FOLİK ASİT VE CHELİDONİC ASİT İLE KO-FONKSİYONELİZE EDİLMİŞ GÜMÜŞ NANOPARTİKÜLLER: HELA VE L929 HÜCRELERİNDE FARKLI SİTOTOKSİK VE OKSİDATİF STRES YANITLARI

Öz

Gümüş nanopartiküller sağlıklı hücrelerde toksisite ve reaktif oksijen türleri (ROS) birikimine neden olmasına rağmen güçlü bir antikanser potansiyel sergilemektedir. Bu çalışmanın amacı, gümüş nanopartiküllerin sağlıklı hücrelerde oluşturduğu toksisiteyi ve ROS aktivitesini azaltırken, kanser hücrelerini seçici olarak hedefleyen bir nanopartikül sistemi geliştirmektir. Bu doğrultuda folik asit (FA) ve chelidonic acid (CA) gümüş nanopartiküllere konjuge edilmiştir. FA, kanser hücrelerinde aşırı eksprese edilen folat reseptörleri aracılığıyla hedeflenmeyi artırarak sitotoksisiteyi güçlendirmiş; CA ise sağlıklı hücrelerde antioksidan kapasiteyi artırarak koruyucu etki sağlamıştır. Böylece çift işlevli bir nanopartikül konjugatı (AgNP/FA/CA) elde edilmiştir. Elde edilen konjugatların fizikokimyasal karakterizasyonu dinamik ışık saçılımı (DLS), taramalı elektron mikroskobu (SEM) ve Fourier dönüşümlü kızılötesi spektroskopi (FT-IR) analizleri ile gerçekleştirilmiştir. DLS sonuçlarına göre AgNP/FA/CA konjugatının hidrodinamik boyutu 178,3 nm, zeta potansiyeli ise −7,5 ± 3,55 mV olarak belirlenmiştir. Partiküllerin kolloidal olarak stabil, monodispers (PDI: 0,1) ve küresel morfolojide olduğu karakterizasyon analizleri ile doğrulanmıştır. FT-IR analizleri, konjugatların FA ve CA ile başarılı şekilde fonksiyonelleştirildiğini göstermiştir. Hücre canlılığı (MTT testi) sonuçlarına göre, AgNP/FA/CA uygulaması L929 fibroblast hücre hattında canlılığı korurken, HeLa kanser hücrelerinde belirgin sitotoksisite oluşturmuştur. Toplam Oksidan Durumu (TOS) ve Toplam Antioksidan Durumu (TAS) verileri kullanılarak hesaplanan Oksidatif Stres İndeksi (OSI) sonuçları, konjugatın hücre tipine bağlı farklı oksidatif stres yanıtları oluşturduğunu göstermiştir. L929 hücrelerinde CA’nın antioksidan etkisiyle OSI değeri kontrol grubunun (118,07) altına düşerek 92,32 olarak belirlenmiş ve hücresel stresin baskılandığı gözlenmiştir. Buna karşılık, HeLa hücrelerinde AgNP/FA/CA konjugatı adaptif antioksidan kapasiteyi aşarak yüksek düzeyde oksidatif stres (OSI: 216,20) indüklemiş ve antitümöral etki göstermiştir. Sonuç olarak AgNP/FA/CA konjugatı, sağlıklı dokularda sistemik toksisiteyi baskılarken kanser hücrelerini seçici olarak hedefleyen, çift işlevli ve potansiyel olarak güvenli bir nanoterapötik yaklaşım sunmaktadır.

Anahtar Kelimeler

• Gümüş Nanopartiküller (AgNP'ler)
• Folik Asit (FA)
• Chelidonik Asit (CA)
• Aktif Hedefleme
• Oksidatif Stres.

CO-FUNCTIONALIZED SILVER NANOPARTICLES WITH FOLIC ACID AND CHELIDONIC ACID: DIFFERENTIAL CYTOTOXIC AND OXIDATIVE STRESS RESPONSES IN HELA AND L929 CELLS

