Fibroblast growth factor 2 (FGF-2) is a key signaling molecule involved in wound healing, tissue remodeling, and the regulation of cell proliferation. Despite its important biological roles, the use of FGF-2 in cell culture and therapeutic settings is limited by its poor stability in aqueous environments. Three different antioxidants were selected and screened in this study for their ability to enhance the stability of FGF-2 in cell culture applications. BALB/3T3 and NIH/3T3 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented individually with disodium ethylenediaminetetraacetic acid dihydrate (EDTA), sodium selenite (Se), or zinc chloride (Zn), each at a final concentration of 0.1 μM. Cytotoxicity and cell proliferation were subsequently assessed using the MTT assay. The cells were then cultured at 37?C for three days to assess the biological effects of antioxidant supplementation. All three antioxidants individually enhanced the proliferation of both cell lines without inducing cytotoxicity. These findings suggest that antioxidants may represent a promising approach for improving vitro cell growth. Therefore, antioxidants were evaluated in parallel with FGF-2 by adding them separately to DMEM and Roswell Park Memorial Institute (RPMI) 1640 serum and cells free media under identical conditions to determine whether their proliferative effects were associated with improved FGF-2 stability. The stability of FGF-2 in each medium was assessed using reversed phase high performance liquid chromatography (RP-HPLC). The results show that, even in the presence of antioxidants, FGF-2 remained unstable at 37?C in both DMEM and RPMI media, indicating that these antioxidants do not confer stability to FGF-2 in either medium.
References
[1]
Powers, C.J., McLeskey, S.W. and Wellstein, A. (2000) Fibroblast Growth Factors, Their Receptors and Signaling. Endocrine-Related Cancer, 7, 165-197. https://doi.org/10.1677/erc.0.0070165
[2]
Akl, M.R., Nagpal, P., Ayoub, N.M., Tai, B., Prabhu, S.A., Capac, C.M., et al. (2016) Molecular and Clinical Significance of Fibroblast Growth Factor 2 (FGF2/bFGF) in Malignancies of Solid and Hematological Cancers for Personalized Therapies. Oncotarget, 7, 44735-44762. https://doi.org/10.18632/oncotarget.8203
[3]
Ornitz, D.M. and Itoh, N. (2001) Fibroblast Growth Factors. Genome Biology, 2, reviews3005.1. https://doi.org/10.1186/gb-2001-2-3-reviews3005
[4]
Katoh, M. (2016) Therapeutics Targeting FGF Signaling Network in Human Diseases. Trends in Pharmacological Sciences, 37, 1081-1096. https://doi.org/10.1016/j.tips.2016.10.003
[5]
Akita, S., Akino, K., Imaizumi, T. and Hirano, A. (2008) Basic Fibroblast Growth Factor Accelerates and Improves Second-Degree Burn Wound Healing. Wound Repair and Regeneration, 16, 635-641. https://doi.org/10.1111/j.1524-475x.2008.00414.x
[6]
Kumagai, M., Marui, A., Tabata, Y., Takeda, T., Yamamoto, M., Yonezawa, A., et al. (2016) Safety and Efficacy of Sustained Release of Basic Fibroblast Growth Factor Using Gelatin Hydrogel in Patients with Critical Limb Ischemia. Heart and Vessels, 31, 713-721. https://doi.org/10.1007/s00380-015-0677-x
[7]
Moya, M.L., Cheng, M., Huang, J., Francis-Sedlak, M.E., Kao, S., Opara, E.C., et al. (2010) The Effect of FGF-1 Loaded Alginate Microbeads on Neovascularization and Adipogenesis in a Vascular Pedicle Model of Adipose Tissue Engineering. Biomaterials, 31, 2816-2826. https://doi.org/10.1016/j.biomaterials.2009.12.053
