The harsh microenvironment, that stem cells are transplanted into, has been a prevailing challenge that scientist are aiming to overcome for improving current stem cell-based therapies, due to the low cell viability seen post-transplantation. Previous approaches have proposed the supplementation of the culture media with different drugs for priming purposes and thus enhanced survival of the post-engrafted cells. However, a recurring worry is the exogenous nature of the supplements used and the effect these might have in multipotent cells because of their susceptibility towards differentiation. In this study, two types of biomimetic iron complexes, DNIC-COOH and DNIC-COOMe, were investigated for their different cellular uptake and later NO release kinetics in mesenchymal stem cells (MSCs), followed by enhanced survival gene expression. Such application was inspired by previous studies showing controlled NO concentrations aiding in the preservation of pluripotency, cell survival, and inducing differentiation in stem cells, all in a dose-dependent manner. It was found that, based on their different charge and sidechains (-R), DNIC-COOMe showed a faster cellular uptake and burst release of NO, while DNIC-COOH was internalized slower by MSCs. These findings offered an explanation for their distinct cytoprotective effects at different concentrations, bringing these complexes closer to a potential application as biomimetic stem cell priming media supplements. Furthermore, geNOps, which referred to cells transduced with a gene encoding for an NO-sensitive protein, were successfully used to obtain a direct, single-cell detection of intracellular NO delivered by different types of DNICs. These findings not only served as prove of concept of an more accurate alternative for detection of NO inside cells, but most significantly demonstrated the capability of nitrosylation of DNICs, even under the presence of oxidating agents, such as hydrogen peroxide, which are compounds commonly found under stress cellular microenvironments. The results obtained highlight the prospects of DNICs as NO prodrugs for the support of MSC-based regenerative cell therapies, by increasing cell viability and potentially aiding in the maintenance of stem cell multipotency.

Abstract--------------------------------------i
Acknowledgements------------------------------ii
Table of Contents-----------------------------iv
List of Figures-------------------------------vi
List of Tables--------------------------------viii
1. Introduction-------------------------------1
1.1 Stem Cell Therapy-------------------------1
1.1-1 History of Stem Cells-------------------1
1.1-2 Mesenchymal Stem Cells (MSC)------------1
1.2 Physiological Nitric Oxide----------------3
1.2-2 NO Detection in biological samples------7
1.3 Dinitrosyl Iron Complexes (DNICs)---------12
1.4 Cellular Uptake of Molecules--------------14
1.4-1 Endocytosis-----------------------------15
1.4-2 Thiol-mediated uptake-------------------18
1.5 Research Motivation-----------------------21
1.5-1 Research Proposal-----------------------22
2. Experimental Section-----------------------23
2.1 Materials---------------------------------23
2.2 Apparatus---------------------------------24
2.3 Preparation of buffer solutions and other reagents---25
2.3.1 DNIC stock solution (100 mM)------------25
2.3.2 Phosphate-buffer saline (PBS)-----------25
2.3.3 Supplemented media----------------------25
2.4 Characterization of DNIC under simulated extracellular and intracellular conditions----------------------26
2.4.1 EPR sample preparation------------------26
2.4.2 Kinetic study of conversion of DNIC in the presence of Cysteine-containing proteins---------------------------26
2.5 Cell culture and cell-related experiments-27
2.5.1 Preparation of complete cell culture media for the culture of different cell-lines--------------------------27
2.5.2 Cell culture environment----------------27
2.5.3 Thawing of cells------------------------27
2.5.4 Cell passaging--------------------------28
2.5.5 Cell counting---------------------------29
2.5.6 Cell freezing---------------------------29
2.5.7 Cell viability assay--------------------29
2.5.8 Kinetic study of cellular uptake and intracellular conversion of DNIC------------------------------------------30
2.5.9 Quantification of NO release by DNIC----31
2.5.10 Intracellular NO release by DNIC-------32
2.5.11 qPCR of DNIC-induced HO-1 gene expression---32
2.5.12 Transfection---------------------------34
2.5.13 Flow cytometry of fluorescence changes in geNOps---34
2.6 Statistical analysis----------------------35
3. Results and discussion---------------------36
3.1 Stability of DNICs in the presence of cysteine or cysteine-containing proteins---------------------------36
3.1-1 NO-release kinetic profile of DNICs-----36
3.1-2 Interconversion between binuclear/mononuclear DNICs and corresponding kinetic stability profile upon conversion to mononuclear DNIC------------------------------------------38
3.2 In vitro investigations on the intracellular presence of DNICs, cellular uptake kinetics and mechanisms by cbMSCs---40
3.2-1 Cell viability of MSCs vs. DNICs--------40
3.2-2 Intracellular mononuclear DNICs — Kinetic curve---41
3.2-3 Cellular uptake mechanisms--------------42
3.2-4 Intracellular NO-release----------------44
3.3 Activation of NO-related signaling pathways by DNICs---45
3.3-1 Regulation of the NO-dependent HO-1 expression-------45
3.3-2 Activation of the sGC/cGMP/HO-1 signaling pathway by NO---47
3.4 Development of fluorescent genetically encoded NO probes (geNOps) for intracellular detection-------------------48
3.4-1 Transfection — Fine-tuning of parameters---49
3.4-2 NO-reactive fluorescent proteins in the presence of NO donors — Transduced cells------------------------------51
4. Conclusion---------------------------------55
References------------------------------------57

[1] J. E. Till, and E. A. McCulloch, “A Direct Measurement of the Radiation Sensitivity of Normal Mouse Bone Marrow Cells,” Radiation Research, vol. 14, no. 2, pp. 213-222, 1961.
[2] M. J. Evans, and M. H. Kaufman, “Establishment in culture of pluripotential cells from mouse embryos,” Nature, vol. 292, no. 5819, pp. 154-156, 1981/07/01, 1981.
