Skip to main content
Log in

Chitosan nanoparticles from Artemia salina inhibit progression of hepatocellular carcinoma in vitro and in vivo

  • Nanotechnology, Nanopollution, Nanotoxicology and Nanomedicine (NNNN)
  • Published:
Environmental Science and Pollution Research Aims and scope Submit manuscript


This study was conducted to evaluate the effect of chitosan nanoparticles (CNPs) isolated from Artemia salina against hepatocellular carcinoma (HCC) both in vitro (HepG2) and in vivo (diethylnitrosamine-induced HCC in rats) and to investigate the involved underlying mechanisms. Administration of CNPs decreased HCC progression as evidenced by (1) induced HepG2 cell death as detected by MTT assay; (2) induced necrosis as indicated by acridine orange/propidium iodide (AO/PI) red staining, annexin V/7-AAD positive staining (detected by flow cytometry), and upregulated expression of necrosis markers (PARP1 and its downstream target, RIP1 genes), but no effect on apoptosis as revealed by insignificant changes in caspase 3 activity and mRNA levels of Bax and AIF; (3) increased intracellular ROS and decreased mitochondrial membrane potential in HepG2; (4) decreased liver relative weight, serum levels of liver enzymes (ALT, AST, and ALP), total bilirubin, and cancer markers (AFP and GGT), number and area of GST-P positive tumor nodules; and (5) reduced oxidative stress (decrease in MDA levels) and increased activities of SOD, CAT, and GPx enzymes in rat liver. The preventive (pre-treatment) effect of CNPs was better than the therapeutic (post-treatment) effect. Collectively, administration of CNPs inhibited HCC progression in vitro and in vivo, possibly through induction of necrosis, rather than apoptosis, and induction of antioxidant enzyme activities in vivo, but with stimulation of ROS production in vitro. Thus, CNPs could be used as a promise agent for treating HCC after application of further confirmatory clinical trials.

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

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others



Apoptosis-inducing factor


Alkaline phosphatase


Alanine transaminase


Analysis of variance


Aspartate transaminase




Diethyl nitosoamine


γ-Glutamyl transferase


Glutathione peroxidase


Hepatocellular carcinoma;


Mitochondrial permeability transition


Mitochondrial membrane potential


Poly(ADP-ribose) polymerase 1


Serine/threonine kinases receptor interacting protein 1


Superoxide dismutase


Tukey’s honestly significant difference


  • Abdelhadya DH, El-Magd MA, Elbialy ZI, Saleh AA, 2017 Bromuconazole-induced hepatotoxicity is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1 gene expression. Toxicol Mech Methods 1–7

  • Abdo W, Hirata A, Sakai H, El-Sawak A, Nikami H, Yanai T (2013) Combined effects of organochlorine pesticides heptachlor and hexachlorobenzene on the promotion stage of hepatocarcinogenesis in rats. Food Chem Toxicol 55:578–585

    Article  CAS  Google Scholar 

  • Abdulkarim A, Isa MT, Abdulsalam S, Muhammad AJ, Ameh AO, 2013. Extraction and characterisation of chitin and chitosan from mussel shell. Extraction 3

  • Ajun W, Yan S, Li G, Huili L (2009) Preparation of aspirin and probucol in combination loaded chitosan nanoparticles and in vitro release study. Carbohydr Polym 75:566–574

    Article  Google Scholar 

  • Douglas DL, Baines CP (2014) PARP1-mediated necrosis is dependent on parallel JNK and ca(2)(+)/calpain pathways. J Cell Sci 127:4134–4145

    Article  CAS  Google Scholar 

  • El-Denshary E, Aljawish A, El-Nekeety A, Hassan N, Saleh R, Rihn B, Abdel-Wahhab M (2015) Possible synergistic effect and antioxidant properties of chitosan nanoparticles and quercetin against carbon tetrachloride-induce hepatotoxicity in rats. Soft Nanoscience Letters 5:36–51

    Article  CAS  Google Scholar 

  • El-Magd MA, Abdo WS, El-Maddaway M, Nasr NM, Gaber RA, El-Shetry ES, Saleh AA, Alzahrani FAA, Abdelhady DH (2017a) High doses of S-methylcysteine cause hypoxia-induced cardiomyocyte apoptosis accompanied by engulfment of mitochondaria by nucleus. Biomed Pharmacother 94:589–597

    Article  CAS  Google Scholar 

  • El-Magd MA, Khalifa SF, Alzahrani FA, Badawy AA, El-Shetry ES, Dawood LM, Alruwaili MM, Alrawaili HA, Risha EF, El-Taweel FM, Marei HE (2018) Incensole acetate prevents beta-amyloid-induced neurotoxicity in human olfactory bulb neural stem cells. Biomed Pharmacother 105:813–823

    Article  CAS  Google Scholar 

  • El-Magd MA, Khamis A, Nasr Eldeen SK, Ibrahim WM, Salama AF (2017b) Trehalose enhances the antitumor potential of methotrexate against mice bearing Ehrlich ascites carcinoma. Biomed Pharmacother 92:870–878

