A growing body of research has shown that misshapen and misfolded alpha-synuclein, the protein culprit behind Parkinson’s disease and its characteristics, travels from the gut to the brain, where it spreads and sticks together in lethal clumps known as Lewy bodies. As these clumps accumulate, they cause brain cell death.
Now, Johns Hopkins Medicine researchers have created an artificial enzyme that stops misfolded alpha-synuclein from spreading and could become the basis for a new treatment for Parkinson’s disease.
The results were announced in a study published online Nov. 20, 2020, in the journal Nano Today.
The artificial enzymes, nanosized (a nanometer is a billionth of a meter) combinations of platinum and copper called PtCu bimetallic nanoalloys, were created by the research team for their strong antioxidant properties.
The antioxidant capability is dependent largely on the alloy composition.
“Oxidative stress caused by reactive oxygen species is inescapable, and increases with age due to mechanistic slowdowns in processes such as protein degradation,” says senior study researcher Xiaobo Mao, Ph.D., assistant professor of neurology at the Johns Hopkins University School of Medicine.
“This indicates the importance of antioxidants, because in Parkinson’s disease, roaming reactive oxygen species promote the spread of misfolded alpha-synuclein, leading to worse symptoms.”
When injected into the brain, the nanozymes scavenge for reactive oxygen species, gobbling them up and preventing them from causing damage to neurons in the brain.
The nanozymes mimic catalase and superoxide dismutase, two enzymes found in our bodies that break down reactive oxygen species. Adding the nanozymes strengthens our body’s response to them.
The study used a research method known as the alpha-synuclein preformed fibril model, which replicates the pathology, spreading and neurodegeneration resulting from Lewy bodies.
The nanozyme was found to decrease alpha-synuclein induced pathology and inhibit neurotoxicity, in addition to decreasing reactive oxygen species. The nanozyme also prevented alpha-synuclein from passing from cell to cell, and from the substantia nigra to the dorsal striatum, two areas in the midbrain that influence movement and cognition.
Mao has long collaborated with fellow Parkinson’s disease expert Ted Dawson, M.D., Ph.D., professor of neurology and director of the Institute for Cell Engineering at the Johns Hopkins University School of Medicine. Dawson recently added to evidence that misfolded alpha-synuclein travels along the vagus nerve from the gut to the brain. Mao hopes that further research can connect the two findings and lead to a Parkinson’s disease treatment that targets the gut.
“We know that the nanoenzymes work when injected directly into the brain,” says Mao. “Now, we’d like to see if the nanoenzymes can block the disease progression induced by pathogenic alpha-synuclein traveling from the gut, across the blood-brain barrier and into the brain.”
Metal Nanoparticles: Overview
Metal-based nanoparticles are the most popular inorganic nanoparticles and represent a promising solution against the resistance to traditional antibiotics. Not only do they use mechanisms of action that are completely different from those described for traditional antibiotics, exhibiting activity against bacteria that have already developed resistance, but they also target multiple biomolecules compromising the development of resistant strains .
Metal-based nanoparticles may be characterized by numerous techniques. These methods provide valuable information about their morphology, physicochemical, and electric properties which are crucial for their in vivo activity. The most relevant properties of nanoparticles include aspects as their size, shape, roughness, and surface energy .
Metal-Based Nanoparticle General Mechanisms
Bacteria have specific characteristics that explain their behaviour in contact with metal nanoparticles. Since the main toxicological effect induced by antimicrobial compounds in bacteria occurs by direct contact with the cell surface, it is important to understand the differences between the cell wall of Gram-positive and Gram-negative bacteria .
Both Gram-positive and Gram-negative bacteria have a negatively charged surface . Gram-positive bacteria have a thick layer of peptidoglycan formed by linear chains alternating residues of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) linked together by a sequence of 3 to 5 amino acids that cross-link each other, forming a cohesive mesh. Additionally, negatively charged teichoic acids (with high levels of phosphate groups) extend from the cell wall to the surface of most Gram-positive bacteria. Gram-negative bacteria, on the other hand, have a slightly more complex structure. In addition to the thin layer of peptidoglycan, Gram-negative bacteria have a phospholipid outer membrane with partially phosphorylated lipopolysaccharides (LPS) that contribute to increase the negative surface charge of their cell envelope .