[3] J. A. Thomson, J. Itskovitz-Eldor, S. S. Shapiro, M. A. Waknitz, J. J. Swiergiel, V. S. Marshall, and J. M. Jones, “Embryonic Stem Cell Lines Derived from Human Blastocysts,” Science, vol. 282, no. 5391, pp. 1145-1147, 1998.
[4] L. Bacakova, J. Zarubova, M. Travnickova, J. Musilkova, J. Pajorova, P. Slepicka, N. S. Kasalkova, V. Svorcik, Z. Kolska, H. Motarjemi, and M. Molitor, “Stem cells: their source, potency and use in regenerative therapies with focus on adipose-derived stem cells – a review,” Biotechnology Advances, vol. 36, no. 4, pp. 1111-1126, 2018/07/01/, 2018.
[5] R. S. Nandoe Tewarie Md, A. Hurtado Md, P. R. H. Bartels Md, P. A. Grotenhuis Md, and M. Oudega PhD, “Stem Cell-Based Therapies for Spinal Cord Injury,” The Journal of Spinal Cord Medicine, vol. 32, no. 2, pp. 105-114, 2009/01/01, 2009.
[6] Z. Shang, M. Wang, B. Zhang, X. Wang, and P. Wanyan, “Clinical translation of stem cell therapy for spinal cord injury still premature: results from a single-arm meta-analysis based on 62 clinical trials,” BMC Medicine, vol. 20, no. 1, pp. 284, 2022/09/05, 2022.
[7] M. I. Nasser, X. Qi, S. Zhu, Y. He, M. Zhao, H. Guo, and P. Zhu, “Current situation and future of stem cells in cardiovascular medicine,” Biomedicine & Pharmacotherapy, vol. 132, pp. 110813, 2020/12/01/, 2020.
[8] D. Chang, T. Fan, S. Gao, Y. Jin, M. Zhang, and M. Ono, “Application of mesenchymal stem cell sheet to treatment of ischemic heart disease,” Stem Cell Research & Therapy, vol. 12, no. 1, pp. 384, 2021/07/07, 2021.
[9] M. A. Ghoneim, M. M. Gabr, S. M. El-Halawani, and A. F. Refaie, “Current status of stem cell therapy for type 1 diabetes: a critique and a prospective consideration,” Stem Cell Research & Therapy, vol. 15, no. 1, pp. 23, 2024/01/29, 2024.
[10] Z. Guan, J. Zhang, N. Jiang, M. Tian, H. Wang, and B. Liang, “Efficacy of mesenchymal stem cell therapy in rodent models of radiation-induced xerostomia and oral mucositis: a systematic review,” Stem Cell Research & Therapy, vol. 14, no. 1, pp. 82, 2023/04/12, 2023.
[11] M. Benderitter, F. Caviggioli, A. Chapel, R. P. Coppes, C. Guha, M. Klinger, O. Malard, F. Stewart, R. Tamarat, P. van Luijk, and C. L. Limoli, “Stem Cell Therapies for the Treatment of Radiation-Induced Normal Tissue Side Effects,” Antioxidants & Redox Signaling, vol. 21, no. 2, pp. 338-355, 2014/07/10, 2013.
[12] U. S. F. a. D. Administration, "Approved Cellular and Gene Therapy Products," 2024.
[13] M. B. LLC, "Current FDA Approved Cell & Gene Therapies," 2023.
[14] S. H. Lee, “The advantages and limitations of mesenchymal stem cells in clinical application for treating human diseases,” Osteoporos Sarcopenia, vol. 4, no. 4, pp. 150, Dec, 2018.
[15] J. Gopalarethinam, A. P. Nair, M. Iyer, B. Vellingiri, and M. D. Subramaniam, “Advantages of mesenchymal stem cell over the other stem cells,” Acta Histochemica, vol. 125, no. 4, pp. 152041, 2023/05/01/, 2023.
[16] J. Zhou, and Y. Shi, “Mesenchymal stem/stromal cells (MSCs): origin, immune regulation, and clinical applications,” Cellular & Molecular Immunology, vol. 20, no. 6, pp. 555-557, 2023/06/01, 2023.
[17] A. Hmadcha, A. Martin-Montalvo, B. R. Gauthier, B. Soria, and V. Capilla-Gonzalez, “Therapeutic Potential of Mesenchymal Stem Cells for Cancer Therapy,” Frontiers in Bioengineering and Biotechnology, vol. 8, 2020-February-05, 2020.
[18] N. Eiro, M. Fraile, S. Fernández-Francos, R. Sánchez, L. A. Costa, and F. J. Vizoso, “Importance of the origin of mesenchymal (stem) stromal cells in cancer biology: “alliance” or “war” in intercellular signals,” Cell & Bioscience, vol. 11, no. 1, pp. 109, 2021/06/10, 2021.
[19] L. A. Cona. "Mesenchymal Stem Cells (MSCs): A Comprehensive Overview of Their Properties and Uses," 04/17/2024; https://www.dvcstem.com/post/what-are-mesenchymal-stem-cells.
[20] N. Stansfield. "FDA Deems Mesoblast’s Data on Remestemcel-L Cell Therapy Sufficient for BLA Submission in Pediatric SR-aGVHD," 04/15/2024; https://www.cgtlive.com/view/fda-deems-mesoblast-data-remestemcel-l-cell-therapy-sufficient-bla-submission-pediatric-sr-agvhd.
[21] M. Ltd. "FDA Notifies Clinical Data Sufficient for Refiling aGVHD BLA," 04/15/2024; https://investorsmedia.mesoblast.com/asx-announcements.
[22] S. Baldari, G. Di Rocco, M. Piccoli, M. Pozzobon, M. Muraca, and G. Toietta, “Challenges and Strategies for Improving the Regenerative Effects of Mesenchymal Stromal Cell-Based Therapies,” International Journal of Molecular Sciences, vol. 18, no. 10, pp. 2087, 2017.
