Adv Sci (Weinh). 2019 Feb 10;6(8):1801233. doi: 10.1002/advs.201801233. eCollection 2019 Apr 17.

Inhibition of CaMKIIα Activity Enhances Antitumor Effect of Fullerene C60 Nanocrystals by Suppression of Autophagic Degradation.

Xu J1, Wang H2, Hu Y3, Zhang YS4, Wen L5, Yin F1, Wang Z1, Zhang Y2, Li S2, Miao Y6, Lin B2, Zuo D2, Wang G2, Mao M2, Zhang T1, Ding J7, Hua Y1, Cai Z1.

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Fullerene C60 nanocrystals (nano-C60) possess various attractive bioactivities, including autophagy induction and calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) activation. CaMKIIα is a multifunctional protein kinase involved in many cellular processes including tumor progression; however, the biological effects of CaMKIIα activity modulated by nano-C60 in tumors have not been reported, and the relationship between CaMKIIα activity and autophagic degradation remains unclear. Herein, nano-C60 is demonstrated to elicit reactive oxygen species (ROS)-dependent cytotoxicity and persistent activation of CaMKIIα in osteosarcoma (OS) cells. CaMKIIα activation, in turn, produces a protective effect against cytotoxicity from nano-C60 itself. Inhibition of CaMKIIα activity by either the chemical inhibitor KN-93 or CaMKIIα knockdown dramatically promotes the anti-OS effect of nano-C60. Moreover, inhibition of CaMKIIα activity causes lysosomal alkalinization and enlargement, and impairs the degradation function of lysosomes, leading to autophagosome accumulation. Importantly, excessive autophagosome accumulation and autophagic degradation blocking are shown to play an important role in KN-93-enhanced-OS cell death. The synergistic anti-OS efficacy of KN-93 and nano-C60 is further revealed in an OS-xenografted murine model.

 * The results demonstrate that CaMKIIα inhibition, along with the suppression of autophagic degradation, presents a promising strategy for improving the antitumor efficacy of nano-C60.

Oxid Med Cell Longev. 2018 Jul 15;2018:1261356. doi: 10.1155/2018/1261356. eCollection 2018.

Protective Effects of Fullerene C60 Nanoparticles and Virgin Olive Oil against Genotoxicity Induced by Cyclophosphamide in Rats.

Aly FM1, Othman A2, Haridy MAM3.

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The potential effects of the fullerene C60 nanoparticle (C60) as well as virgin olive oil (VOO) against the cyclophosphamide- (CP-) induced cytotoxic and mutagenic effects were evaluated by two main methods: molecular intersimple sequence repeat (ISSR) assay and cytogenetic biomarkers.

Thirty adult male rats were divided to five groups (control, CP, C60, CP + C60, and CP + VOO). CP was i.p. injected with a single dose of 200 mg/kg; C60 and VOO were given orally (4 mg/kg dissolved in VOO and 1 ml, resp.) in alternative days for 20 days. The ISSR analysis revealed an increased in the DNA fragmentation level for liver and heart tissues represented by 21.2% and 32.6%, respectively, in the CP group. The DNA polymorphism levels were modulated and improved in CP + C60 (8.9% and 12%) and CP + VOO (9.8% and 12.7%) for hepatic and cardiac tissues, respectively.

The bone marrow cytogenetic analysis revealed that C60 and VOO had significantly decreased the frequency of CP-induced chromosomal aberrations (chromosomal ring, deletion, dicentric chromosome, fragmentation, and polyploidy).

Fullerene C60 and VOO have ability to reduce DNA damage and decrease chromosomal aberrations.

 * In conclusion, fullerene C60 and VOO have protective effects against the CP-induced mutagenicity and genotoxicity. Fullerene C60 and VOO open an interesting field concerning their potential antigenotoxic agents against deleterious side effects of chemotherapeutics.

