Stefanie Klein

Mesoporous and nonmesoporous SiO2@MnFe2O4 nanostructures were loaded with the hypoxia-induciblefactor-1inhibitor acriflavine for combined radiation and hypoxia therapies.The X-ray irradiation of the drug-loaded nanostructures not only triggeredthe release of the acriflavine inside the cells but also initiatedan energy transfer from the nanostructures to surface-adsorbed oxygento generate singlet oxygen. While the drug-loaded mesoporous nanostructuresshowed an initial drug release before the irradiation, the drug wasprimarily released upon X-ray radiation in the case of the nonmesoporousnanostructures. However, the drug loading capacity was less efficientfor the nonmesoporous nanostructures. Both drug-loaded nanostructuresproved to be very efficient in irradiated MCF-7 multicellular tumorspheroids. The damage of these nanostructures toward the nontumorigenicMCF-10A multicellular spheroids was very limited because of the smallnumber of nanostructures that entered the MCF-10A spheroids, whilesimilar concentrations of acriflavine without nanostructures weretoxic for the MCF-10A spheroids.

The X-radiation enhancing effect of caffeic acid-functionalized Au–Fe₃O₄, Pt–Fe₃O₄, and Pd–Fe₃O₄ nanoheterodimers (NHDs) on 2D and 3D breast tumor (MCF-7) and healthy breast epithelial (MCF-10A) cells was comprehensively examined by performing cell viability, reactive oxygen species (ROS) detection, enzyme activity, and clonogenic assays. Intracellular NHDs were observed to cause DNA fragmentation and to enhance superoxide and hydroxyl radical formation in MCF-7 cells when exposed to a single dose of 1 Gy. MCF-7-derived multicellular tumor spheroids (MCF-7 MCTS) and MCF-10A-derived multicellular spheroids (MCF-10A MCS) were incubated with the NHDs and irradiated with five daily doses of 2 Gy. The Au–Fe₃O₄ and Pt–Fe₃O₄ NHD-loaded MCF-7 MCTS significantly decreased in volume after being exposed to fractionated irradiation, whereas intracellular Au–Fe₃O₄ and Pt–Fe₃O₄ NHDs promoted the growth of the irradiated MCF-10A MCS. Moreover, Au–Fe₃O₄ and Pt–Fe₃O₄ NHDs were shown to impede ROS formation and inhibit DNA strand breakage in the noncancerous MCF-10A cells. On the other hand, the Pd–Fe₃O₄ NHDs boosted X-radiation-induced damage of MCF-10 A cells, which was ascribed to impairment of the ROS scavenging enzyme activities. In summary, caffeic acid-functionalized Au–Fe₃O₄ and Pt–Fe₃O₄ NHDs performed as excellent X-radiation enhancing agents in breast tumor cells, while they protect healthy breast epithelial cells against X-radiation.

Au-Fe₃O₄ nanoheterodimers (NHD) were functionalized with the natural and synthetic anticancer drugs caffeic acid (CA), quercetin (Q) and 5-fluorocytidine (5FC). Their X-radiation dose-enhancing potential and chemotherapeutic efficacy for bimodal cancer therapy were investigated by designing multicellular tumor spheroids (MCTS) to in vitro avascular tumor models. MCTS were grown from the breast cancer cell lines MCF-7, MDA-MB-231, and MCF-10A. The MCF-7, MDA-MB-231 and MCF-10A MCTS were incubated with NHD-CA, NHD-Q, or NHD-5FC and then exposed to fractionated X-radiation comprising either a single 10 Gy dose, 2 daily single 5 Gy doses or 5 daily single 2 Gy doses. The NHD-CA, NHD-Q, and NHD-5FC affected the growth of X-ray irradiated and non-irradiated MCTS in a different manner. The impact of the NHDs on the glycolytic metabolism due to oxygen deprivation inside MCTS was assessed by measuring lactate secretion and glucose uptake by the MCTS. The NHD-CA and NHD-Q were found to act as X-radiation dose agents in MCF-7 MCTS and MDA-MB-231 MCTS and served as radioprotector in MCF-10A MCTS. X-ray triggered release of CA and Q inhibited lactate secretion and thereupon disturbed glycolytic reprogramming, whereas 5FC exerted their cytotoxic effects on both, healthy and tumor cells, after their release into the cytosol.