[23] H. Song, M.-J. Cha, B.-W. Song, I.-K. Kim, W. Chang, S. Lim, E. J. Choi, O. Ham, S.-Y. Lee, N. Chung, Y. Jang, and K.-C. Hwang, “Reactive Oxygen Species Inhibit Adhesion of Mesenchymal Stem Cells Implanted into Ischemic Myocardium via Interference of Focal Adhesion Complex,” Stem Cells, vol. 28, no. 3, pp. 555-563, 2010.
[24] W. Chang, B.-W. Song, J.-Y. Moon, M.-J. Cha, O. Ham, S.-Y. Lee, E. Choi, E. Choi, and K.-C. Hwang, “Anti-death strategies against oxidative stress in grafted mesenchymal stem cells,” Histology and histopathology, vol. 28, no. 12, pp. 1529-1536, 2013/12//, 2013.
[25] Z. Hu, Y. Yuan, X. Zhang, Y. Lu, N. Dong, X. Jiang, J. Xu, and D. Zheng, “Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Attenuate Oxygen-Glucose Deprivation/Reperfusion-Induced Microglial Pyroptosis by Promoting FOXO3a-Dependent Mitophagy,” Oxid Med Cell Longev, vol. 2021, pp. 6219715, 2021.
[26] Y. Huang, Z. Bai, and K. Zhang, “A new insight for stem cell therapy: apoptotic stem cells as a key player,” Signal Transduction and Targeted Therapy, vol. 7, no. 1, pp. 299, 2022/08/28, 2022.
[27] I. Kang, B.-C. Lee, S. W. Choi, J. Y. Lee, J.-J. Kim, B.-E. Kim, D.-H. Kim, S. E. Lee, N. Shin, Y. Seo, H.-S. Kim, D.-I. Kim, and K.-S. Kang, “Donor-dependent variation of human umbilical cord blood mesenchymal stem cells in response to hypoxic preconditioning and amelioration of limb ischemia,” Experimental & Molecular Medicine, vol. 50, no. 4, pp. 1-15, 2018/04/01, 2018.
[28] N. Haque, N. H. A. Kasim, and M. T. Rahman, “Optimization of Pre-transplantation Conditions to Enhance the Efficacy of Mesenchymal Stem Cells,” International Journal of Biological Sciences, vol. 11, no. 3, pp. 324-334, 2015.
[29] M. Mebarki, L. Coquelin, P. Layrolle, S. Battaglia, M. Tossou, P. Hernigou, H. Rouard, and N. Chevallier, “Enhanced human bone marrow mesenchymal stromal cell adhesion on scaffolds promotes cell survival and bone formation,” Acta Biomaterialia, vol. 59, pp. 94-107, 2017/09/01/, 2017.
[30] N. Su, P.-L. Gao, K. Wang, J.-Y. Wang, Y. Zhong, and Y. Luo, “Fibrous scaffolds potentiate the paracrine function of mesenchymal stem cells: A new dimension in cell-material interaction,” Biomaterials, vol. 141, pp. 74-85, 2017/10/01/, 2017.
[31] X. Hu, S. P. Yu, J. L. Fraser, Z. Lu, M. E. Ogle, J.-A. Wang, and L. Wei, “Transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis,” The Journal of Thoracic and Cardiovascular Surgery, vol. 135, no. 4, pp. 799-808, 2008/04/01/, 2008.
[32] S. Wisel, M. Khan, M. L. Kuppusamy, I. K. Mohan, S. M. Chacko, B. K. Rivera, B. C. Sun, K. Hideg, and P. Kuppusamy, “Pharmacological Preconditioning of Mesenchymal Stem Cells with Trimetazidine (1-[2,3,4-Trimethoxybenzyl]piperazine) Protects Hypoxic Cells against Oxidative Stress and Enhances Recovery of Myocardial Function in Infarcted Heart through Bcl-2 Expression,” Journal of Pharmacology and Experimental Therapeutics, vol. 329, no. 2, pp. 543-550, 2009.
[33] Y. L. Tang, Y. Tang, Y. C. Zhang, K. Qian, L. Shen, and M. I. Phillips, “Improved Graft Mesenchymal Stem Cell Survival in Ischemic Heart With a Hypoxia-Regulated Heme Oxygenase-1 Vector,” Journal of the American College of Cardiology, vol. 46, no. 7, pp. 1339-1350, 2005.
[34] L. S. Saleh, and S. J. Bryant, “The Host Response in Tissue Engineering: Crosstalk Between Immune cells and Cell-laden Scaffolds,” Curr Opin Biomed Eng, vol. 6, pp. 58-65, Jun, 2018.
[35] C. Wu, and C. E. Dunbar, “Stem cell gene therapy: the risks of insertional mutagenesis and approaches to minimize genotoxicity,” Front Med, vol. 5, no. 4, pp. 356-71, Dec, 2011.
[36] J. L. Spees, C. A. Gregory, H. Singh, H. A. Tucker, A. Peister, P. J. Lynch, S.-C. Hsu, J. Smith, and D. J. Prockop, “Internalized antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy,” Molecular Therapy, vol. 9, no. 5, pp. 747-756, 2004.
[37] D. S. Bredt, “Endogenous nitric oxide synthesis: Biological functions and pathophysiology,” Free Radical Research, vol. 31, no. 6, pp. 577-596, 1999/01/01, 1999.
[38] S. M. Andrabi, N. S. Sharma, A. Karan, S. M. S. Shahriar, B. Cordon, B. Ma, and J. Xie, “Nitric Oxide: Physiological Functions, Delivery, and Biomedical Applications,” Advanced Science, vol. 10, no. 30, pp. 2303259, 2023.
[39] N. K. Hollenberg, “Organ Systems Dependent on Nitric Oxide and the Potential for Nitric Oxide-Targeted Therapies in Related Diseases,” The Journal of Clinical Hypertension, vol. 8, no. s12, pp. 63-76, 2006.
[40] D. L. Rousseau, D. Li, M. Couture, and S.-R. Yeh, “Ligand–protein interactions in nitric oxide synthase,” Journal of Inorganic Biochemistry, vol. 99, no. 1, pp. 306-323, 2005/01/01/, 2005.