[8]
Zhan, X. and Goldfarb, M. (1986) Growth Factor Requirements of Oncogene-Transformed NIH 3T3 and BALB/c 3T3 Cells Cultured in Defined Media. Molecular and Cellular Biology, 6, 3541-3544. https://doi.org/10.1128/mcb.6.10.3541-3544.1986
[9]
Gospodarowicz, D. and Moran, J.S. (1974) Stimulation of Division of Sparse and Confluent 3T3 Cell Populations by a Fibroblast Growth Factor, Dexamethasone, and Insulin. Proceedings of the National Academy of Sciences of the United States of America, 71, 4584-4588. https://doi.org/10.1073/pnas.71.11.4584
[10]
Freudenberg, U., Hermann, A., Welzel, P.B., Stirl, K., Schwarz, S.C., Grimmer, M., et al. (2009) A Star-PEG-Heparin Hydrogel Platform to Aid Cell Replacement Therapies for Neurodegenerative Diseases. Biomaterials, 30, 5049-5060. https://doi.org/10.1016/j.biomaterials.2009.06.002
[11]
Wang, Y.J., Shahrokh, Z., Vemuri, S., Eberlein, G., Beylin, I. and Busch, M. (2002) Characterization, Stability, and Formulations of Basic Fibroblast Growth Factor. In: Pearlman, R. and Wang, J.Y., Eds., Pharmaceutical Biotechnology, Springer, 141-180. https://doi.org/10.1007/0-306-47452-2_2
[12]
Vemuri, S., Beylin, I., Sluzky, V., Stratton, P., Eberlein, G. and Wang, Y.J. (1994) The Stability of bFGF against Thermal Denaturation. Journal of Pharmacy and Pharmacology, 46, 481-486. https://doi.org/10.1111/j.2042-7158.1994.tb03831.x
[13]
Schreck, R. and Baeuerle, P.A. (1991) A Role for Oxygen Radicals as Second Messengers. Trends in Cell Biology, 1, 39-42. https://doi.org/10.1016/0962-8924(91)90072-h
[14]
Kamerzell, T.J., Esfandiary, R., Joshi, S.B., Middaugh, C.R. and Volkin, D.B. (2011) Protein-Excipient Interactions: Mechanisms and Biophysical Characterization Applied to Protein Formulation Development. Advanced Drug Delivery Reviews, 63, 1118-1159. https://doi.org/10.1016/j.addr.2011.07.006
[15]
Rayaprolu, B.M., Strawser, J.J. and Anyarambhatla, G. (2018) Excipients in Parenteral Formulations: Selection Considerations and Effective Utilization with Small Molecules and Biologics. Drug Development and Industrial Pharmacy, 44, 1565-1571. https://doi.org/10.1080/03639045.2018.1483392
[16]
Chu, H., Gao, J., Chen, C., Huard, J. and Wang, Y. (2011) Injectable Fibroblast Growth Factor-2 Coacervate for Persistent Angiogenesis. Proceedings of the National Academy of Sciences of the United States of America, 108, 13444-13449. https://doi.org/10.1073/pnas.1110121108
[17]
Dvorak, P., Bednar, D., Vanacek, P., Balek, L., Eiselleova, L., Stepankova, V., et al. (2018) Computer-Assisted Engineering of Hyperstable Fibroblast Growth Factor 2. Biotechnology and Bioengineering, 115, 850-862. https://doi.org/10.1002/bit.26531
[18]
Benington, L.R., Rajan, G., Locher, C. and Lim, L.Y. (2021) Stabilisation of Recombinant Human Basic Fibroblast Growth Factor (FGF-2) against Stressors Encountered in Medicinal Product Processing and Evaluation. Pharmaceutics, 13, Article 1762. https://doi.org/10.3390/pharmaceutics13111762
[19]
Ohtake, S., Kita, Y. and Arakawa, T. (2011) Interactions of Formulation Excipients with Proteins in Solution and in the Dried State. Advanced Drug Delivery Reviews, 63, 1053-1073. https://doi.org/10.1016/j.addr.2011.06.011
[20]
Ferrero, M.E. (2016) Rationale for the Successful Management of EDTA Chelation Therapy in Human Burden by Toxic Metals. BioMed Research International, 2016, Article 8274504. https://doi.org/10.1155/2016/8274504
