{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T06:07:07Z","timestamp":1785305227678,"version":"3.55.0"},"reference-count":42,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2025,6,10]],"date-time":"2025-06-10T00:00:00Z","timestamp":1749513600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004281","name":"PRELUDIUM grant","doi-asserted-by":"publisher","award":["2017\/27\/N\/NZ7\/02675"],"award-info":[{"award-number":["2017\/27\/N\/NZ7\/02675"]}],"id":[{"id":"10.13039\/501100004281","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>This study examines the cytotoxicity of two silver nanoparticle formulations\u2014AgNPs conjugated with chlorhexidine (AgNPs-CHL) and AgNPs conjugated with polyethylene glycol and metronidazole (AgNPs-PEG-MET)\u2014as examples of the surface functionalization of silver nanoparticles with drugs via sulfur\u2013silver bonds and nitrogen\u2013silver interactions. We previously reported the synthesis of these NPs and their efficiency in periodontitis treatment. Here, we analyze the relationships between the cytotoxic mechanisms of AgNPs and their surface chemistry. UV\u2013Vis spectroscopy, dynamic light scattering (DLS), and scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDX) were used for physicochemical studies of the conjugates in two environments: aqueous solutions and commonly used cell culture media. Cytotoxicity was assessed in human fetal osteoblasts (hFOB 1.19) and human gingival fibroblasts (HGF-1) through BrdU and LDH assays, ROS detection, cell cycle analysis, apoptosis assays, and protein expression studies. AgNPs-CHL showed aggregation and increased hydrodynamic diameters in the culture medium, while AgNPs-PEG-MET remained stable. Both exhibited concentration-dependent cytotoxicity: AgNPs-CHL at 0.4\u201310 \u03bcg\/mL and AgNPs-PEG-MET at 0.75\u201310 \u03bcg\/mL. AgNPs-CHL, in which silver surface functionalization was realized via nitrogen\u2013silver interactions, induced significant ROS generation, LDH release, and necroptosis, marked by increased RIP1, RIP3, and MLKL proteins. In the case of AgNPs-PEG-MET, where sulfur\u2013silver bonds combined the drug via a PEG linker, they triggered apoptosis, as evidenced by elevated caspase-2 levels and flow cytometry. These findings highlight that the type of surface functionalization of silver nanoparticles significantly influences their physicochemical behavior and biological effects. Understanding these mechanisms is crucial in designing safer, more effective nanoparticle-based therapies for periodontal and other inflammatory conditions.<\/jats:p>","DOI":"10.3390\/biom15060850","type":"journal-article","created":{"date-parts":[[2025,6,10]],"date-time":"2025-06-10T12:53:16Z","timestamp":1749559996000},"page":"850","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Functionalization-Dependent Cytotoxicity of Silver Nanoparticles: A Comparative Study of Chlorhexidine and Metronidazole Conjugates"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5353-9849","authenticated-orcid":false,"given":"Karol P.","family":"Steckiewicz","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Pathophysiology, Faculty of Pharmacy, Medical University of Gdansk, 80-211 Gdansk, Poland"},{"name":"Department of Anesthesiology and Intensive Therapy, Faculty of Medicine, Medical University of Gdansk, 80-214 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monika","family":"Dmochowska","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Pathophysiology, Faculty of Pharmacy, Medical University of Gdansk, 80-211 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3422-9017","authenticated-orcid":false,"given":"El\u017cbieta","family":"Megiel","sequence":"additional","affiliation":[{"name":"Faculty of Chemistry, University of Warsaw, 02-093 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ewelina","family":"Barci\u0144ska","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Pathophysiology, Faculty of Pharmacy, Medical University of Gdansk, 80-211 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1676-2723","authenticated-orcid":false,"given":"Iwona","family":"Inkielewicz-St\u0119pniak","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Pathophysiology, Faculty of Pharmacy, Medical University of Gdansk, 80-211 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1931","DOI":"10.1002\/jps.24001","article-title":"Silver Nanoparticles: Therapeutical Uses, Toxicity, and Safety Issues","volume":"103","author":"Seckler","year":"2014","journal-title":"J. Pharm. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1143","DOI":"10.1016\/j.drudis.2015.05.009","article-title":"Metal Nanoparticles: A Theranostic Nanotool against Cancer","volume":"20","author":"Sharma","year":"2015","journal-title":"Drug Discov. Today"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Wu, J., Zheng, Y., Wen, X., Lin, Q., Chen, X., and Wu, Z. (2014). Silver Nanoparticle\/Bacterial Cellulose Gel Membranes for Antibacterial Wound Dressing: Investigation in Vitro and in Vivo. Biomed. Mater. Bristol Engl., 9.","DOI":"10.1088\/1748-6041\/9\/3\/035005"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2561","DOI":"10.1016\/j.biopha.2018.11.116","article-title":"Anti-Inflammatory Mechanism of Various Metal and Metal Oxide Nanoparticles Synthesized Using Plant Extracts: A Review","volume":"109","author":"Agarwal","year":"2019","journal-title":"Biomed. Pharmacother. Biomed. Pharmacother."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1517\/17425247.4.6.621","article-title":"Advances and Challenges of Nanotechnology-Based Drug Delivery Systems","volume":"4","author":"Jiang","year":"2007","journal-title":"Expert Opin. Drug Deliv."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1593","DOI":"10.1007\/s42247-021-00335-x","article-title":"Review on Metal Nanoparticles as Nanocarriers: Current Challenges and Perspectives in Drug Delivery Systems","volume":"5","author":"Chandrakala","year":"2022","journal-title":"Emergent Mater."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1111\/j.1365-2672.2012.05253.x","article-title":"Silver Nanoparticles: The Powerful Nanoweapon against Multidrug-resistant Bacteria","volume":"112","author":"Rai","year":"2012","journal-title":"J. Appl. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"8856","DOI":"10.3390\/molecules20058856","article-title":"Silver Nanoparticles as Potential Antibacterial Agents","volume":"20","author":"Franci","year":"2015","journal-title":"Molecules"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"588","DOI":"10.1016\/j.tibtech.2016.02.006","article-title":"Biological Synthesis of Nanoparticles from Plants and Microorganisms","volume":"34","author":"Singh","year":"2016","journal-title":"Trends Biotechnol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"13608","DOI":"10.1021\/jp712087m","article-title":"Unique Cellular Interaction of Silver Nanoparticles: Size-Dependent Generation of Reactive Oxygen Species","volume":"112","author":"Carlson","year":"2008","journal-title":"J. Phys. Chem. B"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1186\/1471-2121-10-65","article-title":"Anti-Proliferative Activity of Silver Nanoparticles","volume":"10","author":"Asharani","year":"2009","journal-title":"BMC Cell Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"4706","DOI":"10.1016\/j.biomaterials.2014.02.021","article-title":"Cytotoxicity, Oxidative Stress, Apoptosis and the Autophagic Effects of Silver Nanoparticles in Mouse Embryonic Fibroblasts","volume":"35","author":"Lee","year":"2014","journal-title":"Biomaterials"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"573","DOI":"10.3906\/biy-1302-21","article-title":"Silver Nanoparticles: Cytotoxic, Apoptotic, and Necrotic Effects on MCF-7 Cells","volume":"37","author":"Turk","year":"2013","journal-title":"Turk. J. Biol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1016\/j.tiv.2010.11.008","article-title":"ROS-Mediated Genotoxicity Induced by Titanium Dioxide Nanoparticles in Human Epidermal Cells","volume":"25","author":"Shukla","year":"2011","journal-title":"Toxicol. In Vitro"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1841","DOI":"10.1016\/j.cca.2010.08.016","article-title":"Silver Nanoparticle Applications and Human Health","volume":"411","author":"Ahamed","year":"2010","journal-title":"Clin. Chim. Acta"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/1743-8977-11-11","article-title":"Size-Dependent Cytotoxicity of Silver Nanoparticles in Human Lung Cells: The Role of Cellular Uptake, Agglomeration and Ag Release","volume":"11","author":"Gliga","year":"2014","journal-title":"Part. Fibre Toxicol."