Öz

Silver nanoparticles exhibit strong anticancer potential despite causing toxicity and reactive oxygen species (ROS) accumulation in healthy cells. This study aims to develop a nanoparticle system that selectively targets cancer cells while reducing the toxicity and ROS activity of silver nanoparticles in healthy cells. In this context, folic acid (FA) and chelidonic acid (CA) were conjugated to silver nanoparticles. FA enhanced cytotoxicity by increasing targeting via folate receptors overexpressed in cancer cells, while CA provided a protective effect by increasing antioxidant capacity in healthy cells. Thus, a dual-functional nanoparticle conjugate (AgNP/FA/CA) was obtained. The physicochemical characterization of the obtained conjugates was performed using dynamic light scattering (DLS), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR) analyses. According to DLS results, the hydrodynamic size of the AgNP/FA/CA conjugate was determined to be 178.3 nm, and its zeta potential was −7.5 ± 3.55 mV. Characterization analyses confirmed that the particles were colloidally stable, monodisperse (PDI: 0.1), and spherical in morphology. FT-IR analyses demonstrated that the conjugates were successfully functionalized with FA and CA. According to cell viability (MTT assay) results, the AgNP/FA/CA application maintained viability in the L929 fibroblast cell line, while it caused significant cytotoxicity in HeLa cancer cells. Oxidative Stress Index (OSI) results, calculated using Total Oxidant Status (TOS) and Total Antioxidant Status (TAS) data, showed that the conjugate produced different oxidative stress responses depending on the cell type. In L929 cells, the antioxidant effect of CA caused the OSI value to fall below that of the control group (118.07) to 92.32, indicating that cellular stress was suppressed. In contrast, in HeLa cells, the AgNP/FA/CA conjugate induced high levels of oxidative stress (OSI: 216.20) by exceeding the adaptive antioxidant capacity and exhibited antitumor effects. In conclusion, the AgNP/FA/CA conjugate offers a dual-functional and potentially safe nanotherapeutic approach that selectively targets cancer cells while suppressing systemic toxicity in healthy tissues.

Anahtar Kelimeler

• Silver Nanoparticles (AgNPs)
• Folic Acid (FA)
• Chelidonium Acid (CA)
• Active Targeting
• Oxidative Stress.

Kaynakça

1. [1] R. N. A. Kazi et al., “Nanomedicine: The Effective Role of Nanomaterials in Healthcare from Diagnosis to Therapy,” AugPharmaceutics, vol. 17, no. 8, p. 987, Aug. 2025, doi: 10.3390/pharmaceutics17080987.
2. [2] K. A. Altammar, “A review on nanoparticles: characteristics, synthesis, applications, and challenges,” Front. Microbiol., vol. 14, p. 1155622, Apr. 2023, doi: 10.3389/fmicb.2023.1155622.
3. [3] H. Lu, S. Zhang, J. Wang, and Q. Chen, “A Review on Polymer and Lipid-Based Nanocarriers and Its Application to Nano-Pharmaceutical and Food-Based Systems,” Front. Nutr., vol. 8, p. 783831, Dec. 2021, doi: 10.3389/fnut.2021.783831.
4. [4] B. Begines et al., “Polymeric nanoparticles for drug delivery: Recent developments and future prospects,” Nanomaterials, vol. 10, no. 7, p. 1403, Jul. 2020, doi: 10.3390/nano10071403.
5. [5] A. Luceri, R. Francese, D. Lembo, M. Ferraris, and C. Balagna, “Silver Nanoparticles: Review of Antiviral Properties, Mechanism of Action and Applications,” Microorganisms, vol. 11, no. 3, p. 629, Feb. 2023, doi: 10.3390/microorganisms11030629.
6. [6] A. Gibała et al., “Antibacterial and antifungal properties of silver nanoparticles—effect of a surface-stabilizing agent,” Biomolecules, vol. 11, no. 10, Oct. 2021, doi: 10.3390/biom11101481.
7. [7] E. E. Okur, F. Eker, E. Akdaşçi, M. Bechelany, and S. Karav, “Comprehensive Review of Silver Nanoparticles in Food Packaging Applications,” Int. J. Mol. Sci., vol. 26, no. 20, p. 9842, Oct. 2025, doi: 10.3390/ijms26209842.
8. [8] Laib, I., Gheraissa, N., Benaissa, A., Benkhira, L., Azzi, M., Benaissa, Y., ... & Barhoum, A. (2025). Tailoring innovative silver nanoparticles for modern medicine: The importance of size and shape control and functional modifications. Materials Today Bio, 102071.

