We have already written about how FLNP scientists use neutron scattering techniques to study the structural properties of biomimetic membranes that play an important role in the development of conformational diseases, such as Alzheimer’s disease. Today, we bring to your attention material about another area of research in the Frank Laboratory of Neutron Physics of JINR, aimed at finding ways to combat these diseases.

JINR researchers use all possible methods, including neutron scattering, to study the activity of fullerenes in the destruction of amyloid plaques and to reveal the underlying mechanisms. These studies open up a new avenue for the development of anti-amyloid nanodrugs for the treatment of neurodegenerative diseases such as Alzheimer’s disease.

Stages of disaggregation of amyloid fibrils (Reprinted with permission from ACS Applied Materials & Interfaces. Copyright 2020 American Chemical Society) and C₆₀ fullerene (Wikipedia)

Fullerenes as neuroprotective nanoparticles

Amyloidosis is the leading cause of death after cardiovascular and cancer diseases. For unclear reasons, the normal folding of proteins and peptides is disrupted in the body, and amyloid fibrils build up in various organs and tissues. The resulting aggregates have a toxic effect on the surrounding cells and cause a number of diseases, including Alzheimer’s, Parkinson’s, diabetes, systemic amyloidosis, and many others (to date numbering more than fifty). In each disease, the misfolding of different proteins such as tau proteins, Aβ-peptides, insulin, lysozyme or others (today, more than 30 are known) occurs, but the fibrils formed in each case have a common structure and properties. The most common and severe Alzheimer’s disease, for example, is caused by the buildup of a large amount of Aβ-peptides in the brain. The peptides clump together and form plaques that destroy brain cells and contribute to a gradual cognitive decline.

Unfortunately, the mechanism of amyloidogenesis is not fully understood. How to prevent the formation of fibrils, detect them in advance and prescribe effective treatment? In recent years, in search of a promising strategy for the treatment of amyloidosis, researchers are increasingly paying attention to the neuroprotective properties of nanoparticles and their anti-amyloid activity.

More than 30 years have passed since the discovery of fullerenes (carbon nanoparticles). During this time, the discoverers were awarded the Nobel Prize in Chemistry (1996, Robert F. Curl, Jr., Harold W. Kroto, Richard E. Smalley), researchers learned how to produce these nanoparticles in sufficient quantities, and a new field of research “fullerene chemistry” was born and is rapidly developing. The unique properties of, for example, buckminsterfullerene (type of fullerene with the formula C₆₀), the most stable member of the fullerene family, are now widely used in various fields of physics, electrochemistry and medicine. Intensive studies of the antitumor, antioxidant, and antibacterial activity of fullerenes open up new opportunities for their biomedical applications as imaging contrast agents, biosensors, novel pharmaceutical forms, and targeted delivery systems for radiopharmaceuticals and drugs. Depending on their size, charge, shape and composition, nanoparticles can affect the fibrillation of amyloid proteins in different ways, and can also be used to detect amyloid aggregates. The neuroprotective properties and anti-amyloid activity of fullerenes are largely due to their antioxidant properties, small size (about 0.7 nm in diameter), unique structure and ability to cross biological barriers by penetrating the membranes of various types of cells.

Despite active research on the great potential of fullerenes for treating amyloidosis, a complete description of the mechanism of amyloid disaggregation has not yet been provided. There is also insufficient experimental evidence of the ability of fullerenes to destroy amyloid plaques and disassembly various protein aggregates (other than Aβ-peptides). These are the key questions to be answered for the future successful application of fullerenes in the treatment of neurodegenerative and amyloid-related diseases.

The answers to these questions are also the subject of research carried out by JINR scientists together with their colleagues from other Russian and European research centers*, who have been studying the biological and anti-amyloid activity of fullerenes for a number of years.

An aggregate of scientific methods against amyloid aggregates
(fluorescence assay, atomic force microscopy, small-angle neutron scattering)

“Our study pursued two goals: we aimed to evaluate the anti-amyloid activity of aqueous solutions of C₆₀ and C₇₀ fullerenes dispersed in N-methylpyrrolidone (NMP) and to analyze the mechanism of amyloid disassembly. These are important issues for further successful use of fullerenes and other nanoparticles in the treatment of diseases associated with the formation of amyloid fibrils,” explained Mikhail V. Avdeev, a researcher from the Frank Laboratory of Neutron Physics, JINR. For this purpose, the scientists used a combination of several biophysical and biochemical techniques, including small-angle neutron scattering, as well as previous results of joint investigations performed together with the research teams of Peter Kopčanský from the Institute of Experimental Physics of the Slovak Academy of Sciences (IEP SAS) and Leonid A. Bulavin from the Taras Shevchenko National University of Kyiv.