},{"key":"ref_17","first-page":"1","article-title":"Concentration- and Time-Dependent Cytotoxicity of Silver Nanoparticles on Normal Human Skin Fibroblast Cell Line","volume":"20","author":"Paknejadi","year":"2024","journal-title":"Iran. Red Crescent Med. J. IRCMJ"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Conde, J., Dias, J.T., Graz\u00fa, V., Moros, M., Baptista, P.V., and de la Fuente, J.M. (2014). Revisiting 30 Years of Biofunctionalization and Surface Chemistry of Inorganic Nanoparticles for Nanomedicine. Front. Chem., 2.","DOI":"10.3389\/fchem.2014.00048"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2050","DOI":"10.1073\/pnas.0608582104","article-title":"Understanding the Nanoparticle\u2013Protein Corona Using Methods to Quantify Exchange Rates and Affinities of Proteins for Nanoparticles","volume":"104","author":"Cedervall","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"495","DOI":"10.2147\/IJN.S339046","article-title":"Silver Nanoparticles as Chlorhexidine and Metronidazole Drug Delivery Platforms: Their Potential Use in Treating Periodontitis","volume":"17","author":"Steckiewicz","year":"2022","journal-title":"Int. J. Nanomed."},{"key":"ref_21","first-page":"e0403","article-title":"Effect of Metronidazole Microcapsules with Silver Nanoparticles on the Efficacy of Alginate Gel in the Correction of Structural and Functional Periodontium Disorders in Rats with Periodontitis","volume":"4","author":"Savkina","year":"2023","journal-title":"Saratov Med. J."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"354","DOI":"10.1016\/j.identj.2023.01.002","article-title":"A Novel Antimicrobial Hydrogel for the Management of Periodontal Diseases","volume":"73","author":"Wong","year":"2023","journal-title":"Int. Dent. J."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"715","DOI":"10.21608\/edj.2023.237424.2719","article-title":"The Impact of Silver Nanoparticles, 2% Chlorhexidine and Silver Nanoparticles Loaded in Chlorhexidine on Radicular Dentin Microhardness (An In-Vitro Comparative Study)","volume":"70","author":"Fahmy","year":"2024","journal-title":"Egypt. Dent. J."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"58403","DOI":"10.1039\/C5RA09366J","article-title":"Nitroxide-Coated Silver Nanoparticles: Synthesis, Surface Physicochemistry and Antibacterial Activity","volume":"5","author":"Gozdziewska","year":"2015","journal-title":"RSC Adv."},{"key":"ref_25","unstructured":"Hodoroaba, V.-D., Unger, W.E.S., and Shard, A.G. (2020). Chapter 3.2.1\u2014Dynamic Light Scattering (DLS). Characterization of Nanoparticles, Elsevier. Micro and Nano Technologies."},{"key":"ref_26","first-page":"183","article-title":"PEG in Aqueous Salt Solutions. Viscosity and Separation Ability in a TiO2 Suspension","volume":"58","author":"Brunchi","year":"2013","journal-title":"Rev. Roum. Chim"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1007\/s12668-017-0423-1","article-title":"Green Synthesis of Different Shapes of Silver Nanostructures and Evaluation of Their Antibacterial and Cytotoxic Activity","volume":"8","author":"Soleimani","year":"2018","journal-title":"BioNanoScience"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/j.toxlet.2016.03.005","article-title":"The Influence of Citrate or PEG Coating on Silver Nanoparticle Toxicity to a Human Keratinocyte Cell Line","volume":"249","author":"Bastos","year":"2016","journal-title":"Toxicol. Lett."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/j.etap.2017.04.010","article-title":"Silver Nanoparticle Induced Toxicity to Human Sperm by Increasing ROS(Reactive Oxygen Species) Production and DNA Damage","volume":"52","author":"Wang","year":"2017","journal-title":"Environ. Toxicol. Pharmacol."