The Effect of Chitosan-Silver Nanoparticles (Chi-AgNPs) Synthesized Using Clinacanthus nutans Extract on the Viability of MC3T3-E1 Preosteoblast Cells In Vitro

Sinta Candra Wardani*  -  Faculty of Dentistry, Universitas Brawijaya
Muhammad Farand Sheva Rozano  -  Faculty of Dentistry, Universitas Brawijaya
Ariyati Retno Pratiwi  -  Faculty of Dentistry, Universitas Brawijaya
Che Azurahanim Che Abdullah  -  Faculty of Science, Universiti Putra Malaysia
Rosnah Nawang  -  Institute of Nanoscience and Nanotechnology, Universiti Putra Malaysia

(*) Corresponding Author
DOI: 10.30659/odj.13.1.43-50

Background: Chi-AgNPs (chitosan–silver nanoparticles) are antibacterial biomaterials with good biocompatibility potential, but their toxicity to osteoblastic cells still needs to be studied. This study aims to analyze the effect of Chi-AgNPs synthesized using Clinacanthus nutans extract on the viability of MC3T3-E1 preosteoblast cells at several concentrations to determine the safe concentration limit for their use.

Method:  This laboratory experimental study used MC3T3-E1 cells exposed to Chi-AgNPs at concentrations of 12.5 µg/mL, 25 µg/mL, and 50 µg/mL, with a control group as a comparison. Cell viability was measured using the WST assay after 24 hours of incubation, then analyzed using the ANOVA test to see the differences between groups. 

Result: The results showed that a concentration of 12.5 µg/mL increased the average viability value by 30.8%, while a concentration of 25 µg/mL did not show a significant difference in cell viability compared to the control. Conversely, a concentration of 50 µg/mL caused a significant decrease in viability (p < 0.05), indicating toxicity related to the release of Ag⁺ ions and increased oxidative stress due to the small size of the nanoparticles (20 ± 3.93 nm). 

Conclusion: Chi-AgNPs are safe and biocompatible with MC3T3-E1 cells at concentrations below 25 µg/mL

