Passive Diffusion vs Active pH-Dependent Encapsulation of Tyrosine Kinase Inhibitors Vandetanib and Lenvatinib into Folate-Targeted Ferritin Delivery System.

INTRODUCTION: The present study reports on examination of the effects of encapsulating the tyrosine kinase inhibitors (TKIs) vandetanib and lenvatinib into a biomacromolecular ferritin-based delivery system. METHODS: The encapsulation of TKIs was performed via two strategies: i) using an active reversible pH-dependent reassembly of ferritin´s quaternary structure and ii) passive loading of hydrophobic TKIs through the hydrophobic channels at the junctions of ferritin subunits. After encapsulation, ferritins were surface-functionalized with folic acid promoting active-targeting capabilities. RESULTS: The physico-chemical and nanomechanical analyses revealed that despite the comparable encapsulation efficiencies of both protocols, the active loading affects stability and rigidity of ferritins, plausibly due to their imperfect reassembly. Biological experiments with hormone-responsive breast cancer cells (T47-D and MCF-7) confirmed the cytotoxicity of encapsulated and folate-targeted TKIs to folate-receptor positive cancer cells, but only limited cytotoxic effects to healthy breast epithelium. Importantly, the long-term cytotoxic experiments revealed that compared to the pH-dependent encapsulation, the passively-loaded TKIs exert markedly higher anticancer activity, most likely due to undesired influence of harsh acidic environment used for the pH-dependent encapsulation on the TKIs' structural and functional properties. CONCLUSION: Since the passive loading does not require a reassembly step for which acids are needed, the presented investigation serves as a solid basis for future studies focused on encapsulation of small hydrophobic molecules.

Complex cytotoxicity mechanism of bundles formed from self-organised 1-D anodic TiO2 nanotubes layers.

The present study reports on a comprehensive investigation of mechanisms of in vitro cytotoxicity of high aspect ratio (HAR) bundles formed from anodic TiO2 nanotube (TNT) layers. Comparative cytotoxicity studies were performed using two types of HAR TNTs (diameter of ∼110 nm), differing in initial thickness of the nanotubular layer (∼35 µm for TNTs-1 vs. ∼10 µm for TNTs-2). Using two types of epithelial cell lines (MDA-MB-231, HEK-293), it was found that nanotoxicity is highly cell-type dependent and plausibly associates with higher membrane fluidity and decreased rigidity of cancer cells enabling penetration of TNTs to the cell membrane towards disruption of membrane integrity and reorganization of cytoskeletal network. Upon penetration, TNTs dysregulated redox homeostasis followed by DNA fragmentation and apoptotic/necrotic cell death. Both TNTs exhibited haemolytic activity and rapidly activated polarization of RAW 264.7 macrophages. Throughout the whole study, TNTs-2 possessing a lower aspect ratio manifested significantly higher cytotoxic effects. Taken together, this is the first report comprehensively investigating the mechanisms underlying the nanotoxicity of bundles formed from self-organised 1-D anodic TNT layers. Except for description of nanotoxicity of industrially-interesting nanomaterials, the delineation of the nanotoxicity paradigm in cancer cells could serve as solid basis for future efforts in rational engineering of TNTs towards selective anticancer nanomedicine.

Tuning the surface coating of IONs toward efficient sonochemical tethering and sustained liberation of topoisomerase II poisons.

BACKGROUND: Iron oxide nanoparticles (IONs) have been increasingly utilized in a wide spectrum of biomedical applications. Surface coatings of IONs can bestow a number of exceptional properties, including enhanced stability of IONs, increased loading of drugs or their controlled release. METHODS: Using two-step sonochemical protocol, IONs were surface-coated with polyoxyethylene stearate, polyvinylpyrrolidone or chitosan for a loading of two distinct topo II poisons (doxorubicin and ellipticine). The cytotoxic behavior was tested in vitro against breast cancer (MDA-MB-231) and healthy epithelial cells (HEK-293 and HBL-100). In addition, biocompatibility studies (hemotoxicity, protein corona formation, binding of third complement component) were performed. RESULTS: Notably, despite surface-coated IONs exhibited only negligible cytotoxicity, upon tethering with topo II poisons, synergistic or additional enhancement of cytotoxicity was found in MDA-MB-231 cells. Pronounced anti-migratory activity, DNA fragmentation, decrease in expression of procaspase-3 and enhancement of p53 expression were further identified upon exposure to surface-coated IONs with tethered doxorubicin and ellipticine. Moreover, surface-coated IONs nanoformulations of topo II poisons exhibited exceptional stability in human plasma with no protein corona and complement 3 binding, and only a mild induction of hemolysis in human red blood cells. CONCLUSION: The results imply a high potential of an efficient ultrasound-mediated surface functionalization of IONs as delivery vehicles to improve therapeutic efficiency of topo II poisons.