},{"key":"ref_30","first-page":"10293","article-title":"Surface-Coating-Dependent Dissolution, Aggregation, and Reactive Oxygen Species (ROS) Generation of Silver Nanoparticles under Different Irradiation Conditions","volume":"47","author":"Li","year":"2013","journal-title":"Environ. Sci. Technol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1316","DOI":"10.1007\/s12011-020-02255-z","article-title":"Silver Nanoparticles Synthesized Using Carica Papaya Leaf Extract (AgNPs-PLE) Causes Cell Cycle Arrest and Apoptosis in Human Prostate (DU145) Cancer Cells","volume":"199","author":"Singh","year":"2021","journal-title":"Biol. Trace Elem. Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"104084","DOI":"10.1016\/j.jddst.2022.104084","article-title":"ROS Mediated Apoptosis and Cell Cycle Arrest in Human Lung Adenocarcinoma Cell Line by Silver Nanoparticles Synthesized Using Swietenia Macrophylla Seed Extract","volume":"80","author":"Naik","year":"2023","journal-title":"J. Drug Deliv. Sci. Technol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Yang, Y., Guo, L., Wang, Z., Liu, P., Liu, X., Ding, J., and Zhou, W. (2021). Targeted Silver Nanoparticles for Rheumatoid Arthritis Therapy via Macrophage Apoptosis and Re-Polarization. Biomaterials, 264.","DOI":"10.1016\/j.biomaterials.2020.120390"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1016\/j.fct.2015.06.012","article-title":"Flow Cytometry Evaluation of in Vitro Cellular Necrosis and Apoptosis Induced by Silver Nanoparticles","volume":"85","author":"Kumar","year":"2015","journal-title":"Food Chem. Toxicol. Int. J. Publ. Br. Ind. Biol. Res. Assoc."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"5489","DOI":"10.1039\/C6NR08188F","article-title":"Activation of Autophagy by Elevated Reactive Oxygen Species Rather than Released Silver Ions Promotes Cytotoxicity of Polyvinylpyrrolidone-Coated Silver Nanoparticles in Hematopoietic Cells","volume":"9","author":"Zhu","year":"2017","journal-title":"Nanoscale"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"4675","DOI":"10.18632\/oncotarget.22563","article-title":"Silver Nanoparticles of Different Sizes Induce a Mixed Type of Programmed Cell Death in Human Pancreatic Ductal Adenocarcinoma","volume":"9","author":"Zielinska","year":"2017","journal-title":"Oncotarget"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"375102","DOI":"10.1088\/0957-4484\/23\/37\/375102","article-title":"The Influence of the Surface Chemistry of Silver Nanoparticles on Cell Death","volume":"23","author":"Sur","year":"2012","journal-title":"Nanotechnology"},{"key":"ref_38","unstructured":"(2025, May 15). The Role of Interaction between Autophagy and Apoptosis in Tumorigenesis (Review). Available online: https:\/\/www.spandidos-publications.com\/10.3892\/or.2022.8423."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1007\/s10495-021-01663-3","article-title":"Release Mechanisms of Major DAMPs","volume":"26","author":"Murao","year":"2021","journal-title":"Apoptosis"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Sanit\u00e0, G., Carrese, B., and Lamberti, A. (2020). Nanoparticle Surface Functionalization: How to Improve Biocompatibility and Cellular Internalization. Front. Mol. Biosci., 7.","DOI":"10.3389\/fmolb.2020.587012"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1080\/15548627.2018.1509171","article-title":"Necrotic, Apoptotic and Autophagic Cell Fates Triggered by Nanoparticles","volume":"15","author":"Mohammadinejad","year":"2019","journal-title":"Autophagy"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Gieleci\u0144ska, A., Kciuk, M., Yahya, E.-B., Ainane, T., Mujwar, S., and Kontek, R. (2023). Apoptosis, Necroptosis, and Pyroptosis as Alternative Cell Death Pathways Induced by Chemotherapeutic Agents?. Biochim. Biophys. Acta BBA-Rev. Cancer, 1878.","DOI":"10.1016\/j.bbcan.2023.189024"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/15\/6\/850\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:49:36Z","timestamp":1760032176000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/15\/6\/850"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,6,10]]},"references-count":42,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2025,6]]}},"alternative-id":["biom15060850"],"URL":"https:\/\/doi.org\/10.3390\/biom15060850","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,6,10]]}}}