Fullerene C60 Conjugate with Folic Acid and Polyvinylpyrrolidone for Targeted Delivery to Tumor Cells.

The use of targeted drug delivery systems, including those based on selective absorption by certain receptors on the surface of the target cell, can lead to a decrease in the minimum effective dose and the accompanying toxicity of the drug, as well as an increase in therapeutic efficacy. A fullerene C60 conjugate (FA-PVP-C60) with polyvinylpyrrolidone (PVP) as a biocompatible spacer and folic acid (FA) as a targeting ligand for tumor cells with increased expression of folate receptors (FR) was obtained. Using 13C NMR spectroscopy, FT-IR, UV-Vis spectrometry, fluorometry and thermal analysis, the formation of the conjugate was confirmed and the nature of the binding of its components was established. The average particle sizes of the conjugate in aqueous solutions and cell culture medium were determined using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). The FA-PVP-C60 showed antiradical activity against •DPPH, •OH and O2•-, but at the same time, it was shown to generate 1O2. It was found that the conjugate in the studied concentration range (up to 200 µg/mL) is non-toxic in vitro and does not affect the cell cycle. To confirm the ability of the conjugate to selectively accumulate through folate-mediated endocytosis, its uptake into cells was analyzed by flow cytometry and confocal microscopy. It was shown that the conjugate is less absorbed by A549 cells with low FR expression than by HeLa, which has a high level of expression of this receptor.

Reduction of the α-synuclein expression promotes slowing down early neuropathology development in the Drosophila model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease characterised by the formation of Lewy bodies and progressive loss of dopaminergic (DA) neurons in the substantia nigra. Lewy bodies mainly consist of α-synuclein, which plays a critical role in the pathophysiology of PD. The α-synuclein is encoded by the SNCA gene and is the first identified gene associated with hereditary PD. Currently, there are at least six disease-associated mutations in α-synuclein that cause dominantly inherited familial forms of PD. Targeted expression of human SNCA.WT/SNCA.A30P/SNCA.A53T gene in Drosophila melanogaster over specific times employing a temperature-dependent UAS/GAL4 - GAL80 system allows for the evaluation of neurodegenerative processes. In this study, SNCA was expressed only in the adult stage of Drosophila development for 1 or 2 weeks, followed by repression of gene expression for the rest of the fly's life. It was demonstrated that the level of pathology significantly depends on the duration of α-synuclein expression. SNCA gene expression over a longer period of time caused the death of DA neurons, decreased levels of dopamine and locomotor ability. In this case, the observed neurodegenerative processes correlated with the accumulation of α-synuclein in the Drosophila brain. Importantly, repression of α-synuclein expression led to elimination of the soluble protein fraction, in contrast to the insoluble fraction. No further significant development of characteristic signs of pathology was observed after the α-synuclein expression was blocked. Thus, we suggest that reduction of α-synuclein expression alone contributes to slowing down the development of PD-like symptoms.

Fullerenols Prevent Neuron Death and Reduce Oxidative Stress in Drosophila Huntington's Disease Model.

Huntington's disease (HD) is one of the human neurodegenerative diseases for which there is no effective treatment. Therefore, there is a strong demand for a novel neuroprotective agent that can alleviate its course. Fullerene derivatives are considered to be such agents; however, they need to be comprehensively investigated in model organisms. In this work, neuroprotective activity of C60(OH)30 and C120O(OH)44 fullerenols was analyzed for the first time in a Drosophila transgenic model of HD. Lifespan, behavior, oxidative stress level and age-related neurodegeneration were assessed in flies with the pathogenic Huntingtin protein expression in nerve cells. Feed supplementation with hydroxylated C60 fullerene and C120O dimer oxide molecules was shown to diminish the oxidative stress level and neurodegenerative processes in the flies' brains. Thus, fullerenes displayed neuroprotective activity in this model.

Blood-Brain Barrier Penetrating Luminescent Conjugates Based on Cyclometalated Platinum(II) Complexes.

Herein we report on the synthesis, structural characterization and photophysical properties of cyclometalated Pt(II) complexes [Pt(N^C)(PPh2(C6H4COOH))Cl] (where N^C ligands are 2-phenylpyridine, (2-benzofuran-3-yl)pyridine, and (2-benzo[b]tiophen-3-yl)pyridine) and their conjugates with the histidine-containing RRRRRRRRRRHVLPKVQA peptide. This peptide contains the RHVLPKVQA sequence, which is responsible for antiamyloid activity, and the Arg9 RRRRRRRRR domain, which shows improved translocation through cell membranes. The chemistry underpinning the conjugation is regioselective complexation between Pt(II) complexes and histidine residue in the peptide. The prepared conjugates have been characterized using high-resolution mass spectrometry and NMR spectroscopy. It was shown that the conjugates are easily soluble in aqueous media and display emission band profiles essentially similar to those of the starting complexes but considerably higher luminescence quantum yield and much longer phosphorescence lifetime. MTT assay on HeLa cell culture revealed no cytotoxicity up to 10 µM after 24 h of incubation. Ex vivo and in vivo neuroimaging experiments on both wild and amyloid peptide expressing strains of Drosophila melanogaster revealed that the conjugates penetrate the blood-brain barrier and are evenly distributed throughout the brain independently of the strain used.

Human APP Gene Expression Alters Active Zone Distribution and Spontaneous Neurotransmitter Release at the Drosophila Larval Neuromuscular Junction.

This study provides further insight into the molecular mechanisms that control neurotransmitter release. Experiments were performed on larval neuromuscular junctions of transgenic Drosophila melanogaster lines with different levels of human amyloid precursor protein (APP) production. To express human genes in motor neurons of Drosophila, the UAS-GAL4 system was used. Human APP gene expression increased the number of synaptic boutons per neuromuscular junction. The total number of active zones, detected by Bruchpilot protein puncta distribution, remained unchanged; however, the average number of active zones per bouton decreased. These disturbances were accompanied by a decrease in frequency of miniature excitatory junction potentials without alteration in random nature of spontaneous quantal release. Similar structural and functional changes were observed with co-overexpression of human APP and ß-secretase genes. In Drosophila line with expression of human amyloid-ß42 peptide itself, parameters analyzed did not differ from controls, suggesting the specificity of APP effects. These results confirm the involvement of APP in synaptogenesis and provide evidence to suggest that human APP overexpression specifically disturbs the structural and functional organization of active zone and results in altered Bruchpilot distribution and lowered probability of spontaneous neurotransmitter release.

GAPDH binders as potential drugs for the therapy of polyglutamine diseases: design of a new screening assay.

Proteins with long polyglutamine repeats form a complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which enhances aggregation and cytotoxicity in models of Huntington disease. The aim of this study was to develop a novel assay for the screening of anti-aggregation compounds with a focus on the aggregation-promoting capacity of GAPDH. The assay includes a pure Q58 polyglutamine fragment, GAPDH, and a transglutaminase that links the two proteins. The feasibility of the new assay was verified using two GAPDH binders, hydroxynonenal and -(-)deprenyl, and the benzothiazole derivative PGL-135 which exhibits anti-aggregation effect. All three substances were shown to reduce aggregation and cytotoxicity in the cell and in the fly model of Spinocerebellar ataxia.