The challenge of accurately measuring drug permeability

In our seventh NanoBio Lab, Erin Gallagher, a predoctoral candidate from the lab of professor Peter Searson, demonstrated the use of a cell permeability assay as a means of modeling drug diffusion through the blood brain barrier (BBB) endothelium. Assays such as this one enable us to better understand the cellular processes that govern what drug molecules are able to cross the BBB and the role of efflux pumps and transporters. Development of more accurate in vitro models is a highly valuable avenue of research, as it will allow for rational drug design to more effectively treat diseases such as Alzheimer’s, Parkinson’s and mood disorders with potentially fewer side effects.

The blood brain barrier (BBB) presents a challenge for delivery of drug molecules to the central nervous system, as many of the mechanisms it employs were evolved specifically to prevent introduction of dangerous substances into the central nervous system. Understanding the mechanisms by which various substances are able to cross the BBB will allow for more rational design of future generations of drug molecules and carrier systems.

The blood brain barrier (BBB) presents a challenge for delivery of drug molecules to the central nervous system, as many of the mechanisms it employs were evolved specifically to prevent introduction of dangerous substances into the central nervous system. Understanding the mechanisms by which various substances are able to cross the BBB will allow for more rational design of future generations of drug molecules and carrier systems.

For the assay, canine kidney cells (MDCK II) were seeded on transwells in a 24 well plate, 24 hours prior to the assay to allow the cells to form a confluent endothelial layer with functional tight junctions. When cells have formed a confluent endothelial layer, ion movement must occur through the cells themselves instead of through the much higher resistivity tight junctions. As a result, the overall resistivity measured is higher than for non-confluent cells, for which ions are able to simply diffuse through the transwell. Therefore, assessment of the integrity of the endothelial layer was done to measure the conductivity through the layer of cells.

Following assessment of the endothelial layer integrity, we ran a permeability assay for the fluorescent molecule Lucifer Yellow (LY) to determine its apparent permeability as a model for drugs diffusing across the BBB. Utilizing a standard concentration curve of LY fluorescence, the amount of LY that diffused through the layer was determined at specific time points to imply apparent permeability. For more typical non-fluorescent drug molecules, high performance liquid chromatography (HPLC) can be used to measure the amount of drug having diffused.

As a tool, assays modeling the blood brain barrier are indispensible to the pharmaceutical industry, but finding a model system that effectively reproduces in vivo conditions for less expensive, high throughput in vitro testing is a challenge. Permeability models, such as the one used in this lab, also allow development of novel strategies for moving drugs across the BBB. These strategies include molecular engineering of drug molecules to take advantage of cellular active transport mechanisms or peptide engineering that facilitates vesicle transport across the endothelium.

David Wilson is a first year PhD student in biomedical engineering working in the drug delivery laboratory of associate professor Jordan Green in biomedical engineering.

Image Citation:  Wong, A. D., Ye, M., Levy, A. F., Rothstein, J. D., Bergles, D. E., & Searson, P. C. (2013). The blood-brain barrier: an engineering perspective. Frontiers in Neuroengineering, 6(August), 1–22. doi:10.3389/fneng.2013.00007

For all press inquiries regarding INBT, its faculty and programs, contact Mary Spiro, mspiro@jhu.edu or 410-516-4802.

Getting my hands dirty in NanoBio lab

As a second year graduate student, classes take up a non-insignificant part of my day. One of the classes that I had the opportunity to take last spring was NanoBio Laboratory. NanoBio lab is clearly a laboratory class, which is always very exciting for an engineer. I enjoy any opportunity to get my hands dirty and really learn some techniques. And that was exactly what we had the opportunity to do.

NanoBio Lab was our chance to go into many of the labs in The Institute for NanoBioTechnology (INBT) and get an idea of some of the techniques that they use and the general area of research of the lab. Some of the techniques that were demonstrated in this course included gold nanoparticles synthesis, transfecting cells with luciferase (the chemical that makes fireflies glow), and a novel method of analyzing images. While not all of the labs necessarily apply to the work that I am doing, many of them have some relevance and could come in handy in the future.

Through this lab, I have learned techniques that could be useful in my research in the future. Not only have I learned useful techniques, it was also an excellent chance to network within other labs. In this course, we had one or two representatives from many of the labs associated with the INBT instruct us and assist us in learning the techniques. This allowed us to form a relationship with at least one member in the represented labs, which will make it easier to reach out to other labs for help learning new procedures and protocols.

I just found out that I’m going to have to attempt to transfect a cell line, which I have never done outside of the NanoBio lab. Just as all laboratory work I know that it will be difficult, and that I’m likely to fail a number of times before I have any success. Through this class, however, I know someone who I can talk to for advice and assistance as I go through this process.

Moriah Knight is a second year PhD student in Peter Searson’s lab studying Materials Science and Engineering.