Negatively charged bacterial cell walls attract positively charged nanoparticles to their surface due to electrostatic interactions. On the other hand, positively charged metal-based nanoparticles establish a strong bond with membranes, resulting in disruption of cell walls and, consequently, increase their permeability. In addition, nanoparticles can also release metal ions from the extracellular space, capable of entering the cell and disrupt biological processes . Inside the cell, either metal ions or nanoparticles can induce production of reactive oxygen species (ROS). The oxidative stress generated leads to oxidation of glutathione, thus suppressing the antioxidant defence mechanism of bacteria against ROS. The metal ions are then free to interact with cellular structures (e.g., proteins, membranes, DNA), disrupting cell functions . Metal ions can form strong coordination bonds with N, O, or S atoms which are abundant in organic compounds and biomolecules. Since the bond between metal ions and biomolecules is generally non-specific, metal-based nanoparticles generally exhibit a broad spectrum activity .
Synthesis of Metal and Metal Oxide Nanoparticles
Metal-based nanoparticles are not a recent technology. The natural production of metal-based nanoparticles by some microorganisms as a mechanism of heavy metals detoxification has been described. However, the versatility of this technology has only been described over the last decades, with metal-based nanoparticles being widely used in the production of cosmetics and textiles ever since .
Their versatility has arisen the interest of the scientific community, which began an endless search for new compositions, applications, and methods of synthesis. Although research has been expanded over the recent years to other less-common metals, the most widely used materials in metal-based nanoparticles include silver, gold, copper, iron, and zinc [17,18,19].
Transition metals are expected to be the best candidates for the synthesis of metal-based nanoparticles since these have partially filled d-orbitals which make them more redox-active (easier to reduce to zerovalent atoms), a feature that facilitates their nanoparticle aggregation . The various synthesis methods developed can be classified as physical methods, chemical methods, and more recently developed biological methods .
Physical methods use a top-down approach (Figure 2), starting from bulk metal that undergoes fractionation into smaller pieces by mechanical action into successively smaller fragments. Although very simplistic, this technique creates nanoparticles with a fairly dispersed size distribution and is therefore not the most appropriate in the synthesis of metal-based nanoparticles, in which the size is the determining factor for their activity . On the other hand, bottom-up approaches are used in chemical methods involving organic solvents and also in biological methods, which are focussed on green-synthesis processes using different types of microorganisms.
Generally, this technique relies upon the dissociation of organometallic precursors in organic solvents at temperatures generally higher than 100 °C under inert atmosphere to avoid surface oxidation of the nanoparticles . As a disadvantage of this method, reactions are difficult to apply to large-scale synthesis, due to their highly diluted and exothermic conditions. Otherwise, there are other methods for the synthesis of nanoparticles, such as controlled thermolysis of silver alkyl carboxylates, in order to produce silver nanoparticles (AgNPs) without using organic solvents. As an advantage of this method, the controlled thermolysis can be applied to industrial large-scale synthesis with very low cost .
Chemical Reduction Methods
In these methods, a metal precursor dissolved in a solvent is mixed with both a suitable reducing agent and a surfactant in a constantly stirring batch reactor under inert atmosphere. When two or more metal cationic species are present in the solvent, a nanosized phase of variable composition is formed.
This constitutes a promising method to obtain metastable metal nanoparticles. The choice of the reducing agent is very wide, but it could be based on the specific redox thermodynamics. Moreover, in the majority of the cases, the activity of reducing agents is strongly dependent on the pH of the solution . For example, for the preparation of copper nanoparticles (CuNPs), the precursor copper acetate is dissolved in stirring deionized water. Hydrazine, the reducing agent, is added to the solution and the nanoparticles are formed afterwards .