The efficiency of nanoparticle-enhanced radiotherapy was studied by loading MCF-7 and A549 multicellular tumor spheroids (MCTSs) with caffeic acid- and nitrosonium-functionalized Au–Fe₃O₄ nanoheterodimers (Au–Fe₃O₄ NHDs). Transmission electron microscope images of MCTS cross-sectional sections visualized the invasion and distribution of the nitrosonium- and caffeic acid-functionalized Au–Fe₃O₄ NHDs (NO- and CA-NHDs) in the A549 and MCF-7 MCTSs, whereas the iron content of the MCTSs were quantified using the ferrozine assay. The synergistic impact of intracellular NO- and CA-NHDs and X-ray irradiation on the growth dynamics of the A549 and MCF-7 MCTSs was surveyed by monitoring their temporal evolution under a light microscope over a period of 14 days. The emergence of hypoxia during the spheroid growth was followed by detecting the lactate efflux of MCTSs without and with NO- and CA-NHDs. The performance of the NO- and CA-NHDs as X-ray dose-enhancing agents in the A549 and MCF-7 MCTSs was clarified by performing clonogenic cell survival assays and determining the respective dose-modifying factors for X-ray doses of 0, 2, 4, and 6 Gy. The NO- and CA-NHDs were shown to perform as potent X-ray dose-enhancing agents in A549 and MCF-7 MCTSs. Moreover, the CA-NHDs boosted their radio-sensitizing efficacy by inhibiting the lactate efflux as impairing metabolic reprogramming. A synergistic effect on the MCTS destruction was observed for the combination of both NHDs since the surfactants differ in their antitumor effect.

Our research objective was to develop novel drug delivery vehicles consisting of TiO₂ and Al₂O₃ nanoparticles encapsulated by a bilayer shell that allows the reversible embedment of hydrophobic drugs. The first shell is formed by covalent binding of hydrophobic phosphonic acid at the metal oxide surface. The second shell composed of amphiphilic sodium dodecylbenzenesulfonate emerges by self-aggregation driven by hydrophobic interactions between the dodecylbenzene moiety and the hydrophobic first shell. The resulting double layer provides hydrophobic pockets suited for the intake of hydrophobic drugs. The nanoparticles were loaded with the anticancer drugs quercetin and 7-amino-4-methylcoumarin. Irradiation with X-rays was observed to release the potential anticancer drugs into the cytoplasm. In Michigan Cancer Foundation (MCF)-10 A cells, quercetin and 7-amino-4-methylcoumarin acted as antioxidants by protecting the non-tumorigenic cells from harmful radiation effects. In contrast, these agents increased the reactive oxygen species (ROS) formation in cancerous MCF-7 cells. Quercetin and 7-amino-4-methylcoumarin were shown to induce apoptosis via the mitochondrial pathway in cancer cells by determining an increase in TUNEL-positive cells and a decrease in mitochondrial membrane potential after irradiation. After X-ray irradiation, the survival fraction of MCF-7 cells with drug-loaded nanoparticles considerably decreased, which demonstrates the excellent performance of the double-layer stabilized nanoparticles as drug delivery vehicles.

Bifunctional Au-Fe3O4 nanoheterodimers were synthesized by thermally decomposing Fe(III)oleate on gold nanoparticles followed by functionalizing with tiron, 2,3-dihydroxybenzoic acid or caffeic acid. These catechol derivatives are antioxidative and thus are predicted to function as superoxide scavengers. In particular, caffeic acid lost its antioxidant capacity, although it was covalently linked through its carboxyl moiety to the Fe3O4 surface. Tiron was shown to bind via its catechol group to the Au-Fe3O4 nanoheterodimers, and 2,3-dihydroxybenzoic was just physisorbed between the oleic acid surface structures. Caffeic-acid stabilized Au-Fe3O4 nanoheterodimers turned out to act as X-ray protector in healthy cells but as X-ray enhancing agents in cancer cells. Furthermore, these functionalized Au-Fe3O4 nanoheterodimers were found to inhibit the migratory capacity of the cancer cells.

Superparamagnetic iron oxide nanoparticles were shown to exhibit a high performance as X-ray dosage enhancer in tumor cells. The radio-enhancing potential of uncoated and citrate-coated SPIONs was comprehensively studied for tumor and healthy cells. Pristine and citrate-coated SPIONs drastically differ in their water dispersibility and adsorption affinity for proteins. The activities of antioxidant enzymes in the healthy cells were shown to be significantly distinct from those in the tumor cells as containing a much higher H2O2 concentration. Pristine SPIONs catalyzed the Fenton reaction of hydrogen peroxide to the highly reactive hydroxyl radical in all cell types. In contrast, intracellular citrate-stabilized SPIONs were shown t o be non-toxic and to do not affect the formation of reactive oxygen species. X-ray irradiation of citrate-stabilized SPIONs, when internalized by tumor cells, significantly boost the formation of hydroxyl radicals, whereas the healthy cells preserved their initial levels of reactive oxygen species.