[41] U. Förstermann, and W. C. Sessa, “Nitric oxide synthases: regulation and function,” European Heart Journal, vol. 33, no. 7, pp. 829-837, 2011.
[42] M. Król, and M. Kepinska, “Human Nitric Oxide Synthase—Its Functions, Polymorphisms, and Inhibitors in the Context of Inflammation, Diabetes and Cardiovascular Diseases,” International Journal of Molecular Sciences, vol. 22, no. 1, pp. 56, 2021.
[43] M. R. Garren, M. Ashcraft, Y. Qian, M. Douglass, E. J. Brisbois, and H. Handa, “Nitric oxide and viral infection: Recent developments in antiviral therapies and platforms,” Applied Materials Today, vol. 22, pp. 100887, 2021/03/01/, 2021.
[44] D. D. Thomas, L. A. Ridnour, J. S. Isenberg, W. Flores-Santana, C. H. Switzer, S. Donzelli, P. Hussain, C. Vecoli, N. Paolocci, S. Ambs, C. A. Colton, C. C. Harris, D. D. Roberts, and D. A. Wink, “The chemical biology of nitric oxide: Implications in cellular signaling,” Free Radical Biology and Medicine, vol. 45, no. 1, pp. 18-31, 2008/07/01/, 2008.
[45] Y. Wei, H. Jiang, C. Chai, P. Liu, M. Qian, N. Sun, M. Gao, H. Zu, Y. Yu, G. Ji, Y. Zhang, S. Yang, J. He, J. Cheng, J. Tian, and Q. Zhao, “Endothelium-Mimetic Surface Modification Improves Antithrombogenicity and Enhances Patency of Vascular Grafts in Rats and Pigs,” JACC: Basic to Translational Science, vol. 8, no. 7, pp. 843-861, 2023.
[46] J. P. Cooke, and D. W. Losordo, “Nitric Oxide and Angiogenesis,” Circulation, vol. 105, no. 18, pp. 2133-2135, 2002.
[47] M. Luo, R. Tian, and N. Lu, “Nitric oxide protected against NADPH oxidase-derived superoxide generation in vascular endothelium: Critical role for heme oxygenase-1,” Int J Biol Macromol, vol. 126, pp. 549-554, Apr 1, 2019.
[48] A. Koike, I. Minamiguchi, K. Fujimori, and F. Amano, “Nitric oxide is an important regulator of heme oxygenase-1 expression in the lipopolysaccharide and interferon-γ-treated murine macrophage-like cell line J774.1/JA-4,” Biol Pharm Bull, vol. 38, no. 1, pp. 7-16, 2015.
[49] B.-M. Choi, H.-O. Pae, and H.-T. Chung, “Nitric oxide priming protects nitric oxide-mediated apoptosis via HEME OXYGENASE-1 induction,” Free Radical Biology and Medicine, vol. 34, no. 9, pp. 1136-1145, 2003/05/01/, 2003.
[50] T. Polte, A. Abate, P. A. Dennery, and H. Schröder, “Heme oxygenase-1 is a cGMP-inducible endothelial protein and mediates the cytoprotective action of nitric oxide,” Arterioscler Thromb Vasc Biol, vol. 20, no. 5, pp. 1209-15, May, 2000.
[51] J. Li, C. A. Bombeck, S. Yang, Y.-M. Kim, and T. R. Billiar, “Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes,” Journal of Biological Chemistry, vol. 274, no. 24, pp. 17325-17333, 1999.
[52] R. Ahmed, A. Afreen, M. Tariq, A. A. Zahid, M. S. Masoud, M. Ahmed, I. Ali, Z. Akram, and A. Hasan, “Bone marrow mesenchymal stem cells preconditioned with nitric-oxide-releasing chitosan/PVA hydrogel accelerate diabetic wound healing in rabbits,” Biomedical Materials, vol. 16, no. 3, pp. 035014, 2021/03/01, 2021.
[53] M.-L. Kang, H.-S. Kim, J. You, Y. S. Choi, B.-J. Kwon, C. H. Park, W. Baek, M. S. Kim, Y. J. Lee, G.-I. Im, J.-K. Yoon, J. B. Lee, and H.-J. Sung, “Hydrogel cross-linking-programmed release of nitric oxide regulates source-dependent angiogenic behaviors of human mesenchymal stem cell,” Science Advances, vol. 6, no. 9, pp. eaay5413, 2020.
[54] S. Jalnapurkar, R. D. Moirangthem, S. Singh, L. Limaye, and V. Kale, “Microvesicles Secreted by Nitric Oxide-Primed Mesenchymal Stromal Cells Boost the Engraftment Potential of Hematopoietic Stem Cells,” STEM CELLS, vol. 37, no. 1, pp. 128-138, 2019.
[55] G. Ali, S. Mohsin, M. Khan, G. A. Nasir, S. Shams, S. N. Khan, and S. Riazuddin, “Nitric oxide augments mesenchymal stem cell ability to repair liver fibrosis,” Journal of Translational Medicine, vol. 10, pp. 75, 2012-04-25, 2012.
[56] G. Ren, L. Zhang, X. Zhao, G. Xu, Y. Zhang, A. I. Roberts, R. C. Zhao, and Y. Shi, “Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide,” Cell Stem Cell, vol. 2, no. 2, pp. 141-50, Feb 7, 2008.
[57] E. Caballano-Infantes, G. M. Cahuana, F. J. Bedoya, C. Salguero-Aranda, and J. R. Tejedo, “The Role of Nitric Oxide in Stem Cell Biology,” Antioxidants (Basel), vol. 11, no. 3, Mar 3, 2022.
[58] A. Beltran-Povea, E. Caballano-Infantes, C. Salguero-Aranda, F. Martín, B. Soria, F. J. Bedoya, J. R. Tejedo, and G. M. Cahuana, “Role of nitric oxide in the maintenance of pluripotency and regulation of the hypoxia response in stem cells,” World J Stem Cells, vol. 7, no. 3, pp. 605-17, Apr 26, 2015.