For these methods, plants, algae, yeasts, fungi, bacteria, and even viruses have been recently used along with chemical reagents . The growth process is undertaken in intracellular or extracellular environment and it relies upon enzymatic or nonenzymatic reduction processes. Gold and silver nanoparticles could be synthesized by bacteria or fungi with a multiplicity of shapes (cubes, triangles, spheres, plates, or wires) according to the specific host cells and method parameters. Despite several patents reporting the use of these methods, biosynthesis process optimization still remains an unsolved problem .
Electrochemical methods have demonstrated some additional advantages over chemical methods in the synthesis of size-selective or shape-controlled highly pure metal nanomaterials. A metal sheet is anodically dissolved and the intermediate metal salt formed is reduced at the cathode, giving rise to metallic particles stabilized by ammonium salts .
Some authors reported the synthesis of bimetallic Cd-Ag nanoalloys by sequential electrodeposition of two different cations on a carbon electrode . Similarly, palladium metallic nanostructures were obtained via templated-assisted electrodeposition from electrolytes containing salts of the relevant cation precursor . Other authors reported the synthesis of AuNPs via direct electroreduction of gold ions bulk by utilizing polyvinylpyrrolidone (PVP) in enhancing the gold nanoparticle formation and inhibiting the metal deposition on the cathode .
Wave-Assisted Chemical Methods
Sonochemical methods rely upon the use of a source of ultrasounds inducing cavitation in a solution containing a metal precursor mixed with a reducing agent and a surfactant as stabilizer. The formation and further implosion of microcavities in the liquid phase produce local spots with extremely high temperatures (theoretically higher than 3000 °C) that may trigger chemical reactions otherwise unfeasible with traditional techniques .
In radiolytic processes, a metal precursor mixed with a suitable reducing agent is subject to an electromagnetic or particle irradiation, such as an accelerated electron beam , gamma-rays , X-rays , and ultraviolet rays . AgNPs can be prepared by ultrasonic wave assisted synthesis, by reducing AgNO3 with strong reducing agent as sodium borohydride in the presence of ultrasonic waves. A greyish precipitate is formed, which is irradiated ultrasonically and then centrifuged, obtaining the AgNPs .
Over the last years, micro-wave assisted synthesis has been considered as an eco-friendly and fast method. This fact is because the stabilizer and complexing agent can be replaced by less polluting materials, such as chitosan and polymers. Moreover, this method is able to carry out chemical transformations in minutes. For example, AgNPs can be also prepared by complexing PVP and reducing Ag+ ion with N,N-dimethylformamide .
When a strongly electropositive metal A (sacrificial element) is left in contact with a solution containing ions of a less electropositive metal B, the following spontaneous reaction is thermodynamically allowed, and metal B separates in elemental form
A + n/mBm+ = An+ + n/mB (1)
This reaction, which is commonly used in industry to purify solutions in hydrometallurgy, can be used to reduce cations obtaining metal nanoparticles or aggregates with a relatively simple and cheap process . The two main disadvantages of this method are the poor control of nanoparticle agglomeration owing to a sticking of the cemented metal phase B on the surface element A. However, if A contains impurities, they can contaminate B as a consequence of a multicluster surface etching of A.
These problems can be avoided by damped mechanically with a tailored hydrodynamic control . For example, CuNPs can be synthetized with a reduction of copper from a copper nitrate salt in the presence of iron, and to prevent the formation of larger sized CuNPs, the sample was continuously ultra-sonicated. The obtained nanoparticles sizes were recorded between 90 and 150 nm .
Biological methods arose from the need to develop new more environmentally friendly techniques that exclude the use of organic solvents and toxic chemicals (Table 1). They also proved to be safe and economically sustainable alternatives. Critical aspects of the synthesis of metal-based nanoparticles, such as size distribution and crystallinity, can be overcome for example by selecting the strain, incubation temperature and time, concentration of metal precursor, and optimal pH conditions .