Sci Total Environ. 2018 Jul 15;630:750-756. doi: 10.1016/j.scitotenv.2018.02.205. Epub 2018 Feb 27.

Impacts of fullerene C60 and virgin olive oil on cadmium-induced genotoxicity in rats.

Aly FM1, Kotb AM2, Haridy MAM3, Hammad S4.

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Currently, cadmium is considered to be one of the major environmental pollutants. Environmentally, cadmium is released in various forms e.g. oxide, chloride and sulphide. The aim of the present study was to examine the genotoxic impact of fullerene nanoparticles C60 (C60) and virgin olive oil (VOO) on cadmium chloride (CdCl2)-induced genotoxicity in rats. To evaluate these effects on DNA damage and chromosomal frequency, 25 albino rats were randomly assigned to 5 groups (n=5 per group): Group 1 served as a control; Group 2 received a single intraperitoneal dose of CdCl2 (3.5mg/kg); Group 3 animals were treated with C60 (4mg/kg, orally) every other day for 20days; Group 4 received a single intraperitoneal dose of CdCl2 (3.5mg/kg) and an oral dose of C60 (4mg/kg); and Group 5 received a single intraperitoneal dose of CdCl2 (3.5mg/kg) and oral doses of VOO every other day for 20 consecutive days. Genotoxic and anti-genotoxic effects of C60 and VOO were evaluated in the liver, kidney and bone marrow using molecular and cytogenetic assays. As expected, CdCl2 and C60administration was associated with band number alterations in both liver and kidney; however, C60 pretreatment recovered to approximately basal number. Surprisingly, C60 and VOO significantly attenuated the genotoxic effects caused by CdCl2 in livers and kidneys.

In bone marrow, in addition to a reduction in the chromosomal number, several chromosomal aberrations were caused by CdCl2. These chromosomal alterations were also reversed by C60 and VOO.

* In conclusion, molecular and cytogenetic studies showed that C60 and VOO exhibit anti-genotoxic agents against CdCl2-induced genotoxicity in rats. Further studies are needed to investigate the optimal conditions for potential biomedical applications of these anti-genotoxic agents.

J Alzheimers Dis. 2017;58(3):711-724. doi: 10.3233/JAD-161182.

Intrahippocampal Pathways Involved in Learning/Memory Mechanisms are Affected by Intracerebral Infusions of Amyloid-β25-35 Peptide and Hydrated Fullerene C60 in Rats.

Gordon R1, Podolski I2, Makarova E2, Deev A2, Mugantseva E2, Khutsyan S1,2, Sengpiel F3, Murashev A4, Vorobyov V1.

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Primary memory impairments associated with increased level of amyloid-β (Aβ) in the brain have been shown to be linked, partially, with early pathological changes in the entorhinal cortex (EC) which spread on the whole limbic system. While the hippocampus is known to play a key role in learning and memory mechanisms, it is as yet unclear how its structures are involved in the EC pathology. In this study, changes in memory and neuronal morphology in male Wistar rats intrahippocampally injected with Aβ25-35 were correlated on days 14 and 45 after the injection to reveal specific cognitive-structural associations. The main focus was on the dentate gyrus (DG) and hippocampal areas of CA1 and CA3 because of their involvement in afferent flows from EC to the hippocampus through tri-synaptic (EC → DG → CA3 → CA1) and/or mono-synaptic (EC → CA1) pathways. Evident memory impairments were observed at both time points after Aβ25-35 injection. However, on day 14, populations of morphological intact neurons were decreased in CA3 and, drastically, in CA1, and the DG supramedial bundle was significantly damaged. On day 45, this bundle largely and CA1 neurons partially recovered, whereas CA3 neurons remained damaged. We suggest that Aβ25-35 primarily affects the tri-synaptic pathway, destroying the granular cells in the DG supramedial area and neurons in CA3 and, through the Schaffer collaterals, in CA1. Intrahippocampal pretreatment with hydrated fullerene C60 allows the neurons and their connections to survive the amyloidosis, thus supporting the memory mechanisms.