[59] J. R. Tejedo, R. Tapia-Limonchi, S. Mora-Castilla, G. M. Cahuana, A. Hmadcha, F. Martin, F. J. Bedoya, and B. Soria, “Low concentrations of nitric oxide delay the differentiation of embryonic stem cells and promote their survival,” Cell Death & Disease, vol. 1, no. 10, pp. e80-e80, 2010/10/01, 2010.
[60] H.-T. Chung, H.-O. Pae, B.-M. Choi, T. R. Billiar, and Y.-M. Kim, “Nitric Oxide as a Bioregulator of Apoptosis,” Biochemical and Biophysical Research Communications, vol. 282, no. 5, pp. 1075-1079, 2001/04/20/, 2001.
[61] Z. Zhu, Q. Pan, W. Zhao, X. Wu, S. Yu, Q. Shen, J. Zhang, W. Yue, S. Peng, N. Li, S. Zhang, A. Lei, and J. Hua, “BCL2 enhances survival of porcine pluripotent stem cells through promoting FGFR2,” Cell Proliferation, vol. 54, no. 1, pp. e12932, 2021.
[62] E. Caballano-Infantes, I. Díaz, A. B. Hitos, G. M. Cahuana, A. Martínez-Ruiz, B. Soria-Juan, R. Rodríguez-Griñolo, A. Hmadcha, F. Martín, B. Soria, J. R. Tejedo, and F. J. Bedoya, “Stemness of Human Pluripotent Cells: Hypoxia-Like Response Induced by Low Nitric Oxide,” Antioxidants, vol. 10, no. 9, pp. 1408, 2021.
[63] S. Mora-Castilla, J. R. Tejedo, A. Hmadcha, G. M. Cahuana, F. Martín, B. Soria, and F. J. Bedoya, “Nitric oxide repression of Nanog promotes mouse embryonic stem cell differentiation,” Cell Death & Differentiation, vol. 17, no. 6, pp. 1025-1033, 2010/06/01, 2010.
[64] J. C. Wong, and R. R. Fiscus, “Essential roles of the nitric oxide (no)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway and the atrial natriuretic peptide (ANP)/cGMP/PKG-Iα autocrine loop in promoting proliferation and cell survival of OP9 bone marrow stromal cells,” Journal of Cellular Biochemistry, vol. 112, no. 3, pp. 829-839, 2011.
[65] H. Kim, H. Jang, T. W. Kim, B.-H. Kang, S. E. Lee, Y. K. Jeon, D. H. Chung, J. Choi, J. Shin, E.-J. Cho, and H.-D. Youn, “Core Pluripotency Factors Directly Regulate Metabolism in Embryonic Stem Cell to Maintain Pluripotency,” Stem Cells, vol. 33, no. 9, pp. 2699-2711, 2015.
[66] Q.-Y. Wang, Z.-S. Liu, J. Wang, H.-X. Wang, A. Li, Y. Yang, X.-Z. Wang, Y.-Q. Zhao, Q.-Y. Han, H. Cai, B. Liang, N. Song, W.-H. Li, and T. Li, “Glutathione peroxidase-1 is required for self-renewal of murine embryonic stem cells,” Biochemical and Biophysical Research Communications, vol. 448, no. 4, pp. 454-460, 2014/06/13/, 2014.
[67] N. Bandara, S. Gurusinghe, A. Kong, G. Mitchell, L.-X. Wang, S. Y. Lim, and P. Strappe, “Generation of a nitric oxide signaling pathway in mesenchymal stem cells promotes endothelial lineage commitment,” Journal of Cellular Physiology, vol. 234, no. 11, pp. 20392-20407, 2019.
[68] W. Bloch, B. K. Fleischmann, D. E. Lorke, C. Andressen, B. Hops, J. Hescheler, and K. Addicks, “Nitric oxide synthase expression and role during cardiomyogenesis,” Cardiovascular Research, vol. 43, no. 3, pp. 675-684, 1999.
[69] J. Li, A. LoBue, S. K. Heuser, and M. M. Cortese-Krott, “Determination of Nitric Oxide and Its Metabolites in Biological Tissues Using Ozone-Based Chemiluminescence Detection: A State-of-the-Art Review,” Antioxidants, vol. 13, no. 2, pp. 179, 2024.
[70] S. Menon, M. R. Mathew, S. Sam, K. Keerthi, and K. G. Kumar, “Recent advances and challenges in electrochemical biosensors for emerging and re-emerging infectious diseases,” Journal of Electroanalytical Chemistry, vol. 878, pp. 114596, 2020/12/01/, 2020.
[71] D. Tsikas, “Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction: Appraisal of the Griess reaction in the l-arginine/nitric oxide area of research,” Journal of Chromatography B, vol. 851, no. 1, pp. 51-70, 2007/05/15/, 2007.
[72] V. P. R, F. Antunes, J. Pires, A. Silva-Herdade, and M. L. Pinto, “A Comparison of Different Approaches to Quantify Nitric Oxide Release from NO-Releasing Materials in Relevant Biological Media,” Molecules, vol. 25, no. 11, Jun 2, 2020.
[73] J. Li, A. LoBue, S. K. Heuser, F. Leo, and M. M. Cortese-Krott, “Using diaminofluoresceins (DAFs) in nitric oxide research,” Nitric Oxide, vol. 115, pp. 44-54, 2021/10/01/, 2021.
[74] N. Gharavi, and A. El-Kadi, “Measurement of nitric oxide in murine Hepatoma Hepa1c1c7 cells by reversed phase HPLC with fluorescence detection,” Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Société canadienne des sciences pharmaceutiques, vol. 6, pp. 302-7, 05/01, 2003.
[75] S. M. Namin, S. Nofallah, M. S. Joshi, K. Kavallieratos, and N. M. Tsoukias, “Kinetic analysis of DAF-FM activation by NO: toward calibration of a NO-sensitive fluorescent dye,” Nitric Oxide, vol. 28, pp. 39-46, Jan 15, 2013.