[21]
Ebert, R., Ulmer, M., Zeck, S., Meissner‐Weigl, J., Schneider, D., Stopper, H., et al. (2006) Selenium Supplementation Restores the Antioxidative Capacity and Prevents Cell Damage in Bone Marrow Stromal Cells in Vitro. Stem Cells, 24, 1226-1235. https://doi.org/10.1634/stemcells.2005-0117
[22]
Prasad, A.S. (2014) Zinc Is an Antioxidant and Anti-Inflammatory Agent: Its Role in Human Health. Frontiers in Nutrition, 1, Article 14. https://doi.org/10.3389/fnut.2014.00014
[23]
Sluzky, V., Shahrokh, Z., Stratton, P., Eberlein, G. and Wang, Y.J. (1994) Chromatographic Methods for Quantitative Analysis of Native, Denatured, and Aggregated Basic Fibroblast Growth Factor in Solution Formulations. Pharmaceutical Research, 11, 485-490. https://doi.org/10.1023/a:1018946011652
[24]
Sauer, D.G., Mosor, M., Jungbauer, A. and Dürauer, A. (2021) Separation of Truncated Basic Fibroblast Growth Factor from the Full-Length Protein by Hydrophobic Interaction Chromatography. Separation and Purification Technology, 254, Article ID: 117564. https://doi.org/10.1016/j.seppur.2020.117564
[25]
Strober, W. (2015) Trypan Blue Exclusion Test of Cell Viability. Current Protocols in Immunology, 111, A3.B.1-A3.B.3. https://doi.org/10.1002/0471142735.ima03bs111
[26]
Gowda, A.S.P., Schaefer, A.D. and Schuck, T.K. (2025) Epidermal Growth Factor Stability and Cell Proliferation Enhanced by Antioxidants. Advances in Bioscience and Biotechnology, 16, 65-89. https://doi.org/10.4236/abb.2025.163004
[27]
Marins, J.S.R., Sassone, L.M., Fidel, S.R. and Ribeiro, D.A. (2012) In Vitro Genotoxicity and Cytotoxicity in Murine Fibroblasts Exposed to EDTA, NaOCl, MTAD and Citric Acid. Brazilian Dental Journal, 23, 527-533. https://doi.org/10.1590/s0103-64402012000500010
[28]
Feril Jr., L.B., Ogawa, K., Watanabe, A., Ogawa, R., et al. (2017) Anticancer Potential of EDTA: A Preliminary in Vitro Study. Mathews Journal of Cancer Science, 2, Article 9.
[29]
Abedelahi, A., Salehnia, M. and Allameh, A.A. (2008) The Effects of Different Concentrations of Sodium Selenite on the in Vitro Maturation of Preantral Follicles in Serum-Free and Serum Supplemented Media. Journal of Assisted Reproduction and Genetics, 25, 483-488. https://doi.org/10.1007/s10815-008-9252-z
[30]
Selenius, M., Rundl？f, A., Olm, E., Fernandes, A.P. and Bj？rnstedt, M. (2010) Selenium and the Selenoprotein Thioredoxin Reductase in the Prevention, Treatment and Diagnostics of Cancer. Antioxidants & Redox Signaling, 12, 867-880. https://doi.org/10.1089/ars.2009.2884
[31]
Bozym, R.A., Chimienti, F., Giblin, L.J., Gross, G.W., Korichneva, I., Li, Y., et al. (2010) Free Zinc Ions Outside a Narrow Concentration Range Are Toxic to a Variety of Cells in Vitro. Experimental Biology and Medicine, 235, 741-750. https://doi.org/10.1258/ebm.2010.009258
[32]
Du, Y., Guo, D., Wu, Q., Liu, D. and Bi, H. (2014) Zinc Chloride Inhibits Human Lens Epithelial Cell Migration and Proliferation Involved in TGF-β1 and TNF-α Signaling Pathways in HLE B-3 Cells. Biological Trace Element Research, 159, 425-433. https://doi.org/10.1007/s12011-014-9979-6
[33]
Ye, J., Wu, H., Wu, Y., Wang, C., Zhang, H., Shi, X., et al. (2012) High Molecular Weight Hyaluronan Decreases Oxidative DNA Damage Induced by EDTA in Human Corneal Epithelial Cells. Eye, 26, 1012-1020. https://doi.org/10.1038/eye.2012.89