Prevalent anatase crystalline phase increases the cytotoxicity of biphasic titanium dioxide nanoparticles in mammalian cells.

Nanoparticular form of titanium dioxide (TiO2 NPs) belongs to important industrial material. Despite being widely used, serious contradictions regarding biosafety of TiO2 NPs remain. We anticipate that such discrepancies could be due to a lack of understanding of a linkage between TiO2 NPs phase composition and cytotoxicity. Therefore, we synthesized two types of biphasic TiO2 NPs differing in an anatase-brookite phase composition. The study presents an array of in vitro data suggesting that TiO2 NPs with a prevailing anatase phase composition possess higher cytotoxicity compared to TiO2 NPs with an equal anatase-brookite crystallinity. This phenomenon was evidenced by significantly higher inhibition of metabolic activity and growth of epithelial and neuroblast-like cells. Moreover, anatase-prevailing TiO2 NPs tend to produce higher amount of reactive oxygen species resulting in DNA fragmentation. Further insights into the molecular aspects of cytotoxicity of anatase-prevailing TiO2 NPs were obtained by comparative proteomics delineating that TiO2 NPs deregulate expression of a variety of proteins and associated pathways. This inevitably results in a decreased cellular ability to detoxify reactive oxygen species and respond to various stress conditions. The study provides novel data that add another piece to the jigsaw of the relation between structural features of NPs and biosafety.

Synthesis and structural characterization of antimicrobial binuclear copper(II) coordination compounds bridged by hydroxy- and/or thiodipropionic acid.

In the present study, two binuclear copper(II) coordination compounds bridged by hydroxy- and thiodipropionic acid have been synthesized. The structure of compounds was determined by X-ray crystallography. The central copper atoms exist in square pyramidal surroundings. Basal plane is formed by nitrogen atoms of amines and oxygen atoms of bridges, whereas apical positions are occupied by oxygen atoms of coordinated water molecules. Temperature dependence study of magnetic susceptibility proved strong antiferromagnetic exchange between copper atoms in hydroxy-bridged complex. These coordination compounds were also tested for their biological activities in vitro. Both coordination compounds exhibit pronounced cytocompatibility in mammalian epithelial cells with no induction of oxidative stress and DNA fragmentation. Moreover, synthesized compounds are hemocompatible and do not alter expression of a marker of multiple cellular stress, p53. On the other hand, both compounds had stimulatory effect on expression of metallothioneins (MT-1/2 and MT-3). Antimicrobial testing on Escherichia coli, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus revealed that both copper compounds exhibit antibacterial activity regardless the cell wall composition. Overall, current work presents a synthesis of Cu(II) coordination compounds with interesting biological behavior and with a promising potential to be further tested in pre-clinical models.

Improving cytocompatibility of CdTe quantum dots by Schiff-base-coordinated lanthanides surface doping.

BACKGROUND: Suitable fluorophores are the core of fluorescence imaging. Among the most exciting, yet controversial, labels are quantum dots (QDs) with their unique optical and chemical properties, but also considerable toxicity. This hinders QDs applicability in living systems. Surface chemistry has a profound impact on biological behavior of QDs. This study describes a two-step synthesis of QDs formed by CdTe core doped with Schiff base ligand for lanthanides [Ln (Yb3+, Tb3+ and Gd3+)] as novel cytocompatible fluorophores. RESULTS: Microwave-assisted synthesis resulted in water-soluble nanocrystals with high colloidal and fluorescence stability with quantum yields of 40.9-58.0%. Despite induction of endocytosis and cytoplasm accumulation of Yb- and TbQDs, surface doping resulted in significant enhancement in cytocompatibility when compared to the un-doped CdTe QDs. Furthermore, only negligible antimigratory properties without triggering formation of reactive oxygen species were found, particularly for TbQDs. Ln-doped QDs did not cause observable hemolysis, adsorbed only a low degree of plasma proteins onto their surface and did not possess significant genotoxicity. To validate the applicability of Ln-doped QDs for in vitro visualization of receptor status of living cells, we performed a site-directed conjugation of antibodies towards immuno-labeling of clinically relevant target-human norepinephrine transporter (hNET), over-expressed in neuroendocrine tumors like neuroblastoma. Immuno-performance of modified TbQDs was successfully tested in distinct types of cells varying in hNET expression and also in neuroblastoma cells with hNET expression up-regulated by vorinostat. CONCLUSION: For the first time we show that Ln-doping of CdTe QDs can significantly alleviate their cytotoxic effects. The obtained results imply great potential of Ln-doped QDs as cytocompatible and stable fluorophores for various bio-labeling applications.