Development of Biocompatible Fe3O4@SiO2 Nanoparticles as Subcellular Delivery Platform for Glucosamine-6-phosphate Synthase Inhibitors
Numerous inhibitors of glucoseamine-6-phophate synthase (GlcN-6-P), the enzyme responsible from catalysis of the first step of metabolic pathway leading to metabolism 5’-diphospho-N-acetyl-D- glucosamine, were reported as effective agents for inhibiting the growth of various fungal pathogens. Among the reported inhibitors, N3-4-(methoxyfumaroyl)-L-2,3- diaminopropanoic acid (FMDP) was reported to be the most effective inhibitor of GlcN-6-P. However, poor in vitro inhibitory concentrations recorded for FMDP in free molecular form required further research on development of more effective derivatives of this valuable inhibitor. Despite the 35-years of intensive research on the topic and numerous reports on new peptide and prodrug forms of FMDP, poor inhibitory effects of prodrugs and the rapid cellular resistance acquired against FMDP-peptides required the other areas of the antifungal development to be discovered. Among these areas, nanoparticle mediated delivery of FMDP constitutes the topic of this thesis. Within this concept, iron oxide-silica core- shell nanoparticles (CSNPs) as the only Food and Drug Administration (FDA) approved nanomaterials to the date has been further developed to function as biocompatible drug delivery platforms for GlcN-6-P enzyme. In order to develop the preparation of CSNPs, thesis deeply explores the cetyltrimethylammonium (CTA+) directed silica coating methods of the oleic-acid capped iron oxide nanoparticles (OA-IONPs) initialized under near-neutral pH conditions. It is demonstrated that the initial alkaline hydrolysis of ethyl acetate in the presence of CTA+ and OA-IONPs induces an unusual ligand exchange mechanism between oleic acid and in situ formed acetate ions. The acetate induced mechanism is shown to be catalysing the formation of ∼20-25 nm thick amorphous silica shells around the individual iron oxide nanoparticles resulting in highly mono-dispersed CSNPs. Synergetic effects of the iron oxide nanoparticle clusters (IONPCs) and the in situ formed CTAB vesicles encapsulating the silica precursors formed in high [CTAB]/[IONP] ratio conditions are demonstrated. Time-resolved HR- TEM and cryo-TEM observations demonstrates a unique micellar fusion mechanism inducing the formation of mono-dispersed core-shell nanoparticles. Detailed electron energy-loss spectroscopy (EELS), Fourier transform-infrared (FT-IR), Raman and NMR spectroscopy further supports the micellar fusion mechanism behind the formation of CSNPs. Thesis further presents the superparamagnetic properties of aqueously well dispersible CSNPs prepared by reported nanoemulsion procedure. Measurement of the magnetic properties, confirming the superparamagnetic nature of CSNPs by DC magnetometry and paramagnetic nature of the particles confirmed by AC magnetometry, are discussed in detail. In vitro biological activities of CSNP-GlcN-6-P inhibitor conjugates against human cervical cancer (HeLa), hypopharyngeal carcinoma (FaDu), human fibroblast (MSU1.1) cell lines andCandida albicans demonstrates that the cancer cells almost completely loose their viability in the presence of CSNP-GlcN- 6-P inhibitor conjugates, whereas, the growth of Candida albicans is reduced by only 30%. Nevertheless, cellular internalization studies performed with polyethyleneglycol functionalized core-shell nanoparticles (OH-PEG-CSNPs) in the absence of GlcN-6-P inhibitors by TEM imaging evidences the excellent biocompatibility of OH-PEG-CSNPs. Biological TEM studies suggest that, elevated internalization of OH- PEG-CSNPs by human cells in contrast to inefficient internalization by Candida albicans could be the explanation for ineffective inhibition of fungal cells. Overall, the thesis questions the validity and meaning of “biocompatibility” approach in nanoparticle formulations of antifungal agent.
Findings of this thesis have been published in:
1. Kertmen, A.; Torruella, P.; Coy, E.; Yate, L.; Nowaczyk, G.; Gapiński, J.; Vogt, C.; Toprak, M.; Estradé, S.; Peiró, F.; Milewski, S.; Jurga, S.; Andruszkiewicz, R. Acetate-Induced Disassembly of Spherical Iron Oxide Nanoparticle Clusters into Monodispersed Core-Shell Structures upon Nanoemulsion Fusion. Langmuir 2017, 33 (39), 10351–10365. https://doi.org/10.1021/acs.langmuir.7b02743.
2. Tadyszak, K.; Kertmen, A.; Coy, E.; Andruszkiewicz, R.; Milewski, S.; Kardava, I.; Scheibe, B.; Jurga, S.; Chybczyńska, K. Spectroscopic and Magnetic Studies of Highly Dispersible Superparamagnetic Silica Coated Magnetite Nanoparticles. J. Magn. Magn. Mater. 2017, 433, 254–261. https://doi.org/10.1016/j.jmmm.2017.03.025.
3. Kertmen, A.; Przysiecka, L.; Coy, E.; Popenda, L.; Andruszkiewicz, R.; Jurga, S.; Milewski, S. Emerging Anticancer Activity of Candidal Glucoseamine-6-Phosphate Synthase Inhibitors upon Nanoparticle-Mediated Delivery. Langmuir 2019, 35 (15), 5281–5293. https://doi.org/10.1021/acs.langmuir.8b04250.
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Emerging Anticancer Activity of Candidal Glucoseamine-6-Phosphate Synthase Inhibitors upon Nanoparticle-mediated Delivery usuń
- A. Kertmen,
- Ł. Przysiecka,
- E. Coy
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