[34]
Slepchenko, K.G., Lu, Q. and Li, Y.V. (2017) Cross Talk between Increased Intracellular Zinc (Zn2+) and Accumulation of Reactive Oxygen Species in Chemical Ischemia. American Journal of Physiology-Cell Physiology, 313, C448-C459. https://doi.org/10.1152/ajpcell.00048.2017
[35]
Manning, M.C., Patel, K. and Borchardt, R.T. (1989) Stability of Protein Pharmaceuticals. Pharmaceutical Research, 6, 903-918. https://doi.org/10.1023/a:1015929109894
[36]
Wang, W. (1999) Instability, Stabilization, and Formulation of Liquid Protein Pharmaceuticals. International Journal of Pharmaceutics, 185, 129-188. https://doi.org/10.1016/s0378-5173(99)00152-0
[37]
Son, D.H., Yang, D.J., Sun, J.S., Kim, S.K., Kang, N., Kang, J.Y., et al. (2018) A Novel Peptide, Nicotinyl-Isoleucine-Valine-Histidine (NA-IVH), Promotes Antioxidant Gene Expression and Wound Healing in HaCaT Cells. Marine Drugs, 16, Article 262. https://doi.org/10.3390/md16080262
[38]
Estapé, D., Heuvel, J.V.D. and Rinas, U. (1998) Susceptibility Towards Intramolecular Disulphide-Bond Formation Affects Conformational Stability and Folding of Human Basic Fibroblast Growth Factor. Biochemical Journal, 335, 343-349. https://doi.org/10.1042/bj3350343
[39]
Caccia, P., Nitti, G., Cletini, O., Pucci, P., Ruoppolo, M., Bertolero, F., et al. (1992) Stabilization of Recombinant Human Basic Fibroblast Growth Factor by Chemical Modifications of Cysteine Residues. European Journal of Biochemistry, 204, 649-655. https://doi.org/10.1111/j.1432-1033.1992.tb16678.x
[40]
Sommer, A. and Rifkin, D.B. (1989) Interaction of Heparin with Human Basic Fibroblast Growth Factor: Protection of the Angiogenic Protein from Proteolytic Degradation by a Glycosaminoglycan. Journal of Cellular Physiology, 138, 215-220. https://doi.org/10.1002/jcp.1041380129
[41]
Manning, M.C., Chou, D.K., Murphy, B.M., Payne, R.W. and Katayama, D.S. (2010) Stability of Protein Pharmaceuticals: An Update. Pharmaceutical Research, 27, 544-575. https://doi.org/10.1007/s11095-009-0045-6
[42]
Ribeiro, D.A., Matsumoto, M.A., Duarte, M.A.H., Marques, M.E.A. and Salvadori, D.M.F. (2005) In Vitro Biocompatibility Tests of Two Commercial Types of Mineral Trioxide Aggregate. Brazilian Oral Research, 19, 183-187. https://doi.org/10.1590/s1806-83242005000300005
[43]
Kasugai, S., Hasegawa, N. and Ogura, H. (1990) A Simple in Vito Cytotoxicity Test Using the MTT (3-(4,5)-Dimethylthiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide) Colorimetric Assay: Analysis of Eugenol Toxicity on Dental Pulp Cells (RPC-C2A). Japanese Journal of Pharmacology, 52, 95-100. https://doi.org/10.1254/jjp.52.95
[44]
Wang, H.P., Qian, S.Y., Schafer, F.Q., Domann, F.E., Oberley, L.W. and Buettner, G.R. (2001) Phospholipid Hydroperoxide Glutathione Peroxidase Protects against Singlet Oxygen-Induced Cell Damage of Photodynamic Therapy. Free Radical Biology and Medicine, 30, 825-835. https://doi.org/10.1016/s0891-5849(01)00469-5
[45]
Hartikainen, H., Xue, T. and Piironen, V. (2000) Selenium as an Anti-Oxidant and Pro-Oxidant in Ryegrass. Plant and Soil, 225, 193-200. https://doi.org/10.1023/a:1026512921026
[46]
Holmes, A.M., Mackenzie, L. and Roberts, M.S. (2020) Disposition and Measured Toxicity of Zinc Oxide Nanoparticles and Zinc Ions against Keratinocytes in Cell Culture and Viable Human Epidermis. Nanotoxicology, 14, 263-274. https://doi.org/10.1080/17435390.2019.1692382
