Newly developed chitosan-silver hybrid nanoparticles: biosafety and apoptosis induction in HepG2 cells

  • Ibrahim M. El-SherbinyEmail author
  • Ehab Salih
  • Abdelrahman M. Yassin
  • Elsayed E. Hafez
Research Paper


The present study reports the biosafety assessment, the exact molecular effects, and apoptosis induction of newly developed chitosan-silver hybrid nanoparticles (Cs–Ag NPs) in HepG2 cells. The investigated hybrid NPs were green synthesized using Cs/grape leaves aqueous extract (Cs/GLE) or Cs/GLE NPs as reducing and stabilizing agents. The successful formation of Cs/GLE NPs and Cs–Ag hybrid NPs has been confirmed by UV–Vis spectrophotometry, FTIR spectroscopy, XRD, and HRTEM. From the TEM analysis, the prepared Cs/GLE NPs are uniform and spherical with an average size of 150 nm, and the AgNPs (5–10 nm) were formed mainly on their surface. The UV–Vis spectra of Cs–Ag NPs showed a surface plasmon resonance (SPR) peak at about 450 nm confirming their formation. The synthesized Cs–Ag NPs were found to be crystalline as shown by XRD patterns with fcc phase oriented along the (111), (200), (220), and (311) planes. The cytotoxicity patterns, the antiproliferative activities, and the possible mechanisms of anticancer activity at molecular level of the newly developed Cs–Ag hybrid NPs were investigated. Cytotoxicity patterns of all the preparations demonstrated that the nontoxic treatment concentrations are ranged from 0.39 to 50 %, and many of the newly prepared Cs–Ag hybrid NPs showed high anticancer activities against HpG2 cells, and induced cellular apoptosis by downregulating BCL2 gene and upregulating P53.

Graphical Abstract


Chitosan Silver Nanoparticles Hepatocellular Carcinoma Cytotoxicity Health effects 


  1. Baharara J, Namvar F, Ramezani T, Mousavi M, Mohamad R (2015) Silver Nanoparticles Biosynthesized using Achillea biebersteinii flower extract: apoptosis induction in MCF-7 Cells via caspase activation and regulation of bax and bcl-2. Gene Expr Mol 20:2693–2706. doi: 10.3390/molecules20022693 Google Scholar
  2. Berrada M, Serreqi A, Dabbarh F, Owusu A, Gupta A, Lehnert S (2005) A novel non-toxic camptothecin formulation for cancer chemotherapy. Biomaterials 26:2115–2120. doi: 10.1016/j.biomaterials.2004.06.013 CrossRefGoogle Scholar
  3. Creighton JR, Coltrin ME, Figiel JJ (2008) Observations of gas-phase nanoparticles during InGaN metal-organic chemical vapor deposition. Appl Phys Lett 93:171906. doi: 10.1063/1.3009291 CrossRefGoogle Scholar
  4. Das S, Chaudhury A (2011) Recent advances in lipid nanoparticle formulations with solid matrix for oral drug delivery. AAPS Pharm Sci Tech 12:62–76. doi: 10.1208/s12249-010-9563-0 CrossRefGoogle Scholar
  5. de Freitas VA, Glories Y, Bourgeois G, Vitry C (1998) Characterisation of oligomeric and polymeric procyanidins from grape seeds by liquid secondary ion mass spectrometry. Phytochemistry 49:1435–1441. doi: 10.1016/S0031-9422(98)00107-1 CrossRefGoogle Scholar
  6. Du J, El-Sherbiny IM, Smyth HD (2014) Swellable ciprofloxacin-loaded nano-in-micro hydrogel particles for local lung drug delivery. AAPS PharmSciTech 15:1535–1544. doi: 10.1208/s12249-014-0176-x CrossRefGoogle Scholar