[76] B. Piknova, M. T. Gladwin, A. N. Schechter, and N. Hogg, “Electron paramagnetic resonance analysis of nitrosylhemoglobin in humans during NO inhalation,” J Biol Chem, vol. 280, no. 49, pp. 40583-8, Dec 9, 2005.
[77] N. Hogg, “Detection of nitric oxide by electron paramagnetic resonance spectroscopy,” Free Radic Biol Med, vol. 49, no. 2, pp. 122-9, Jul 15, 2010.
[78] A. Vishwakarma, A. Wany, S. Pandey, M. Bulle, A. Kumari, R. Kishorekumar, A. U. Igamberdiev, L. A. J. Mur, and K. J. Gupta, “Current approaches to measure nitric oxide in plants,” Journal of Experimental Botany, vol. 70, no. 17, pp. 4333-4343, 2019.
[79] K. B. Shumaev, I. V. Gorudko, O. V. Kosmachevskaya, D. V. Grigorieva, О. M. Panasenko, A. F. Vanin, A. F. Topunov, M. S. Terekhova, A. V. Sokolov, S. N. Cherenkevich, and E. K. Ruuge, “Protective Effect of Dinitrosyl Iron Complexes with Glutathione in Red Blood Cell Lysis Induced by Hypochlorous Acid,” Oxidative Medicine and Cellular Longevity, vol. 2019, pp. 2798154, 2019/04/08, 2019.
[80] D. R. Truzzi, N. M. Medeiros, O. Augusto, and P. C. Ford, “Dinitrosyl Iron Complexes (DNICs). From Spontaneous Assembly to Biological Roles,” Inorganic Chemistry, vol. 60, no. 21, pp. 15835-15845, 2021/11/01, 2021.
[81] A. F. Vanin, “Physico-Chemistry of Dinitrosyl Iron Complexes as a Determinant of Their Biological Activity,” International Journal of Molecular Sciences, vol. 22, no. 19, pp. 10356, 2021.
[82] Y.-C. Chen, Y.-H. Chen, H. Chiu, Y.-H. Ko, R.-T. Wang, W.-P. Wang, Y.-J. Chuang, C.-C. Huang, and T.-T. Lu, “Cell-Penetrating Delivery of Nitric Oxide by Biocompatible Dinitrosyl Iron Complex and Its Dermato-Physiological Implications,” International Journal of Molecular Sciences, vol. 22, no. 18, pp. 10101, 2021.
[83] H.-T. Hsieh, H.-C. Huang, C.-W. Chung, C.-C. Chiang, T. Hsia, H.-F. Wu, R.-L. Huang, C.-S. Chiang, J. Wang, T.-T. Lu, and Y. Chen, “CXCR4-targeted nitric oxide nanoparticles deliver PD-L1 siRNA for immunotherapy against glioblastoma,” Journal of Controlled Release, vol. 352, pp. 920-930, 2022/12/01/, 2022.
[84] H.-C. Huang, Y.-C. Sung, C.-P. Li, D. Wan, P.-H. Chao, Y.-T. Tseng, B.-W. Liao, H.-T. Cheng, F.-F. Hsu, C.-C. Huang, Y.-T. Chen, Y.-H. Liao, H. T. Hsieh, Y.-C. Shih, I.-J. Liu, H.-C. Wu, T.-T. Lu, J. Wang, and Y. Chen, “Reversal of pancreatic desmoplasia by a tumour stroma-targeted nitric oxide nanogel overcomes TRAIL resistance in pancreatic tumours,” Gut, vol. 71, no. 9, pp. 1843-1855, 2022.
[85] W.-H. Chuang, Y.-T. Chou, Y.-H. Chen, T.-H. Kuo, W.-F. Liaw, T.-T. Lu, C.-F. Kao, and Y.-M. Wang, “Neuroprotective Effect of NO-Delivery Dinitrosyl Iron Complexes (DNICs) on Amyloid Pathology in the Alzheimer’s Disease Cell Model,” ACS Chemical Neuroscience, vol. 14, no. 16, pp. 2922-2934, 2023/08/16, 2023.
[86] C.-R. Wu, Y.-D. Huang, Y.-H. Hong, Y.-H. Liu, M. Narwane, Y.-H. Chang, T. K. Dinh, H.-T. Hsieh, Y.-J. Hseuh, P.-C. Wu, C.-W. Pao, T.-S. Chan, I. J. Hsu, Y. Chen, H.-C. Chen, T.-Y. Chin, and T.-T. Lu, “Endogenous Conjugation of Biomimetic Dinitrosyl Iron Complex with Protein Vehicles for Oral Delivery of Nitric Oxide to Brain and Activation of Hippocampal Neurogenesis,” JACS Au, vol. 1, no. 7, pp. 998-1013, 2021/07/26, 2021.
[87] C.-W. Chung, B.-W. Liao, S.-W. Huang, S.-J. Chiou, C.-H. Chang, S.-J. Lin, B.-H. Chen, W.-L. Liu, S.-H. Hu, Y.-C. Chuang, C.-H. Lin, I. J. Hsu, C.-M. Cheng, C.-C. Huang, and T.-T. Lu, “Magnetic Responsive Release of Nitric Oxide from an MOF-Derived Fe3O4@PLGA Microsphere for the Treatment of Bacteria-Infected Cutaneous Wound,” ACS Applied Materials & Interfaces, vol. 14, no. 5, pp. 6343-6357, 2022/02/09, 2022.
[88] Y.-J. Chen, S.-C. Wu, H.-C. Wang, T.-H. Wu, S.-S. F. Yuan, T.-T. Lu, W.-F. Liaw, and Y.-M. Wang, “Activation of Angiogenesis and Wound Healing in Diabetic Mice Using NO-Delivery Dinitrosyl Iron Complexes,” Molecular Pharmaceutics, vol. 16, no. 10, pp. 4241-4251, 2019/10/07, 2019.