Examples of green synthesis of alternative metal-based nanoparticles with potential antibacterial activity, with respective minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values.
|Specie||Microorganism||Morphology||Synthesis||Average Size (nm)||Activity||MIB and MIC Values||References|
|Trichoderma hamatum||fungus||spherical, pentagonal and hexagonal||extracellular||5–30||P. aeruginosa; Serratia sp.; B. subtilis; S.aureus||Data not shown|||
|Alternanthera bettzickiana||plant extract||spherical||extracellular||80–120||S. typhi; P. aeruginosa; E. Aerogenes; S. aureus; B. subtilis; M. luteus||MIC values (expressed in µL of AuNPs):|
10 µL B. subtilis
20 µL S. aureus
30 µL M. luteus
40 µL E. aerogenes, S. typhi and P. aeruginosa
|Deinococcus radiodurans||bacteria||spherical, triangular and irregular||intra- and extracellular||~43.75||E. coli; S. aureus||Data not shown|||
|Pseudomonas veronii AS41G||bacteria||irregular||extracellular||5–25||E. coli; S. aureus (+)||Data not shown|||
|E. coli; P. aeroginosa; B. subtilis||Values not shown|||
|Fusarium oxysporum f. sp. cubense JT1||fungus||n.a.0F||extracellular||~22||Pseudomonas sp.||Data not shown|||
|Stoechospermum marginatum||algae||spherical to irregular||extracellular||18.7–93.7||P. aeruginosa; V. cholerae; V. parahaemoluticus; S. paratyphi; P. vulgaris; S. typhi; K. pneumoniae; K. oxytoca; E. faecalis(+);||AuNPs more effective against E. faecalis > K. pneumoniae. Non-effective against E. coli|||
|Streptomyces viridogens (HM10)||bacteria||spherical and rod||intracellular||18–20||E. coli; S. aureus||Data not shown|||
|Shewanella loihica PV-4||bacteria||spherical||extracellular||10–16||E. coli||100 µg/mL Cu-NPs inhibits 86% of the bacteria|||
|Enterococcus faecalis||bacteria||spherical||extracellular||29–195 (~99)||S. aureus (no observed activity against P. aeruginosa, B. subtilis and E. coli)||Data not shown|||
|Glycosmis pentaphylla||plant extract||spherical||extracellular||32–36||S. dysenteriae; S. paratyphi;|
S. aureus; B. cereus
|At 100 µg/mL maximum inhibition is observed|||
|Suaeda aegyptiaca||plant extract||spherical||extracellular||~60||P. aeruginosa; E. coli;|
S. aureus; B. subtilis
MIC and MBC: 0.19–0.78 mg/mL
MIC: 1.56–12.50 mg/mL
MBC: 6.25–12.50 mg/mL
MIC and MBC: 0.39–1.56 mg/mL
MIC: 0.19–0.39 mg/mL
MBC: 0.78–12.50 mg/mL
|Pichia kudriavzevii||fungus||hexagonal||extracellular||10–61||E. coli(+); S. marcescens;|
B. subtilis(+); S. aureus (+); S. epidermis (++)
|Data not shown|||
|Jacaranda mimosifolia||plant extract||spherical||extracellular||2–4||E. coli;|
|Data not shown|||
|Cystoseira trinodis||algae||spherical||intracellular||6–7.8||E. coli; S. typhi; E. faecalis; S. aureus; B. subtilis; S. faecalis||E. coli and S. aureus|
MIC: 2.5 μg/mL
MIC: 5 μg/mL
MIC: 10 μg/mL
Biological methods take advantage of the defence mechanisms present in specific organisms (against high concentrations of metal ions) to produce metal-based nanoparticles. These methods include intracellular (e.g., bioaccumulation) or extracellular mechanisms (e.g., bioabsorption, biomineralization, complexation or precipitation) .
The use of fungi in the production of metal-based nanoparticles offers advantages for industrial scale production when compared to bacteria since these organisms have a higher resistance against the flow pressure and agitation of the bioreactors .
However, in recent years, most of the studies report the use of plant extracts because, in addition to the advantages mentioned above, its use facilitates the treatment of samples, the scale-up production, and the collection of the product of interest.
reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075170/
More information: Yu-Qing Liu et al. Nanozyme scavenging ROS for prevention of pathologic α-synuclein transmission in Parkinson’s disease, Nano Today (2020). DOI: 10.1016/j.nantod.2020.101027