Background

Parkinson’s disease is a neurodegenerative disease that is the result of degeneration of dopaminergic neurons, leading to loss of basic motor functions.

The degeneration of these dopaminergic neurons can be attributed to oxidative stress, which is the build up of radical oxygen species (ROS), such as hydrogen peroxide, building up in the brain. This is a progressive disease that can be genetically inherited, but most often it occurs over time. Therefore, it can be prevented if the central problem is addressed early on. Since the central problem lies in the build up of ROS, being able to eliminate the ROS from the brain can potentially prevent the degeneration of dopaminergic neurons.

Nanoparticles as Treatment

In talks of using nanoparticles as the new innovative form of gene therapy and gene delivery, this project aims at using nanoparticles to break down the reactive oxygen species or take the radical electron away in order to make it not toxic. 

Carboxyfullerene (C60) was the first nanoparticle that was looked at due to its property to scavenge free radicals. C60 has piqued the interest of many researchers in medicine because it is lipid soluble, able to pick up free electrons, and is completely symmetrical. The fact that it is lipid soluble is important because it is then able to pass through the blood brain barrier. However, its most important property is its antioxidant properties. The numerous conjugated double bonds in the structure of C60 is easily able to accept electrons. It is able to take up many radical electrons and then be reused over again once the electrons are given away. C60 stays embedded in the mitochondrial membrane and then quenches radicals when needed. Carboxyfulleres are also able to localize to areas of high radical species such as the mitochondria, which makes localization not an issue when targeting certain cells.

Although carboxyfullerene itself is an excellent source for eliminating radical oxygen species, more benefits can be taken out of these nanoparticles by coating them with different functional groups. After much research, cystine coated carboxyfullerenes has been performed before on PC12 cells as well non-human primates. Cystine is water soluble, which is good in terms of biocompatibility in the blood. Additionally, cystine is readily converted to cysteine once taken up by cells. Cysteine is beneficial to the body in several different ways, but in neurodegenerative diseases it is especially beneficial for its role in glutathione synthesis. Glutathione is the body’s antioxidant that is in charge of removing ROS. Therefore, bringing the body’s natural antioxidant back to normal function will allow the brain to make a healthy amount of hydrogen peroxide that can still be broken down properly. Cystine coating will provide many benefits to the brain while the carboxyfullerene is carrying out its normal function.

Selenium coating is another functionalization that seemed promising for its antioxodiant properties as well. This could be administered if the level of ROS seem very high and perhaps more antioxidants need to be present. Selenium also has properties that are beneficial to the body such as its redox ability, detoxification, and immune-system protection. This compound is more beneficial to patients with Alzheimer’s disease and therefore can be further examined in the Alzheimer’s page.

Experimentation

In this project the different experimental groups that would want to be tested are:

• neurons as a control group
• neurons with hydrogen peroxide as the negative control group
• neurons with hydrogen peroxide and N-Acetyl-Cysteine (NAC) as positive control
• neurons with hydrogen peroxide and C60
• neurons with hydrogen peroxide and cystine coated C60
• neurons with hydrogen peroxide and selenium coated C60

N-acetyl-cysteine provides cysteine to the brain, and as mentioned before it assists in the synthesis of the body’s antioxidant, glutathione. This compound serves as a control because this is what is already in the brain and is being used to combat oxidative stress on its own. Therefore, by comparing the effectiveness of the nanoparticles with this naturally occurring process, the benefit of nanoparticles can be observed.

Each of these experimental groups should have at least 4 trials in order to avoid any construing data.

Analysis

There are two potential ways this experiment can be analyzed, and both forms of analysis would be preferred in order to obtain the more results that can be interpreted.

The first would be to use micro electrode arrays (MEA) in order to see the difference in firing rates between all the experimental groups. In comparing the firing rates and action potentials across all the groups, the cultures with the most number of live neurons can be detected. If the firing rates increase for the cultures with nanoparticles when compared to the negative control, it is evident that the nanoparticles were successful in eliminating the ROS from the neurons, allowing it to fire normally and preventing apoptosis. It would be interesting to see if the firing rates increased for the nanoparticles when compared to the NAC, because that would mean that the nanoparticles are performing a better job compared to the body’s own system of eliminating reactive oxygen species.

Another form of analyses would be to use flow cytometry. After speaking with a member of Dr. Kehn-Hall’s lab in Manassas, the application for a flow cut-meter works perfectly in detecting ROS levels. The machinery works by detecting the apoptosis rate as well detecting the levels of ROS species in each sample. It will be interesting to see how all of these levels compare and if one nanoparticle works better than the others or whether they truly work in eliminating ROS.

Setbacks

A major setback that this project faces is obtaining neurons. Neurons would be necessary to mimic what is happening in the brain. Additionally it is able to fire action potentials which can be picked up by the MEAs. The issue is that there are no brain dissections currently going on, and therefore requires a lot of money to be spent in order to have a couple of brain pieces. Astrocytes were suggested as an alternative to neurons because they are easily available and easy to take care of. However, astrocytes are a form of glial cells and therefore their function is to provide nutrients for the neurons. As a result, they themselves are not able to fire action potentials and cannot be analyzed using MEAs.

Another setback is obtaining the coated C60. Coating the nanoparticles takes a lot of special equipment that this lab does not have access to, and therefore would have to be done professionally in order to be constructed with precision and accuracy. The small budget available makes this a challenge as well.

Since this a novel area of study, not many protocols have been developed. As a result, the protocols must be designed from scratch and with guidance from professionals.

Bibliography:

Bakry, R., Vallant, R. M., Najam-ul-Haq, M., Rainer, M., Szabo, Z., Huck, C. W., & Bonn, G. K. (2007). Medicinal applications of fullerenes. International Journal of Nanomedicine, 2(4), 639–649.

Hu, Z., Guan, W., Wang, W., Huang, L., Xing, H., & Zhu, Z. (2007). Protective effect of a novel cystine C60 derivative on hydrogen peroxide-induced apoptosis in rat pheochromocytoma PC12 cells. Chemico-Biological Interactions, 167(2), 135–144. https://doi.org/10.1016/j.cbi.2007.02.009