[89] J. Mosquera, I. García, and L. M. Liz-Marzán, “Cellular Uptake of Nanoparticles versus Small Molecules: A Matter of Size,” Accounts of Chemical Research, vol. 51, no. 9, pp. 2305-2313, 2018/09/18, 2018.
[90] C. A. Lipinski, F. Lombardo, B. W. Dominy, and P. J. Feeney, “Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings,” Advanced Drug Delivery Reviews, vol. 23, no. 1, pp. 3-25, 1997/01/15/, 1997.
[91] A. A. Ramahi, and R. L. Ruff, "Membrane Potential," Encyclopedia of the Neurological Sciences (Second Edition), M. J. Aminoff and R. B. Daroff, eds., pp. 1034-1035, Oxford: Academic Press, 2014.
[92] LibreTexts. "Facilitated Diffusion," 04/17/2024; https://bio.libretexts.org/@go/page/6460.
[93] B. Online. "Active transport," 04/17/2024; https://www.biologyonline.com/dictionary/active-transport.
[94] G. M. Cooper, "The Cell: A Molecular Approach. 2nd edition.," S. M. S. Associates, ed., 2000.
[95] LibreTexts. "Exocytosis and Endocytosis," 04/17/2024; https://bio.libretexts.org/@go/page/6463.
[96] L. Nordgård, “Survival and uptake of feed-derived DNA in the mammalian intestinal tract,” 09/13, 2009.
[97] J. Gong, H. X. Wang, and K. W. Leong, “Determination of Cellular Uptake and Endocytic Pathways,” Bio Protoc, vol. 9, no. 4, pp. e3169, Feb 20, 2019.
[98] M. C. Kerr, and R. D. Teasdale, “Defining Macropinocytosis,” Traffic, vol. 10, no. 4, pp. 364-371, 2009.
[99] L. Guo. "Pinocytosis - What Is It, How It Occurs, and More," https://www.osmosis.org/answers/pinocytosis#:~:text=Pinocytotic%20vesicles%20are%20very%20small,an%20electron%20microscope%20for%20visualization.
[100] J. Canton, “Macropinocytosis: New Insights Into Its Underappreciated Role in Innate Immune Cell Surveillance,” Frontiers in Immunology, vol. 9, 2018-October-02, 2018.
[101] E. Uribe-Querol, and C. Rosales, “Phagocytosis: Our Current Understanding of a Universal Biological Process,” Frontiers in Immunology, vol. 11, 2020-June-02, 2020.
[102] R. Levin, S. Grinstein, and J. Canton, “The life cycle of phagosomes: formation, maturation, and resolution,” Immunological Reviews, vol. 273, no. 1, pp. 156-179, 2016.
[103] J. Canton, “Phagosome maturation in polarized macrophages,” Journal of Leukocyte Biology, vol. 96, no. 5, pp. 729-738, 2014.
[104] M. Au - Horsthemke, J. Au - Wilden, A. C. Au - Bachg, and P. J. Au - Hanley, “Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages,” JoVE, no. 140, pp. e57566, 2018/10/19/, 2018.
[105] V. Bitsikas, I. R. Corrêa, Jr., and B. J. Nichols, “Clathrin-independent pathways do not contribute significantly to endocytic flux,” eLife, vol. 3, pp. e03970, 2014/09/17, 2014.
[106] M. Kaksonen, and A. Roux, “Mechanisms of clathrin-mediated endocytosis,” Nature Reviews Molecular Cell Biology, vol. 19, no. 5, pp. 313-326, 2018/05/01, 2018.
[107] T. dos Santos, J. Varela, I. Lynch, A. Salvati, and K. A. Dawson, “Effects of transport inhibitors on the cellular uptake of carboxylated polystyrene nanoparticles in different cell lines,” PLoS One, vol. 6, no. 9, pp. e24438, 2011.
[108] L. H. Wang, K. G. Rothberg, and R. G. Anderson, “Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation,” Journal of Cell Biology, vol. 123, no. 5, pp. 1107-1117, 1993.
[109] D. Vercauteren, R. E. Vandenbroucke, A. T. Jones, J. Rejman, J. Demeester, S. C. De Smedt, N. N. Sanders, and K. Braeckmans, “The Use of Inhibitors to Study Endocytic Pathways of Gene Carriers: Optimization and Pitfalls,” Molecular Therapy, vol. 18, no. 3, pp. 561-569, 2010/03/01/, 2010.
[110] R. G. Anderson, “The caveolae membrane system,” Annual review of biochemistry, vol. 67, no. 1, pp. 199-225, 1998.
[111] A. L. Kiss, and E. Botos, “Endocytosis via caveolae: alternative pathway with distinct cellular compartments to avoid lysosomal degradation?,” J Cell Mol Med, vol. 13, no. 7, pp. 1228-37, Jul, 2009.
[112] B. Razani, S. E. Woodman, and M. P. Lisanti, “Caveolae: from cell biology to animal physiology,” Pharmacological reviews, vol. 54, no. 3, pp. 431-467, 2002.
[113] K. Sandvig, S. Kavaliauskiene, and T. Skotland, “Clathrin-independent endocytosis: an increasing degree of complexity,” Histochemistry and Cell Biology, vol. 150, no. 2, pp. 107-118, 2018/08/01, 2018.
[114] H. X. Wang, Z. Song, Y. H. Lao, X. Xu, J. Gong, D. Cheng, S. Chakraborty, J. S. Park, M. Li, D. Huang, L. Yin, J. Cheng, and K. W. Leong, “Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide,” Proc Natl Acad Sci U S A, vol. 115, no. 19, pp. 4903-4908, May 8, 2018.
[115] A. G. Torres, and M. J. Gait, “Exploiting cell surface thiols to enhance cellular uptake,” Trends in Biotechnology, vol. 30, no. 4, pp. 185-190, 2012/04/01/, 2012.
[116] Y. Cheng, A.-T. Pham, T. Kato, B. Lim, D. Moreau, J. López-Andarias, L. Zong, N. Sakai, and S. Matile, “Inhibitors of thiol-mediated uptake,” Chemical Science, vol. 12, no. 2, pp. 626-631, 2021.