J Colloid Interface Sci. 2016 Oct 15;480:69-75. doi: 10.1016/j.jcis.2016.07.008. Epub 2016 Jul 6.

Effect of fullerene on the dispersibility of nanostructured lipid particles and encapsulation in sterically stabilized emulsions.

Kulkarni CV1, Moinuddin Z2, Agarwal Y3.

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We report on the effect of fullerenes (C60) on the stability of nanostructured lipid emulsions. These (oil-in-water) emulsions are essentially aqueous dispersions of lipid particles exhibiting self-assembled nanostructures at their cores. The majority of previous studies on fullerenes were focused on planar and spherical lipid bilayer systems including pure lipids and liposomes. In this work, fullerenes were interacted with a lipid that forms nanostructured dispersions of non-lamellar self-assemblies. A range of parameters including the composition of emulsions and sonication parameters were examined to determine the influence of fullerenes on in-situ and pre-stabilized lipid emulsions. We found that fullerenes mutually stabilize very low concentrations of lipid molecules, while other concentration emulsions struggle to stay stable or even to form at first instance; we provide hypotheses to support these observations. Interestingly though, we were able to encapsulate varying amounts of fullerenes in sterically stabilized emulsions. This step has a significant positive impact, as we could effectively control an inherent aggregation tendency of fullerenes in aqueous environments. These novel hybrid nanomaterials may open a range of avenues for biotechnological and biomedical applications exploiting properties of both lipid and fullerene nanostructures.

J Nanosci Nanotechnol. 2012 Jan;12(1):119-26.

Fullerene C60 prevents neurotoxicity induced by intrahippocampal microinjection of amyloid-beta peptide.

Makarova EG1, Gordon RYPodolski IY.

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The dynamics of the state of hippocampal pyramidal neurons after intrahippocampal microinjections of (1) amyloid-beta25-35 (1.6 nmol/1 microl), (2) an aqueous molecule-colloidal solution of C60 (0.46 nmol/1 microl) and (3) an aqueous molecule-colloidal solution of C60 before amyloid-beta25-35 administration were analysed in rats. This model allowed us to study the role of amyloid-beta25-35 in the pathogenesis of Alzheimer's disease and to test anti-amyloid substances. Methods of fluorescent (acridine orange) and brightfield (cresyl violet and immunohistochemistry) microscopy were used. Acridine orange staining indicated changes in protein synthesis intensity due to alterations in the rRNA state of neuron ribosomes. One day after administration of amyloid-beta25-35, the intensity of protein synthesis in the population of morphologically intact cells decreased by 45%. By day 14, degeneration occurred in the majority of pyramidal cells, and amyloid-beta25-35 deposits were observed in the neuronal cytoplasm. In necrotic cells, acridine orange staining of the cytoplasm was drastically increased as a result of RNA degradation rather than due to an increase in protein synthesis. Because amyloid-beta25-35 administration provoked oxidative stress, we assumed that an aqueous molecule-colloidal solution of C60 administered before amyloid-beta25-35 prevented protein synthesis changes on day 1, while acting as an antioxidant, and by day 14 it inhibited neurodegeneration and amyloid-beta25-35 accumulation. Based on the data that an aqueous molecule-colloidal solution of C60 prevented amyloid-beta25-35 aggregation in in vitro experiments and based on our present evidence on the antitoxicity of an aqueous molecule-colloidal solution of C60, we suggest that functionalised C60 prevents/diminishes amyloid-beta25-35 aggregation in vivo as well. Thus, an aqueous molecule-colloidal solution of C60 administered at a low concentration before amyloid-beta2-35, prevented disturbances in protein synthesis, neurodegeneration and formation amyloid-beta25-35 deposits in hippocampal pyramidal neurons in vivo.

 * This evidence gives promise that functionalised C60 can be used to develop anti-amyloid drugs combining antioxidant and anti-aggregative properties.

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