  7. El-Deeb NM, El-Sherbiny IM, El-Aassara MR, Hafez EE (2015) Novel trend in colon cancer therapy using silver nanoparticles synthesized by honey bee. J Nanomed Nanotechnol 6:2. doi: 10.4172/2157-7439.1000265 Google Scholar
  8. El-Sherbiny IM (2010) Enhanced pH-responsive carrier system based on alginate and chemically modified carboxymethyl chitosan for oral delivery of protein drugs: preparation and in vitro assessment. Carbohydr Polym 80:1125–1136. doi: 10.1016/j.carbpol.2010.01.034 CrossRefGoogle Scholar
  9. El-Sherbiny IM, Smyth HD (2010) Biodegradable nano-micro carrier systems for sustained pulmonary drug delivery:(I) self-assembled nanoparticles encapsulated in respirable/swellable semi-IPN microspheres. Intl J pharm 395:132–141. doi: 10.1016/j.ijpharm.2010.05.032 CrossRefGoogle Scholar
  10. El-Sherbiny IM, Salih E, Reicha FM (2013) Green synthesis of densely dispersed and stable silver nanoparticles using myrrh extract and evaluation of their antibacterial activity. J Nanostructure Chem 3:1–7. doi: 10.1186/2193-8865-3-8 CrossRefGoogle Scholar
  11. El-Sherbiny I, Salih E, Reicha F (2015) New trimethyl chitosan-based composite nanoparticles as promising antibacterial agents. Drug Dev Ind Pharm 42:720–729. doi: 10.3109/03639045.2015.1075035 CrossRefGoogle Scholar
  12. El-Sherbiny IM, El-Shibiny A, Salih E (2016a) Photo-induced green synthesis and antimicrobial efficacy of poly (ɛ-caprolactone)/curcumin/grape leaf extract-silver hybrid nanoparticles. J Photochem Photobiol B: Biol 160:355–363. doi: 10.1016/j.jphotobiol.2016.04.029 CrossRefGoogle Scholar
  13. El-Sherbiny IM, Hefnawy A, Salih E (2016b) New core–shell hyperbranched chitosan-based nanoparticles as optical sensor for ammonia detection. Intl J biol macromol 86:782–788. doi: 10.1016/j.ijbiomac.2016.01.118 CrossRefGoogle Scholar
  14. Fan P, Lou H (2004) Effects of polyphenols from grape seeds on oxidative damage to cellular DNA. Mol Cell Biochem 267:67–74. doi: 10.1023/B:MCBI.0000049366.75461.00 CrossRefGoogle Scholar
  15. Gittins DI, Bethell D, Schiffrin DJ, Nichols RJ (2000) A nanometre-scale electronic switch consisting of a metal cluster and redox-addressable groups. Nature 408:67–69. doi: 10.1038/35040518 CrossRefGoogle Scholar
  16. Govender R, Phulukdaree A, Gengan RM, Anand K, Chuturgoon AA (2013) Silver nanoparticles of Albizia adianthifolia: the induction of apoptosis in human lung carcinoma cell line. J Nanobiotechnol 5:11. doi: 10.1186/1477-3155-11-5 Google Scholar
  17. Gurunathan S et al (2013) Green synthesis of anisotropic silver nanoparticles and its potential cytotoxicity in human breast cancer cells (MCF-7). J Ind Eng Chem 19:1600–1605. doi: 10.1016/j.jiec.2013.01.029 CrossRefGoogle Scholar
  18. Hsin Y-H, Chen C-F, Huang S, Shih T-S, Lai P-S, Chueh PJ (2008) The apoptotic effect of nanosilver is mediated by a ROS-and JNK-dependent mechanism involving the mitochondrial pathway in NIH3T3 cells. Toxicol Lett 179:130–139. doi: 10.1016/j.toxlet.2008.04.015 CrossRefGoogle Scholar
  19. Huang NM, Lim HN, Radiman S, Khiew PS, Chiu WS, Hashim R, Chia CH (2010) Sucrose ester micellar-mediated synthesis of Ag nanoparticles and the antibacterial properties. Coll Surf Asp 353:69–76. doi: 10.1016/j.colsurfa.2009.10.023 CrossRefGoogle Scholar
  20. Ishikawa Y, Shibata N, Fukatsu S (1997) Highly oriented Si nanoparticles in SiO2 created by Si molecular beam epitaxy with oxygen implantation. Thin sol film 294:227–230. doi: 10.1016/S0040-6090(96)09215-2 CrossRefGoogle Scholar
  21. Jacob SJP, Finub J, Narayanan A (2012) Synthesis of silver nanoparticles using Piper longum leaf extracts and its cytotoxic activity against Hep-2 cell line. Coll Surf 91:212–214. doi: 10.1016/j.colsurfb.2011.11.001 CrossRefGoogle Scholar
  22. Kanchana A, Balakrishna M (2011) Anti-cancer effect of saponins isolated from solanum trilobatum leaf extract and induction of apoptosis in human larynx cancer cell lines. Intl J pharm pharm sci 3:356–364Google Scholar
  23. Kaviya S, Santhanalakshmi J, Viswanathan B, Muthumary J, Srinivasan K (2011) Biosynthesis of silver nanoparticles using Citrus sinensis peel extract and its antibacterial activity. Spectrochim Acta Part A Mol Biomol Spectrosc 79:594–598. doi: 10.1016/j.saa.2011.03.040 CrossRefGoogle Scholar
  24. Miculescu M, Thakur VK, Miculescu F, Voicu SI (2016) Graphene-based polymer nanocomposite membranes: a review. Polym Adv Technol. doi: 10.1002/pat.3751 Google Scholar
  25. Mukherjee P et al (2008) Green synthesis of highly stabilized nanocrystalline silver particles by a non-pathogenic and agriculturally important fungus T. asperellum. Nanotechnology 19:075103. doi: 10.1088/0957-4484/19/7/075103 CrossRefGoogle Scholar
  26. Mulvaney P (1996) Surface plasmon spectroscopy of nanosized metal particles. Langmuir 12:788–800. doi: 10.1021/la9502711 CrossRefGoogle Scholar
  27. Muzzarelli RA (2011) Potential of chitin/chitosan-bearing materials for uranium recovery: an interdisciplinary review. Carbohydr Polym 84:54–63. doi: 10.1016/j.carbpol.2010.12.025 CrossRefGoogle Scholar
  28. Muzzarelli R, Tarsi R, Filippini O, Giovanetti E, Biagini G, Varaldo P (1990) Antimicrobial properties of N-carboxybutyl chitosan. Antimicrob agents chemotherap 34:2019–2023. doi: 10.1128/AAC.34.10.2019 CrossRefGoogle Scholar
  29. No HK, Park NY, Lee SH, Meyers SP (2002) Antibacterial activity of chitosans and chitosan oligomers with different molecular weights. Intl J Food microbiol 74:65–72. doi: 10.1016/S0168-1605(01)00717-6 CrossRefGoogle Scholar
  30. Park Y (2014) New paradigm shift for the green synthesis of antibacterial silver nanoparticles utilizing plant extracts. Toxicol res 30:169. doi: 10.5487/TR.2014.30.3.169 CrossRefGoogle Scholar
  31. Park S et al (2007) Cellular toxicity of various inhalable metal nanoparticles on human alveolar epithelial cells. Inhal toxicol 19:59–65. doi: 10.1080/08958370701493282 CrossRefGoogle Scholar
  32. Pastrana-Bonilla E, Akoh CC, Sellappan S, Krewer G (2003) Phenolic content and antioxidant capacity of muscadine grapes. J Agric Food Chem 51:5497–5503. doi: 10.1021/jf030113c CrossRefGoogle Scholar
  33. Prabhu D, Arulvasu C, Babu G, Manikandan R, Srinivasan P (2013) Biologically synthesized green silver nanoparticles from leaf extract of Vitex negundo L. induce growth-inhibitory effect on human colon cancer cell line HCT15. Process Biochem 48:317–324. doi: 10.1016/j.procbio.2012.12.013 CrossRefGoogle Scholar
  34. Renwick L, Brown D, Clouter A, Donaldson K (2004) Increased inflammation and altered macrophage chemotactic responses caused by two ultrafine particle types. Occup Environ Med 61:442–447. doi: 10.1136/oem.2003.008227 CrossRefGoogle Scholar
  35. Salih E, Reicha F, El-Sherbiny I (2016) Electrochemical synthesis of new silver–chitosan/polyvinyl alcohol hybrid nanoparticles and evaluation of their antibacterial activities. J Nanosci Technol 2:94–96Google Scholar
  36. Seo JA, Koh JH, Roh DK, Kim JH (2009) Preparation and characterization of crosslinked proton conducting membranes based on chitosan and PSSA-MA copolymer. Sol State Ion 180:998–1002. doi: 10.1016/j.ssi.2009.04.003 CrossRefGoogle Scholar
  37. Shameli K, Ahmad MB, Yunus WMZW, Rustaiyan A, Ibrahim NA, Zargar M, Abdollahi Y (2010) Green synthesis of silver/montmorillonite/chitosan bionanocomposites using the UV irradiation method and evaluation of antibacterial activity. Intl J Nanomed 5:875–887. doi: 10.2147/IJN.S13632 CrossRefGoogle Scholar
  38. Thakur VK, Gupta RK (2016) recent progress on ferroelectric polymer-based nanocomposites for high energy density capacitors: synthesis, dielectric properties, and future aspects. Chem Rev 116:4260–4317. doi: 10.1021/acs.chemrev.5b00495 CrossRefGoogle Scholar
  39. Thakur VK, Kessler MR (2015) Self-healing polymer nanocomposite materials: a review. Polymer 69:369–383. doi: 10.1016/j.polymer.2015.04.086 CrossRefGoogle Scholar
  40. Thakur VK, Thakur MK (2014) Recent advances in graft copolymerization and applications of chitosan: a review. ACS Sustain Chem Eng 2:2637–2652. doi: 10.1021/sc500634p CrossRefGoogle Scholar
  41. Thakur VK, Voicu SI (2016) Recent advances in cellulose and chitosan based membranes for water purification: a concise review. Carbohydr Polym 146:148–165. doi: 10.1016/j.carbpol.2016.03.030 CrossRefGoogle Scholar
  42. Thomas V, Yallapu MM, Sreedhar B, Bajpai S (2009) Fabrication, characterization of chitosan/nanosilver film and its potential antibacterial application. J Biomat Sci Polym Ed 20:2129–2144. doi: 10.1163/156856209X410102 CrossRefGoogle Scholar
  43. Vigneshwaran N, Kathe AA, Varadarajan PV, Nachane RP, Balasubramanya RH (2007) Silver-protein (core-shell) nanoparticle production using spent mushroom substrate. Langmuir 23:7113–7117. doi: 10.1021/la063627p CrossRefGoogle Scholar
  44. Xia E-Q, Deng G-F, Guo Y-J, Li H-B (2010) Biological activities of polyphenols from grapes. Intl J mol Sci 11:622–646. doi: 10.3390/ijms11020622 CrossRefGoogle Scholar
  45. Xu H, Yao L, Sun H, Wu Y (2009) Chemical composition and antitumor activity of different polysaccharides from the roots of Actinidia eriantha. Carbohydr Polym 78:316–322. doi: 10.1016/j.carbpol.2009.04.007 CrossRefGoogle Scholar
  46. Yilmaz M, Turkdemir H, Kilic MA, Bayram E, Cicek A, Mete A, Ulug B (2011) Biosynthesis of silver nanoparticles using leaves of Stevia rebaudiana. Mater Chem Phys 130:1195–1202. doi: 10.1016/j.matchemphys.2011.08.068 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Ibrahim M. El-Sherbiny
    • 1
    Email author
  • Ehab Salih
    • 1
  • Abdelrahman M. Yassin
    • 2
  • Elsayed E. Hafez
    • 3
  1. 1.Center for Materials ScienceZewail City of Science and TechnologyGizaEgypt
  2. 2.Biopharmaceutical Product Research DepartmentGenetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technology ApplicationsAlexandriaEgypt
  3. 3.Plant Protection and Biomolecular Diagnosis DepartmentCity of Scientific Research and Technology ApplicationsAlexandriaEgypt

Personalised recommendations