Keywords: Cell viability; chitosan–silver nanoparticles; Cytotoxicity; MC3T3-E1

  1. Bruna T, Maldonado-Bravo F, Jara P, Caro N. Silver nanoparticles and their antibacterial applications. International Journal of Molecular Sciences. MDPI; 2021. doi:10.3390/ijms22137202 PubMed PMID: 34281254.
  2. Wahjuningrum DA, Subijanto A, Taqiya AK, Anggini FD, Hasib A, Mooduto L, et al. Cytotoxicity of Chitosan Derived from Shrimp for Bone Scaffold on Adipose Tissue-Derived Mesenchymal Stem Cells. Asia Pac J Med Toxicol [Internet]. 2019 Dec;115–7. Available from: http://apjmt.mums.ac.ir
  3. Meher A, Tandi A, Moharana S, Chakroborty S, Mohapatra SS, Mondal A, et al. Silver nanoparticle for biomedical applications: A review. Hybrid Advances. Elsevier B.V.; 2024. doi:10.1016/j.hybadv.2024.100184
  4. Monika A, Nari Ratih D, Hadriyanto W. Effect of Final Irrigation Materials and Techniques on Apical Sealing Ability of Bioceramic Sealer. Odonto : Dental Journal. 2025;12(3). doi:10.30659/odj.12.300-309
  5. Ali W, Ordoño J, Kopp A, González C, Echeverry-Rendón M, LLorca J. Cytocompatibility, cell-material interaction, and osteogenic differentiation of MC3T3-E1 pre-osteoblasts in contact with engineered Mg/PLA composites. J Biomed Mater Res A. 2024 Dec 1;112(12):2136–48. doi:10.1002/jbm.a.37767 PubMed PMID: 38899796.
  6. Nahian A, Davis DD. Histology, Osteoprogenitor Cells. StatPearls Publishing LLC; 2022.
  7. Afhkami F, Ahmadi P, Rostami G. Cytotoxicity of Different Concentrations of Silver Nanoparticles and Calcium Hydroxide for MC3T3-E1 Preosteoblast Cell Line. Clin Exp Dent Res. 2025 Feb 1;11(1). doi:10.1002/cre2.70075 PubMed PMID: 39967043.
  8. Kasimoglu Y, Tabakcilar D, A. Guclu Z, Yamamoto-Nemoto S, B. Tuna E, Ozen B, et al. Nanomaterials and Nanorobotics in Dentistry: A Review. Journal of Dentistry Indonesia. 2020 Aug 31;27(2). doi:10.14693/jdi.v27i2.1154
  9. Wardani SC, Sujuti H, Mustamsir E, Hapsari DN. Proliferation and viability of preosteoblast cells treated with Katsuwonus pelamis bone hydroxyapatite. In: AIP Conference Proceedings. American Institute of Physics Inc.; 2022. doi:10.1063/5.0098998
  10. Yakop F, Abd Ghafar SA, Yong YK, Saiful Yazan L, Mohamad Hanafiah R, Lim V, et al. Silver nanoparticles Clinacanthus Nutans leaves extract induced apoptosis towards oral squamous cell carcinoma cell lines. Artif Cells Nanomed Biotechnol. 2018 Nov 5;46(sup2):131–9. doi:10.1080/21691401.2018.1452750 PubMed PMID: 29561182.
  11. Chiu HI, Che Mood CNA, Mohamad Zain NN, Ramachandran MR, Yahaya N, Nik Mohamed Kamal NNS, et al. Biogenic Silver Nanoparticles of Clinacanthus nutans as Antioxidant with Antimicrobial and Cytotoxic Effects. Bioinorg Chem Appl. 2021;2021. doi:10.1155/2021/9920890
  12. Devasvaran K, Alallam B, Lee C, Yong YK, Lim V. Clinacanthus nutans crude polysaccharide extract as a green platform for microwave-assisted synthesis of silver nanoparticles: Optimization, characterization, and evaluation of bioactivities. Int J Biol Macromol. 2024 Oct 1;278. doi:10.1016/j.ijbiomac.2024.134893 PubMed PMID: 39168213.
  13. Haugen HJ, Makhtari S, Ahmadi S, Hussain B. The Antibacterial and Cytotoxic Effects of Silver Nanoparticles Coated Titanium Implants: A Narrative Review. Materials. MDPI; 2022. doi:10.3390/ma15145025
  14. Victor R de S, Santos AM da C, de Sousa BV, Neves G de A, Santana LN de L, Menezes RR. A review on Chitosan’s uses as biomaterial: Tissue engineering, drug delivery systems and cancer treatment. Materials. MDPI AG; 2020. p. 1–71. doi:10.3390/ma13214995
  15. Shehabeldine AM, Salem SS, Ali OM, Abd-Elsalam KA, Elkady FM, Hashem AH. Multifunctional Silver Nanoparticles Based on Chitosan: Antibacterial, Antibiofilm, Antifungal, Antioxidant, and Wound-Healing Activities. Journal of Fungi. 2022 Jun 1;8(6). doi:10.3390/jof8060612
  16. Pachghare P, Zambre D, Malode D, Kale M, Wankhede N, Trivedi R, et al. Combating multi-drug resistance with silver nanoparticles: A systematic review. Microbe (Netherlands). 2025 Dec 1;9. doi:10.1016/j.microb.2025.100608
  17. Anees Ahmad S, Sachi Das S, Khatoon A, Tahir Ansari M, Afzal M, Saquib Hasnain M, et al. Bactericidal activity of silver nanoparticles: A mechanistic review. Materials Science for Energy Technologies. KeAi Communications Co.; 2020. p. 756–69. doi:10.1016/j.mset.2020.09.002
  18. Thakkar AB, Subramanian RB, Thakkar VR, Bhatt S V., Chaki S, Vaidya YH, et al. Apoptosis induction capability of silver nanoparticles capped with Acorus calamus L. and Dalbergia sissoo Roxb. Ex DC. against lung carcinoma cells. Heliyon. 2024 Jan 30;10(2). doi:10.1016/j.heliyon.2024.e24400
  19. Quevedo AC, Lynch I, Valsami-Jones E. Mechanisms of silver nanoparticle uptake by embryonic zebrafish cells. Nanomaterials. 2021 Oct 1;11(10). doi:10.3390/nano11102699
  20. Martínez-Cisterna D, Chen L, Bardehle L, Hermosilla E, Tortella G, Chacón-Fuentes M, et al. Chitosan-Coated Silver Nanocomposites: Biosynthesis, Mechanical Properties, and Ag+ Release in Liquid and Biofilm Forms. Int J Mol Sci. 2025 May 1;26(9). doi:10.3390/ijms26094130 PubMed PMID: 40362370.
  21. Holmila RJ, Vance SA, King SB, Tsang AW, Singh R, Furdui CM. Silver nanoparticles induce mitochondrial protein oxidation in lung cells impacting cell cycle and proliferation. Antioxidants. 2019 Nov 1;8(11). doi:10.3390/antiox8110552
  22. He J, Ma Y, Niu X, Pei J, Yan R, Xu F, et al. Silver nanoparticles induce endothelial cytotoxicity through ROS-mediated mitochondria-lysosome damage and autophagy perturbation: The protective role of N-acetylcysteine. Toxicology. 2024 Feb 1;502. doi:10.1016/j.tox.2024.153734 PubMed PMID: 38290605.
  23. Ferdous Z, Nemmar A. Health impact of silver nanoparticles: A review of the biodistribution and toxicity following various routes of exposure. International Journal of Molecular Sciences. MDPI AG; 2020. doi:10.3390/ijms21072375 PubMed PMID: 32235542.
  24. Gurunathan S, Qasim M, Park C, Yoo H, Choi DY, Song H, et al. Cytotoxicity and transcriptomic analysis of silver nanoparticles in mouse embryonic fibroblast cells. Int J Mol Sci. 2018 Nov 16;19(11). doi:10.3390/ijms19113618 PubMed PMID: 30453526.
  25. Chang X, Niu S, Shang M, Li J, Guo M, Zhang W, et al. ROS-Drp1-mediated mitochondria fission contributes to hippocampal HT22 cell apoptosis induced by silver nanoparticles. Redox Biol. 2023 Jul 1;63. doi:10.1016/j.redox.2023.102739 PubMed PMID: 37187014.

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