar
  24. 24.
    Jiang M, Ouyang H, Ruan P, Zhao H, Pi Z, Huang S, et al. Chitosan derivatives inhibit cell proliferation and induce apoptosis in breast cancer cells. Anticancer Res. 2011;31(4):1321–8.PubMedGoogle Scholar
  25. 25.
    Salah R, Michaud P, Mati F, Harrat Z, Lounici H, Abdi N, et al. Anticancer activity of chemically prepared shrimp low molecular weight chitin evaluation with the human monocyte leukaemia cell line, THP-1. Int J Biol Macromol. 2013;52:333–9.CrossRefPubMedGoogle Scholar
  26. 26.
    Xu YL, et al. Chitosan nanoparticles inhibit the growth of human hepatocellular carcinoma xenografts through an antiangiogenic mechanism. Anticancer Res. 2009;29(12):5103–9.PubMedGoogle Scholar
  27. 27.
    Hasegawa M, Yagi K, Iwakawa S, Hirai M. Chitosan induces apoptosis via caspase-3 activation in bladder tumor cells. Jpn J Cancer Res. 2001;92(4):459–66.CrossRefPubMedGoogle Scholar
  28. 28.
    Takimoto H, Hasegawa M, Yagi K, Nakamura T, Sakaeda T, Hirai M. Proapoptotic effect of a dietary supplement: water soluble chitosan activates caspase-8 and modulating death receptor expression. Drug Metab Pharmacokinet. 2004;19(1):76–82.CrossRefPubMedGoogle Scholar
  29. 29.
    Amidi M, Mastrobattista E, Jiskoot W, Hennink WE. Chitosan-based delivery systems for protein therapeutics and antigens. Adv Drug Deliv Rev. 2010;62(1):59–82.CrossRefPubMedGoogle Scholar
  30. 30.
    Kean T, Thanou M. Biodegradation, biodistribution and toxicity of chitosan. Adv Drug Deliv Rev. 2010;62(1):3–11.CrossRefPubMedGoogle Scholar
  31. 31.
    Cavalli R, Leone F, Minelli R, Fantozzi R, Dianzani C. New chitosan nanospheres for the delivery of 5-fluorouracil: preparation, characterization and in vitro studies. Curr Drug Deliv. 2014;11(2):270–8.CrossRefPubMedGoogle Scholar
  32. 32.
    Wilhelm C, Billotey C, Roger J, Pons JN, Bacri JC, Gazeau F. Intracellular uptake of anionic superparamagnetic nanoparticles as a function of their surface coating. Biomaterials. 2003;24(6):1001–11.CrossRefPubMedGoogle Scholar
  33. 33.
    Lee JK, Lim HS, Kim JH. Cytotoxic activity of aminoderivatized cationic chitosan derivatives. Bioorg Med Chem Lett. 2002;12(20):2949–51.CrossRefPubMedGoogle Scholar
  34. 34.
    Edinger AL, Thompson CB. Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol. 2004;16(6):663–9.CrossRefPubMedGoogle Scholar
  35. 35.
    Görlach A, Dimova EY, Petry A, Martínez-Ruiz A, Hernansanz-Agustín P, Rolo AP, et al. Reactive oxygen species, nutrition, hypoxia and diseases: problems solved? Redox Biol. 2015;6:372–85.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Dunn JD, et al. Reactive oxygen species and mitochondria: a nexus of cellular homeostasis. Redox Biol. 2015;6:472–85.CrossRefGoogle Scholar
  37. 37.
    Hsin YH, Chen CF, Huang S, Shih TS, Lai PS, Chueh PJ. The apoptotic effect of nanosilver is mediated by a ROS- and JNK-dependent mechanism involving the mitochondrial pathway in NIH3T3 cells. Toxicol Lett. 2008;179(3):130–9.CrossRefPubMedGoogle Scholar
  38. 38.
    AshaRani PV, Low Kah Mun G, Hande MP, Valiyaveettil S. Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS Nano. 2009;3(2):279–90.CrossRefPubMedGoogle Scholar
  39. 39.
    Hussain SM, Hess KL, Gearhart JM, Geiss KT, Schlager JJ. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol in Vitro. 2005;19(7):975–83.CrossRefPubMedGoogle Scholar
  40. 40.
    Limbach LK, Wick P, Manser P, Grass RN, Bruinink A, Stark WJ. Exposure of engineered nanoparticles to human lung epithelial cells: influence of chemical composition and catalytic activity on oxidative stress. Environ Sci Technol. 2007;41(11):4158–63.CrossRefPubMedGoogle Scholar
  41. 41.
    Park EJ, Choi J, Park YK, Park K. Oxidative stress induced by cerium oxide nanoparticles in cultured BEAS-2B cells. Toxicology. 2008;245(1–2):90–100.CrossRefPubMedGoogle Scholar
  42. 42.
    Kehrer JP, Klotz L. Free radicals and related reactive species as mediators of tissue injury and disease: implications for health. Crit Rev Toxicol. 2015;45(9):765–98.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Amina Khadjavi
    • 1
  • Ilaria Stura
    • 2
  • Mauro Prato
    • 2
  • Valerio Giacomo Minero
    • 3
  • Alice Panariti
    • 4
  • Ilaria Rivolta
    • 4
  • Giulia Rossana Gulino
    • 5
  • Federica Bessone
    • 6
  • Giuliana Giribaldi
    • 5
  • Elena Quaglino
    • 3
  • Roberta Cavalli
    • 6
  • Federica Cavallo
    • 3
  • Caterina Guiot
    • 1
  1. 1.Dipartimento di NeuroscienzeUniversità di TorinoTorinoItaly
  2. 2.Dipartimento di Scienze della Sanità Pubblica e PediatricheUniversità di TorinoTorinoItaly
  3. 3.Dipartimento di Biotecnologie Molecolari e Scienze per la SaluteUniversità di TorinoTorinoItaly
  4. 4.Dipartimento di Medicina SperimentaleUniversità Milano BicoccaMonzaItaly
  5. 5.Dipartimento di OncologiaUniversità di TorinoTorinoItaly
  6. 6.Dipartimento di Scienze e Tecnologia del FarmacoUniversità di TorinoTorinoItaly

Personalised recommendations