    Article  CAS  Google Scholar 

  • Forner A, Llovet JM, Bruix J (2012) Hepatocellular carcinoma. Lancet 379:1245–1255

    Article  Google Scholar 

  • Foster KA, Yazdanian M, Audus KL (2001) Microparticulate uptake mechanisms of in-vitro cell culture models of the respiratory epithelium. J Pharm Pharmacol 53:57–66

    Article  CAS  Google Scholar 

  • Huang G-J, Deng J-S, Chiu C-S, Liao J-C, Hsieh W-T, Sheu M-J, Wu C-H (2012) Hispolon protects against acute liver damage in the rat by inhibiting lipid peroxidation, proinflammatory cytokine, and oxidative stress and downregulating the expressions of iNOS, COX-2, and MMP-9. Evid Based Complement Alternat Med 2012:12

    Google Scholar 

  • Huang M, Khor E, Lim LY (2004) Uptake and cytotoxicity of chitosan molecules and nanoparticles: effects of molecular weight and degree of deacetylation. Pharm Res 21:344–353

    Article  CAS  Google Scholar 

  • Loh JW, Yeoh G, Saunders M, Lim L-Y (2010) Uptake and cytotoxicity of chitosan nanoparticles in human liver cells. Toxicol Appl Pharmacol 249:148–157

    Article  CAS  Google Scholar 

  • Loutfy S, Alam El-Din H, Elberry M, Allam N, Hasanin M, Abdellah A (2016) Synthesis, characterization and cytotoxic evaluation of chitosan nanoparticles: in vitro liver cancer model. Adv Nat Sci Nanosci Nanotechnol 7:035008

    Article  Google Scholar 

  • Nel A, Xia T, Madler L, Li N (2006) Toxic potential of materials at the nanolevel. Science (New York, NY) 311:622–627

    Article  CAS  Google Scholar 

  • Puvvada YS, Vankayalapati S, Sukhavasi S (2012) Extraction of chitin from chitosan from exoskeleton of shrimp for application in the pharmaceutical industry. Int Curr Pharm J 1:258–263

    Article  CAS  Google Scholar 

  • Qi L, Xu Z, Chen M (2007) In vitro and in vivo suppression of hepatocellular carcinoma growth by chitosan nanoparticles. Eur J Cancer (Oxford, England 1990) 43:184–193

    Article  CAS  Google Scholar 

  • Qi LF, Xu ZR, Li Y, Jiang X, Han XY (2005) In vitro effects of chitosan nanoparticles on proliferation of human gastric carcinoma cell line MGC803 cells. World J Gastroenterol 11:5136–5141

    CAS  Google Scholar 

  • Subhapradha N, Shanmugam A (2017) Fabrication of β-chitosan nanoparticles and its anticancer potential against human hepatoma cells. Int J Biol Macromol 94:194–201

    Article  CAS  Google Scholar 

  • Subhapradha N, Shanmugam V, Shanmugam A (2017) Chitosan nanoparticles from marine squid protect liver cells against N-diethylnitrosoamine-induced hepatocellular carcinoma. Carbohydr Polym 171:18–26

    Article  CAS  Google Scholar 

  • Torchilin VP (2006) Micellar nanocarriers: pharmaceutical perspectives. Pharm Res 24(1):1–16

    Article  Google Scholar 

  • Tsai GJ, Su WH (1999) Antibacterial activity of shrimp chitosan against Escherichia coli. J Food Prot 62:239–243

    Article  CAS  Google Scholar 

  • Wang H-Q, Sun X-B, Xu Y-X, Zhao H, Zhu Q-Y, Zhu C-Q (2010a) Astaxanthin upregulates heme oxygenase-1 expression through ERK1/2 pathway and its protective effect against beta-amyloid-induced cytotoxicity in SH-SY5Y cells. Brain Res 1360:159–167

    Article  CAS  Google Scholar 

  • Wang W, Shi J, Xie WF (2010b) Transarterial chemoembolization in combination with percutaneous ablation therapy in unresectable hepatocellular carcinoma: a meta-analysis. Liver Int 30:741–749

    Article  Google Scholar 

  • Wong RS (2011) Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin Cancer Res 30:87

    Article  CAS  Google Scholar 

  • Xu Y, Wen Z, Xu Z (2009) Chitosan nanoparticles inhibit the growth of human hepatocellular carcinoma xenografts through an antiangiogenic mechanism. Anticancer Res 29:5103–5109

    CAS  Google Scholar 

  • Zhao K, Zhao GM, Wu D, Soong Y, Birk AV, Schiller PW, Szeto HH (2004) Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury. J Biol Chem 279:34682–34690

    Article  CAS  Google Scholar 

  • Zuhorn IS, Kalicharan R, Hoekstra D (2002) Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent Clathrin-mediated pathway of endocytosis. J Biol Chem 277:18021–18028

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to Mohammed A. El-Magd.

Additional information

Responsible editor: Philippe Garrigues

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Elkeiy, M.M., Khamis, A.A., El-Gamal, M.M. et al. Chitosan nanoparticles from Artemia salina inhibit progression of hepatocellular carcinoma in vitro and in vivo. Environ Sci Pollut Res 27, 19016–19028 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: