Judges’ Queries and Presenter’s Replies

  • Icon for: Qiaobing Xu

    Qiaobing Xu

    Faculty
    May 20, 2013 | 11:11 p.m.

    interesting work. why is the antibody coating necessary? any toxicity observed from the nanoparticle form of PTx?

  • Icon for: David Cochran

    David Cochran

    Presenter
    May 21, 2013 | 09:37 a.m.

    Hello Qiaobing Xu! Good questions!
    The antibody coating allows us to specifically direct where these particles accumulate inside the body. Normally if these particles were injected into the body, they would circulate and be filtered out, thus provide no localized benefit.
    In this specific case, we are using an antibody coating that adheres to vascular cells. The concept is we can easily substitute the coating to any other antibody to target a multitude of other cell types (Inflamed cells, cancer cells, etc)
    A few examples of this concept are listed here:
    http://jpet.aspetjournals.org/content/317/3/116...
    http://www.sciencedirect.com/science/article/pi...

    As for your other question, we have previously published on the uncoated nanoparticle form of PTx. We saw no toxicity up to 2 mg/mL in our endothelial cell line, whereas we DID see toxicity with trolox (the monomer, it is a modification of vitamin E) at lower concentrations. Most interestingly we saw that PTx can inhibit protein carbonyl formation, a symptom of oxidative stress injury, that we did not observe in the native form of trolox.
    The full article is listed here: http://onlinelibrary.wiley.com/doi/10.1002/jbm....

  • Icon for: Aparna Baskaran

    Aparna Baskaran

    Faculty
    May 21, 2013 | 10:52 a.m.

    As I understand it, you have a culture of endothelial vascular cells that you treat with both the Iron oxide and the therapeutic nanoparticles. Am I right? The data shows that this therapeutic agent binds better than a non specific one (IgG). If yes, how will we demonstrate that in vivo, this targeting holds?, i.e., in the presence of other endothelial/epithelial cells. Is there an in vitro experiment we can design that will be a proof of principle demonstration of this specific targeting?

  • Icon for: David Cochran

    David Cochran

    Presenter
    May 21, 2013 | 11:59 a.m.

    Hi Aparna. This is correct, we have a culture of vascular cells, and show that our anti-PECAM-1 antibody coated particles adhere specifically, as opposed to a non specific IgG.
    There are a couple of ways to demonstrate in vivo. We can look either directly at particle adhesion, or downstream effects.
    To look directly, we can utilize a radioactive label on the antibody (Similar to our cell culture studies), and analyze tissue samples directly to correlate distribution. We have done this in the past in animal studies to determine distribution in the blood brain barrier. To be even more precise, there are methods to separate blood vessels from tissue.
    For an indirect method, we can excise tissue of interest, and observe the antioxidant effects through the use of assays such as DCF (A dye that becomes fluorescent in the presence of free radicals, treated areas would be less fluorescent due to the scavenging ability of the PTx), or a Trolox Equivalent Antioxidant Capacity (TEAC), which can measure the antioxidant potential of the tissue.
    This has been done in a previous publication using targeting in vivo listed here:
    http://www.sciencedirect.com/science/article/pi...

    As for an in vitro experiment demonstrating targeting, we have actually inadvertently done this. In utilizing these nanoparticles for a different cell culture application, we discovered we were using a blood brain barrier epithelial cell culture line that did not express PECAM-1. As a result, these nanoparticles (specific to PECAM-1), did not adhere at all to those BBB epithelial cells. In contrast, vascular endothelial cells express PECAM-1, thus can adhere with this targeting method.

  • May 21, 2013 | 06:05 p.m.

    Good work! I wonder, in your work what is the diameter of a nanoparticle before surface functionalization? Can it be done with ultra-small nanoparticles? For some MRI methods (T1-based), ultra-small particles ( ~ 5 nm) can provide certain advantages. It would be good to make them safer.

  • Icon for: David Cochran

    David Cochran

    Presenter
    May 21, 2013 | 08:13 p.m.

    Hi Natalia. We have found that the antibody coating increases the nanoparticle size by 15-20 nm. This has been consistent with a few of the nanoparticle systems we have used.
    As for your second question, that is an interesting idea and this does tie into the first question.
    The physical size of the antibody (by X-ray crystallography) is roughly 5nm x 5nm x 10nm. Because of this size, I don’t think this would be possible to coat ultra-small particles of 5 nm.
    In fact a quick calculation I did based on the surface area of a particle that size and the smallest surface area (5nm x 5nm) of an antibody would yield 0.62 antibodies per particle.
    However! With that being said, I think it would be possible using just the Fab fragments of an antibody (The portion of the antibody responsible for binding), rather than the entire molecule.
    Additionally, it would also be possible to develop an peptide aptamer-type system, which consists of very small sequence of peptides to target the sites we want, which also would be significantly smaller than a whole antibody molecule.

    See this link for an easier visual representation of the Fab fragments if you are unfamiliar:
    http://en.wikipedia.org/wiki/Fragment_antigen-b...

  • Icon for: Hyunjoon Kong

    Hyunjoon Kong

    Faculty
    May 21, 2013 | 06:35 p.m.

    Interesting idea. Have you examined stability of antioxidant nanoparticles in a media simulating blood?

  • Icon for: David Cochran

    David Cochran

    Presenter
    May 21, 2013 | 07:59 p.m.

    Hello Hyunjoon! Great question!
    While we haven’t looked at stability of THIS specific antioxidant nanoparticle system, we do have a publication coming out on another particle system using the same exact antibody coating.
    In this other publication, I had incubated the nanoparticles in whole blood taken from our sample animals. I found that the particles remained stable, along with the antibody coating, for up to 6 hours in blood. After 24 hours, the particles were still stable in terms of suspension, but approximately 50% of the coating had dissociated (And probably replaced with serum proteins).

  • Icon for: Qi-Huo Wei

    Qi-Huo Wei

    Faculty
    May 21, 2013 | 09:26 p.m.

    To lower/eliminate toxicity, can ion dioxide nanoparticles be made bio-degradable?

  • Icon for: David Cochran

    David Cochran

    Presenter
    May 22, 2013 | 11:31 a.m.

    Hi Qi-Huo, good question! This is an interesting concept in that the term “biocompatibility” always changing.
    Currently, iron oxide nanoparticles are already deemed as biodegradable in literature. Indeed this is the case for particles circulating in the blood stream. Through IV injections (Such as in MRI applications), the particles will eventually be deposited in the liver, where Kupffer cells can metabolize the nanoparticles. The digested iron is then kept in the body’s iron stores. There have been reports in literature that even these cells experience a severe rise in oxidative stress, resulting in toxicity.
    The issue, however, comes to light when these particles are injected or accumulate in a specific area of tissue (Such as in cancer applications). Endothelial cells have been shown to be able to metabolize iron oxide, but at a slower rate than Kupffer cells, with a significant generation of oxidative stress.
    A well written review about iron oxide metabolism is listed here: http://www.sciencedirect.com/science/article/pi...

    In order to “increase” biodegradability, many groups have utilized biocompatible coatings or polymer encapsulating delivery systems. While this does not directly influence how fast the iron can be metabolized, some of the coatings do influence how quickly the particles can be cleared, thus potentially lowering long term toxicity.

  • Further posting is closed as the competition has ended.

Poster Discussion

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    Ray Davidson

    Guest
    May 20, 2013 | 08:29 p.m.

    Very cool! Chemical Engineers FTW!

  • Icon for: Hainsworth Shin

    Hainsworth Shin

    Faculty
    May 21, 2013 | 08:08 p.m.

    Great job with the poster and video.

  • Icon for: David Cochran

    David Cochran

    Presenter
    May 21, 2013 | 08:15 p.m.

    Thank you Dr. Shin!

  • Icon for: Terri La Count

    Terri La Count

    Trainee
    May 23, 2013 | 09:28 a.m.

    I enjoyed your video on your work that will yield important benefits in the medical world.

  • Small_default_profile

    Stefan Kwiatkowski

    Guest
    May 24, 2013 | 08:17 a.m.

    It will be nice to see the whole poster after listening to your introduction which was quite interesting.

  • Icon for: David Cochran

    David Cochran

    Presenter
    May 24, 2013 | 10:32 a.m.

    Hi Dr. Kwiatkowski! I think you should be able to view the poster.
    At the top of the video, there should be two tabs, one that says view video, the other view poster. If you click view poster, it should come up for viewing.

  • Further posting is closed as the competition has ended.

Icon for: David Cochran

DAVID COCHRAN

Presenter’s IGERT
University of Kentucky
Years in Grad School: 4

Fighting “Fire with Fire”: Targeted Antioxidant Polymer Nanoparticles Suppress Iron Oxide Nanoparticle Toxicity in Vascular Endothelial Cells.

Owing to their unique imaging and responsive properties, magnetic iron oxide nanoparticles have been of considerable interest as drug carriers and contrast agents for biomedical applications. Recently, however, there has been growing concern on the potential health effects these particles may pose. Iron oxide toxicity has been demonstrated in in vitro and in vivo models, with oxidative stress being implicated as playing a central role in this pathology. One of the key cell types implicated in this injury is the vascular endothelial cells. Here we report on the development of a targeted polymeric antioxidant, poly(trolox ester), nanoparticle that can suppress oxidative damage. As the polymer undergoes enzymatic hydrolysis, active trolox is locally released, providing a long term protection against pro-oxidant agents. In this work, poly(trolox) nanoparticles are targeted to platelet endothelial cell adhesion molecules (PECAM-1), which are able to bind to and internalize in endothelial cells and provide localized protection against the cytotoxicity caused by iron oxide nanoparticles. These results indicate the potential of using poly(trolox ester) as a means of mitigating iron oxide toxicity, potentially expanding the clinical use and relevance of these exciting systems.