9. [9] A. K. Suresh, D. A. Pelletier, W. Wang, J. L. Morrell-Falvey, B. Gu, and M. J. Doktycz, “Cytotoxicity induced by engineered silver nanocrystallites is dependent on surface coatings and cell types,” Langmuir, vol. 28, no. 5, pp. 2727–2735, Feb. 2012, doi: 10.1021/la2042058.
10. [10] C. Liao, Y. Li, and S. C. Tjong, “Bactericidal and cytotoxic properties of silver nanoparticles,” Int. J. Mol. Sci., vol. 20, no. 2, p. 449, Jan. 2019, doi: 10.3390/ijms20020449.
11. [11] M. Noga, J. Milan, A. Frydrych, and K. Jurowski, “Toxicological Aspects, Safety Assessment, and Green Toxicology of Silver Nanoparticles (AgNPs)—Critical Review: State of the Art,” Int. J. Mol. Sci., vol. 24, no. 6, p. 5133, Mar. 2023, doi: 10.3390/ijms24065133.
12. [12] T. Gonzalez, M. Muminovic, O. Nano, and M. Vulfovich, “Folate Receptor Alpha—A Novel Approach to Cancer Therapy,” Int. J. Mol. Sci., vol. 25, no. 2, p. 1046, Jan. 2024, doi: 10.3390/ijms25021046.
13. [13] M. Ramezani Farani et al., “Folic Acid-Adorned Curcumin-Loaded Iron Oxide Nanoparticles for Cervical Cancer,” ACS Appl. Bio Mater., vol. 5, no. 3, pp. 1305–1318, Mar. 2022, doi: 10.1021/acsabm.1c01311.
14. [14] K. Harish, S. M. Sivasankaran, S. Manoharan, and S. M. Sakthisankaran, “A Brief Review on Pharmacological Efficacy of Chelidonic Acid,” Journal of Drug Delivery and Therapeutics, vol. 15, no. 9, pp. 115–123, Sep. 2025, doi: 10.22270/jddt.v15i9.7352.
15. [15] F. Mahboubi, J. Mohammadnejad, and S. Khaleghi, “Bifunctional folic acid targeted biopolymer Ag@NMOF nanocomposite [{Zn2 (1,4-bdc) 2 (DABCO)} n] as a novel theranostic agent for molecular imaging of colon cancer by SERS,” Heliyon, vol. 10, no. 8, Apr. 2024, doi: 10.1016/j.heliyon.2024.e29876.
16. [16] F. C. KIRMIZITAŞ, M. Demirbilek, and N. T. Laçin, “Engineering Folat Targeted Octadecylamine-Based Solid Lipid Nanoparticles Containing Il-6 Cytokine Encoding Plasmid to Enhance Immunotherapeutic Response,” Preprint, SSRN 4875267, 2024. [Online]. Available: https://ssrn.com/abstract=4875267
17. [17] P. Singh and I. Mijakovic, “Strong Antimicrobial Activity of Silver Nanoparticles Obtained by the Green Synthesis in Viridibacillus sp. Extracts,” Front. Microbiol., vol. 13, Feb. 2022, doi: 10.3389/fmicb.2022.820048. [18] H. M. Azwatul et al., “Plant-based green synthesis of silver nanoparticle via chemical bonding analysis,” Mater. Today Proc., 2023, doi: 10.1016/j.matpr.2023.01.005.
18. [19] S. Revathi et al., “Green synthesis and characterization of silver nanoparticles (AgNP) using Acacia nilotica plant extract and their anti-bacterial activity,” Food Chemistry Advances, vol. 4, Jun. 2024, doi: 10.1016/j.focha.2024.100680.
19. [20] K. SenthilKannan, G. Flora, S. Gunasekaran, H. A. J. Ali, M. Vimalan, and S. Balasubramanian, “Extraction of Silver Nanoparticles (Ag-NPs) by Green Synthesis from Aqueous Extract of Seaweeds and their consequences on HeLa Cell Line and their utility on soil by spectroscopic tools,” in Nanotechnology for Advances in Medical Microbiology, Springer, 2021, pp. 119–138.
20. [21] N. Gogoi, P. J. Babu, C. Mahanta, and U. Bora, “Green synthesis and characterization of silver nanoparticles using alcoholic flower extract of Nyctanthes arbortristis and in vitro investigation of their antibacterial and cytotoxic activities,” Materials Science and Engineering: C, vol. 46, pp. 463–469, 2015.
21. [22] T. Atici and D. A. Çolak, “Combination of Silver Nanoparticles Synthesized Using Karaerik Extract and Cisplatin: Effects on Breast Cancer Cells,” Kahramanmaraş Sütçü İmam Üniversitesi Tarım ve Doğa Dergisi, vol. 28, no. 5, pp. 1229–1240, 2025.
22. [23] S. S. Malaganvi, J. Tonannavar, and J. Tonannavar, “Experimental, DFT dimeric modeling and AIM study of H-bond-mediated composite vibrational structure of Chelidonic acid,” Heliyon, vol. 5, no. 5, 2019.