For the study, they chose two proteins: human insulin and lysozyme (from chicken egg white). Amyloid fibrils were exposed to concentrated NMP-based aqueous solutions of C₆₀ and C₇₀ fullerenes. On the one hand, NMP dissolves and stabilizes fullerene molecules, as well as controls the size of their aggregates. On the other, it is miscible with water and used as a solubilizer in medicines.

The rate of fibril disaggregation can be monitored by using the Thioflavin-T fluorescence assay, since the fluorescence intensity correlates with the degree of amyloid aggregation. Based on the results obtained, at least two important conclusions can be drawn, said M.V.Avdeev. First, the selected fullerenes are capable of disassembling preformed amyloid fibrils at low (micrograms) concentrations and low protein-to-fullerene ratio. Second, the fullerene concentration values at which amyloid disassembly occurs were found to be quite similar for both proteins.

A gradual change in the structure and morphology of amyloid fibrils upon interaction with fullerenes was detected first by atomic force microscopy (AFM). Insulin as well as lysozyme amyloid deposits look like bundles of unbranched fibrils with an average height of 10 nm and average length of up to several micrometers. The addition of fullerenes in increasing concentrations led to the gradual disappearance of most of the amyloid fibrils of both proteins. While the data of the AFM experiments were collected with dried diluted samples, the small-angle neutron scattering technique allowed the researchers to follow the morphology of fibrils directly in bulk concentrated solutions. The experiments carried out using the YuMO small-angle neutron scattering instrument at the IBR-2 pulsed reactor (JINR, Dubna) make it possible to make assumptions about the mechanism of amyloid disaggregation activity of fullerenes. First, fullerenes adsorb on the surface of the protein with the formation of large branched agglomerates of fullerenes and fibrils. Then, fibril disassembly occurs via detachment of the fibril structural units from the aggregates, which leads to a reduction in the size of the branched agglomerates and, finally, to their complete disappearance. Despite the use of two different proteins and two different fullerenes, the fibril disaggregation mechanism was found to be common. At a low concentration of fullerenes, no active destruction of fibrils is observed. However, this concentration is sufficient for the interaction of fullerenes to coat fibrils and initiate the formation of more branched bundles of fibril agglomerates, the sizes of which exceed the sizes of protein aggregates in the untreated samples. As the concentration of fullerenes increases, the agglomerated bunches of fibrils increase in size and begin to break down due to the detachment of the fibril structural units from the aggregates.

SANS: focusing on biomedicine

Scientists note the uniqueness of the small-angle neutron scattering (SANS) method for studying complex multicomponent solutions that one has to deal with in real life when solving biomedical problems. In the study, the Guinier-Porod empirical model was used for the detailed structural analysis of the fibrils in solutions with and without fullerenes. The model allows determining the size and dimensionality of the scattering particles, including asymmetric objects such as rods or platelets, and more importantly for shapes intermediate between spheres and asymmetric objects.

The experimental conditions, in particular the concentration of fullerenes as well as the contrast between the solvent (heavy water, D2O) and fullerenes, were set in such a way that the neutron scattering signal from fullerenes was at the background level. In this case, the detected signal (background-subtracted) is associated only with amyloid fibrils, which makes it possible to accurately trace changes in the structure of fibrils under the action of fullerene solutions.

The researchers believe that the selected solutions of C₆₀ and C₇₀ fullerenes dispersed in N-methylpyrrolidone, which have both hydrophobic and hydrophilic properties, are unique and promising anti-amyloid aggregation nanoparticle-based agents.

“Our work demonstrates how important small-angle scattering techniques are for studying the formation and growth of amyloid aggregates as well as their destruction. Our experimental data provide valuable information on the molecular mechanism by which fullerenes are able to disassemble unwanted amyloid aggregates. Therefore, they can be used to design nanosized materials with a potent anti-amyloid effect”, summed up Andrey Musatov, a researcher from the Department of Biophysics of IEP SAS.

Whether fullerenes are able to destroy other types of amyloid aggregates has yet to be explored.

*Katarina Siposova, Viktor I. Petrenko, Oleksandr I. Ivankov, Andrey Musatov, Leonid A. Bulavin, Mikhail V. Avdeev, and Olena A. Kyzyma. ACS Applied Materials & Interfaces 2020 12 (29), 32410-32419 DOI: 10.1021/acsami.0c07964

Olga Baklitskaya-Kameneva