[117] E. P. Feener, W. C. Shen, and H. J. Ryser, “Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes,” Journal of Biological Chemistry, vol. 265, no. 31, pp. 18780-18785, 1990/11/05/, 1990.
[118] H. J. Ryser, E. M. Levy, R. Mandel, and G. J. DiSciullo, “Inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction,” Proceedings of the National Academy of Sciences, vol. 91, no. 10, pp. 4559-4563, 1994.
[119] D. Thiry, R. Francq, D. Cossement, M. Guillaume, J. Cornil, and R. Snyders, “A Detailed Description of the Chemistry of Thiol Supporting Plasma Polymer Films,” Plasma Processes and Polymers, vol. 11, no. 6, pp. 606-615, 2014.
[120] J. D. Gregory, “The Stability of N-Ethylmaleimide and its Reaction with Sulfhydryl Groups,” Journal of the American Chemical Society, vol. 77, no. 14, pp. 3922-3923, 1955/07/01, 1955.
[121] B. Wenge, and H. Bönisch, “N-Ethylmaleimide differentially inhibits substrate uptake by and ligand binding to the noradrenaline transporter,” Naunyn Schmiedebergs Arch Pharmacol, vol. 377, no. 3, pp. 255-65, May, 2008.
[122] E. Eroglu, S. Charoensin, H. Bischof, J. Ramadani, B. Gottschalk, M. R. Depaoli, M. Waldeck-Weiermair, W. F. Graier, and R. Malli, “Genetic biosensors for imaging nitric oxide in single cells,” Free Radical Biology and Medicine, vol. 128, pp. 50-58, 2018/11/20/, 2018.
[123] E. Eroglu, B. Gottschalk, S. Charoensin, S. Blass, H. Bischof, R. Rost, C. T. Madreiter-Sokolowski, B. Pelzmann, E. Bernhart, W. Sattler, S. Hallström, T. Malinski, M. Waldeck-Weiermair, W. F. Graier, and R. Malli, “Development of novel FP-based probes for live-cell imaging of nitric oxide dynamics,” Nature Communications, vol. 7, no. 1, pp. 10623, 2016/02/04, 2016.
[124] H. Chiu, A. Chau Fang, Y.-H. Chen, R. X. Koi, K.-C. Yu, L.-H. Hsieh, Y.-M. Shyu, T. A. M. Amer, Y.-J. Hsueh, Y.-T. Tsao, Y.-J. Shen, Y.-M. Wang, H.-C. Chen, Y.-J. Lu, C.-C. Huang, and T.-T. Lu, “Mechanistic and Kinetic Insights into Cellular Uptake of Biomimetic Dinitrosyl Iron Complexes and Intracellular Delivery of NO for Activation of Cytoprotective HO-1,” JACS Au, vol. 4, no. 4, pp. 1550-1569, 2024/04/22, 2024.
[125] G. L. Gstraunthaler, Toni, Zell- und Gewebekultur, Heidelberg, Germany: Springer Spektrum, 2013.
[126] K. Hirayama, S. Akashi, M. Furuya, and K. Fukuhara, “Rapid confirmation and revision of the primary structure of bovine serum albumin by ESIMS and Frit-FAB LC/MS,” Biochem Biophys Res Commun, vol. 173, no. 2, pp. 639-46, Dec 14, 1990.
[127] M. Boese, P. I. Mordvintcev, A. F. Vanin, R. Busse, and A. Mülsch, “S-nitrosation of serum albumin by dinitrosyl-iron complex,” J Biol Chem, vol. 270, no. 49, pp. 29244-9, Dec 8, 1995.
[128] Y.-T. Tseng, C.-H. Chen, J.-Y. Lin, B.-H. Li, Y.-H. Lu, C.-H. Lin, H.-T. Chen, T.-C. Weng, D. Sokaras, H.-Y. Chen, Y.-L. Soo, and T.-T. Lu, “To Transfer or Not to Transfer? Development of a Dinitrosyl Iron Complex as a Nitroxyl Donor for the Nitroxylation of an FeIII–Porphyrin Center,” Chemistry – A European Journal, vol. 21, no. 49, pp. 17570-17573, 2015.
[129] T.-T. Lu, T.-C. Weng, and W.-F. Liaw, “X-Ray Emission Spectroscopy: A Spectroscopic Measure for the Determination of NO Oxidation States in Fe–NO Complexes,” Angewandte Chemie International Edition, vol. 53, no. 43, pp. 11562-11566, 2014.
[130] R. Meanti, L. Rizzi, E. Bresciani, L. Molteni, V. Locatelli, S. Coco, R. J. Omeljaniuk, and A. Torsello, “Hexarelin Modulation of MAPK and PI3K/Akt Pathways in Neuro-2A Cells Inhibits Hydrogen Peroxide—Induced Apoptotic Toxicity,” Pharmaceuticals, vol. 14, no. 5, pp. 444, 2021.
[131] J. Wu, F. Wang, Z. Su, J. Liu, S. Hu, H. Li, P. Hu, and D. Wu, “Role of ataxia-telangiectasia mutated in hydrogen peroxide preconditioning against oxidative stress in Neuro-2a cells,” Mol Med Rep, vol. 15, no. 6, pp. 4280-4285, 2017/06/01, 2017.
[132] A. E. Maczurek, R. Wild, D. Laurenti, M. L. Steele, L. Ooi, and G. Münch, “Generation of hydrogen peroxide-resistant murine neuroblastoma cells: a target discovery platform for novel neuroprotective genes,” Journal of Neural Transmission, vol. 120, no. 8, pp. 1171-1178, 2013/08/01, 2013.
[133] C. M. Hardaway, R. B. Badisa, and K. F. A. Soliman, “Effect of ascorbic acid and hydrogen peroxide on mouse neuroblastoma cells,” Mol Med Rep, vol. 5, no. 6, pp. 1449-1452, 2012/06/01, 2012.