23. [24] F.-M. Wang, K. Wagari Guji, A. Ramar, L. Merinda, and W.-C. Chien, “Synthesis and Characterization of Chelidonic Acid and Chelidamic Acid as Organic Anode Materials for Energy Storage,” ACS Sustain. Chem. Eng., vol. 9, no. 36, pp. 12286–12299, 2021.
24. [25] A. R. Chowdhuri et al., “Theoretical and experimental study of folic acid conjugated silver nanoparticles through electrostatic interaction for enhance antibacterial activity,” RSC Adv., vol. 5, no. 28, pp. 21515–21524, 2015.
25. [26] A. Eren and M. F. Baran, “Synthesis, characterization and investigation of antimicrobial activity of silver nanoparticles (AgNP) from pistachio (Pistacia vera L.) leaf.,” Kahramanmaraş Sütçü İmam Üniversitesi Tarım ve Doğa Dergisi, vol. 22, no. 5, pp. 844–855, 2019, doi: 10.18016/ksutarimdoga.vi.585834.
26. [27] N. Makieieva et al., “Experimental and theoretical characterization of chelidonic acid structure,” Struct. Chem., vol. 33, no. 6, pp. 2133–2145, 2022.
27. [28] R. Safarpour, M. Pooresmaeil, and H. Namazi, “Folic acid functionalized Ag@ MOF (Ag) decorated carboxymethyl starch nanoparticles as a new doxorubicin delivery system with inherent antibacterial activity,” Int. J. Biol. Macromol., vol. 282, p. 137096, 2024.
28. [29] A. Secme, B. M. Bozer, A. Y. Kocaman, R. Erenler, and M. H. Calimli, “Synthesis, characterization, and anticancer properties of Ag nanoparticles derived from walnut leaves tested on cells of L929, MCF-7 and H1299,” J. Drug Deliv. Sci. Technol., vol. 94, p. 105478, 2024.
29. [30] Ö. Erdoğan, S. Paşa, and O. Cevik, “Green synthesis and characterization of anticancer effected silver nanoparticles with silverberry (Elaeagnus angustifolia) fruit aqueous extract,” International Journal of Pure and Applied Sciences, vol. 7, no. 3, pp. 391–400, 2021.
30. [31] R. Bhanumathi, M. Manivannan, R. Thangaraj, and S. Kannan, “Drug-carrying capacity and anticancer effect of the folic acid-and berberine-loaded silver nanomaterial to regulate the AKT-ERK pathway in breast cancer,” ACS Omega, vol. 3, no. 7, pp. 8317–8328, 2018.
31. [32] Z. Ferdous and A. Nemmar, “Health impact of silver nanoparticles: a review of the biodistribution and toxicity following various routes of exposure,” Int. J. Mol. Sci., vol. 21, no. 7, p. 2375, 2020.
32. [33] S. I. Khairnar, Y. A. Kulkarni, and K. Singh, “Mitigation of cisplatin-induced nephrotoxicity by chelidonic acid in Wistar rats,” Journal of Trace Elements in Medicine and Biology, vol. 81, p. 127321, 2024.
33. [34] G. Raja, Y.-K. Jang, J.-S. Suh, H.-S. Kim, S. H. Ahn, and T.-J. Kim, “Microcellular environmental regulation of silver nanoparticles in cancer therapy: a critical review,” Cancers (Basel)., vol. 12, no. 3, p. 664, 2020.
34. [35] S. Khorrami, A. Zarrabi, M. Khaleghi, M. Danaei, and M. R. Mozafari, “Selective cytotoxicity of green synthesized silver nanoparticles against the MCF-7 tumor cell line and their enhanced antioxidant and antimicrobial properties,” Int. J. Nanomedicine, pp. 8013–8024, 2018.
35. [36] P. V AshaRani, G. Low Kah Mun, M. P. Hande, and S. Valiyaveettil, “Cytotoxicity and genotoxicity of silver nanoparticles in human cells,” ACS Nano, vol. 3, no. 2, pp. 279–290, 2009.
36. [37] F. N. Sinan, E. SERDAROĞLU KAŞIKÇI, and B. Cevreli, “Chelidonic acid abrogates oxidative stress and memory dysfunction in experimental aging rats,” Turkish Journal of Biology, vol. 48, no. 6, pp. 432–441, 2024.
37. [38] R. Romanu et al., “Chelidonium majus L.: A current perspective on isoquinoline alkaloids, emerging phytochemicals, alkaloid biosynthesis, and biological activities,” Plants, vol. 14, no. 17, p. 2627, 2025.