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Pharmacology

A key to unlock the brain

Carbon-based nanomaterial may help medication reach the central nervous system

It’s not easy to get medicine into the brain. The blood vessels that irrigate the central nervous system are covered with a special structure composed of three types of cells that act together as a very selective filter. Called the hematoencephalic, or blood-brain barrier, this structure only allows some compounds necessary for proper brain functioning to pass through, such as nutrients, hormones and gases. This selectiveness protects the central nervous system from toxic molecules found in the blood; however, it also prevents medications consumed orally or injected into the bloodstream from reaching the brain, even when this is necessary. At the University of Campinas (Unicamp), a group led by biologist Maria Alice da Cruz-Höfling is currently testing the possibility of using reduced graphene oxide, a nanostructured compound formed by carbon atoms, to open the barrier and enable certain medications to reach the brain with fewer side effects that those caused by the compounds currently in use.

Initial tests with reduced graphene oxide have been promising. Experiments conducted with cells and with laboratory animals indicate that it temporarily opens the barrier and is apparently not toxic to the organism, at the doses evaluated. “We work with this compound because nanomaterials in the graphene family showed the potential to interact with the nervous system, since graphene is an excellent conductor of electricity, and nerve cells communicate through electrical impulses,” explains pharmacist and biochemist Monique Mendonça, a post-doctoral researcher at the Unicamp Institute of Biology and the first author of the articles that described these results, published in 2015 and 2016 in the Journal of Nanobiotechnology and in Molecular Pharmaceutics.

Composed of a single layer of carbon atoms organized into regular hexagons, graphene is 200 times more resistant than steel, and is one of the best electrical conductors known. However, pure graphene has limited biological uses, since it is not very soluble in water. On the other hand, reduced graphene oxide can be diluted in water and maintains similar electrical properties to those of graphene.

Mendonça learned about reduced graphene oxide in 2013, in a conversation with researchers at the Nanoengineering and Diamond Laboratory of the School of Electrical Engineering and Computer Science (FEEC) at Unicamp, and decided to evaluate its potential to cross through the barrier. “We adapted the production process for this material to synthesize it without the need for intermediary chemical processes, and increased its purity to around 99%,” says researcher Helder Ceragioli, of the FEEC. The production methods described in the scientific literature normally leave behind impurities (iron, tungsten or nickel atoms), which can be toxic. According to the theoretical predictions, the purer the reduced graphene oxide, the lower the risk of causing damage to living tissue.

In these experiments, Mendonça injected reduced graphene oxide into the bloodstream of rats, and using techniques that allow the compound to be tracked in the organism, and observed that an hour later it had already penetrated into brain structures such as the hippocampus and thalamus. Since she detected a reduction in the level of the proteins that hold together the cells that line the blood vessels, she concluded that reduced graphene oxide had opened the barrier by creating space between the cells that make it up. Mendonça also noted that a few hours after the compound is injected, the barrier closed again. The group from Unicamp is currently investigating possible biochemical mechanisms that are activated in the cells to open the barrier.

Preserved neurons
Seven days after application, most of the compound had already been eliminated from the organism, suggesting that it does not tend to accumulate and become toxic to cells. Other exams found that the neurons in the treated rodents did not die, and that brain morphology remained intact. This material also did not cause any harm to blood cells or to organs such as the liver and kidneys. Moreover, reduced graphene oxide seems to offer advantages over compounds such as mannitol, used by doctors to open the blood-brain barrier.

“Reduced graphene oxide is potentially safer than mannitol, which alters the flow of liquids in the central nervous system and can leave neurons susceptible to damage, besides harming kidney function,” comments physician Licio Velloso, a professor at the Unicamp School of Medical Sciences (FCM). Velloso is currently investigating alterations in organisms that change the permeability of the barrier; he considers reduced graphene oxide to be a promising candidate for this function. “Nevertheless,” he points out, “the pharmacological use of nanoparticles is still in the initial phase, and more studies are needed to determine whether they cause any long-term side effects.”

“The results obtained so far indicate that reduced graphene oxide has the potential to carry medication to the brain or to penetrate the barrier and allow other substances to transport the medications there,” says Cruz-Höfling, who began to investigate ways to penetrate the blood-brain barrier 20 years ago, when she studied the effect of the venom of spiders of the genus Phoneutria, called armed spiders. “Since people bitten by these spiders showed neurotoxic symptoms, I assumed that the venom could cross through the barrier,” she recalls. Later she noted that low doses opened the barrier in rats. Given the difficulty in isolating the compound in the venom that is responsible for this effect, Cruz-Höfling began to test other compounds.

In spite of the encouraging results, it is still early to say whether reduced graphene oxide will be able to be used in clinical practice. First it must be determined whether it is safe for human use, and whether it actually enables other compounds to reach the brain more effectively.

Project
Graphene oxide and the central nervous system: Evaluation of effects on blood brain barrier and nanotoxicological profile (No. 12/24782-5); Grant Mechanism Doctoral Research Grant; Principal Investigator  Maria Alice da Cruz Höfling (Unicamp); Grant Recipient Monique Culturato Padilha Mendonça; Investment R$135,835.83 and R$31,276.24 (BEPE).

Scientific Articles
MENDONÇA, M. C. P. et al. PEGylation of reduced graphene oxide induces toxicity in cells of the blood-brain barrier: An in Vitro and in Vivo Study. Molecular Pharmaceutics. V. 13 (11). 18 Oct. 2016.
MENDONÇA, M. C. P. et al. Reduced graphene oxide: Nanotoxicological profile in rats. Journal of Nanobiotechnology. V. 14 (53). 24 Jun. 2016.
MENDONÇA, M. C. P. et al. Reduced graphene oxide induces transient blood-brain barrier opening: An in vivo study. Journal of Nanobiotechnology. V. 13 (78). 30 Oct. 2015.
DE PAULA LE SUEUR, L. et al. Breakdown of the blood-brain barrier and neuropathological changes induced by Phoneutria nigriventer spider venom. Acta Neuropathologica. V. 105 (2), p. 125-34. Feb. 2003.

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