A facile one-pot synthesis route for the preparation of water-soluble, biocompatible patchy Fe₃O₄-Au nanoparticles (Fe₃O₄-Au pNPs) was developed. Biocompatibility was attained through surface functionalization with 1-methyl-3-(dodecylphosphonic acid) imidazolium bromide. The morphology, composition, crystal structure and magnetic properties of the Fe₃O₄-Au pNPs were investigated by conducting experiments with transmission electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction and superconducting quantum interference device, respectively. Internalization of the Fe₃O₄-Au pNPs by MCF-7 cells occurred via endocytosis. The performance of the Fe₃O₄-Au pNPs as X-ray radiosensitizer in tumor cells was compared with that of gold nanocluster and Fe₃O₄ NPs. For this reason, MCF-7, A549 and MCF-10A cells were loaded with the respective kind of nanoparticles and treated with X-rays at doses of 1, 2 or 3 Gy. The nanoparticle-induced changes of the concentration of the reactive oxygen species (ROS) were detected using specific assays, and the cell survival under X-ray exposure was assessed employing the clonogenic assay. In comparison with the gold nanocluster and Fe₃O₄ NPs, the Fe₃O₄-Au pNPs exhibited the highest catalytic capacity for ROS generation in MCF-7 and A549 cells, whereas in the X-ray-induced ROS formation in healthy MCF-10A cells was hardly enhanced by the Fe₃O₄ NPs and Fe₃O₄-Au pNPs. Moreover, the excellent performance of Fe₃O₄-Au pNPs as X-ray radiosensitizers was verified by the quickly decaying radiation dose survival curve of the nanoparticle-loaded MCF-7 and A549 cells and corroborated by the small values of the associated dose-modifying factors.

Au-Fe3O4 nanoheterodimers were obtained by thermally decomposing iron oleate on pre-synthesized gold nanoparticles. Water solubility as well as surface charges were achieved by encapsulating the initially hydrophobic Au-Fe3O4 nanoheterodimers in a self-assembled bilayer shell formed either by 1-octadecylpyridinium providing positive surface charges or by 4-dodecylbenzenesulfonate yielding a negatively charged surface. The surface charge and surface architecture were shown to control both, the cellular entry and the intracellular trafficking of the Au-Fe3O4 nanoheterodimers. The positively charged (1-octylpyridinium-terminated) Au-Fe3O4 nanoheterodimers were internalized by both, breast cancer cells (MCF-7) and epithelial cells (MCF-10 A), wherein they were electrostatically bound at the negatively charged membranes of the cell organelles and in particular, adsorbed onto the mitochondrial membrane. The treatment of MCF-7 and MCF-10 cells with a fractional X-radiation dose of 1 Gy resulted into a large increase of superoxide production which arose from the Au-Fe3O4 nanoheterodimer-induced mitochondrial depolarization. In contrast, the negatively charged (4-dodecylbenzenesulfonate-terminated) Au-Fe3O4 nanoheterodimers preferentially invaded the cancerous MCF-7 cells by direct permeation. X-ray treatment of MCF-7 cells, loaded with anionic Au-Fe3O4 nanoheterodimers, yielded the increase of both, the hydroxyl radical and cytoplasmic superoxide formation. The X-radiation induced activation of the Fe3O4 surfaces, as consisting of Fe2+ and Fe3+ cations, triggered the catalysis of the hydroxyl radical production, whereas superoxide formation presumably occurred through X-ray-induced photoelectron emission near the Au surface. Since hydroxyl radicals are highly cytotoxic and the negatively charged Au-Fe3O4 NHDs spare the healthy MCF-10A cells, these Au-Fe3O4 nanoheterodimers exhibit a higher potential for radiation therapy than the positively charged Au-Fe3O4 nanoheterodimers. Encouraging results from the clonogenic cell survival assay and DMF calculations corroborate the excellent performance of the anionic Au-Fe3O4 nanoheterodimers as X-ray dosage enhancer.