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.

Vredenburg Scholarship winner headed to Japan

For the last two years, I have worked at INBT as a member of Peter Searson’s laboratory in the Department of Materials Sciences and Engineering at Johns Hopkins University. I have primarily studied the response of glioma cells exposed to direct current electric field. In this work and from listening to and observing others, I have developed an understanding of the brain’s support network including micro-blood vessels and the characteristics of glial cells.

In my classes this year, as a Junior in Biomedical Engineering, System BioEngineering has introduced me to a networks and bioelectrical examination of the brain’s computing parts, its neurons. In this class we learned the basics of neuroscience and how to model the processes of the brain using computer simulations. Getting to study this organ from a structural as well as computational perspective has been intriguing and insightful.

Benjamin Wheeler

Benjamin Wheeler

I have been awarded the Vredenburg Scholarship, which will allow me to study the brain further and work in Dr. Masashi Yanagisawa’s laboratory at the International Institute for Integrative Sleep Medicine at the University of Tsukuba in Japan. Dr. Yanagisawa and others working at IIIS are investigating neuroscience’s remaining black box: sleep. They are addressing questions such as why do we sleep, how is sleep controlled, and what it is the cause of sleepiness. This kind of work has many different applications and uses. Understanding which receptors and circuits are involved in sleep and wakefulness could be used to design drugs and develop treatments for various sleep disorders. For instance, the neuropeptide Orexin is one of the primary molecules involved in sleep/wake regulation. Those at IIIS are pursuing a small molecule agonist to the orexin receptor as a possible way to treat narcolepsy, the disease caused by abnormalities in the orexin-signaling pathway.

The project I will work on involves examining the role of specific receptors in regulating the behavior of neural circuits in the hypothalamus. To do so, I will use genetically altered mice whose receptors no longer respond to their natural agonist. However, these receptors will respond to a normally inert drug whose only effect is on the altered receptors. This will allow me to investigate how the activity of these receptors affects the behavior of the entire animal. Carrying out these experiments will expose me to new techniques such as patch clamp measurements and calcium recording. This will help me to develop an understanding of neuroscience on a scale smaller and a scale larger than I had been previously exposed. Additionally, the grant will allow me to travel and learn the language and culture of Japan. I am very excited to see the beautiful country and also gain a wider view of the global human condition, while getting to investigate some of neuroscience’s most pertinent questions.

About the author: Benjamin Wheeler is a third year undergraduate student at Johns Hopkins University Department of Biomedical Engineering, currently working in professor Peter Searson’s lab.

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

 

Poster presenters sought for Neuro X symposium

Johns Hopkins Institute for NanoBioTechnology (INBT) hosts its ninth annual symposium on May 1, 2015 in the Owens Auditorium on the Johns Hopkins medical campus. The theme for the speakers this year is Neuro X, where X stands for medicine, nanotechnology, engineering, science and more! Posters on any multidisciplinary theme are now being accepted. You do not have to be a member of an INBT affiliated laboratory to participate. Undergraduates, graduate students and postdoctoral fellows welcome. The event is free for Johns Hopkins associated persons. There is a fee for those outside of JHU/JHMI/JHH and is listed on the registration form.

Full details on poster guidelines and current information on the symposium can be found on the Neuro X website. To submit a poster or to simply register to attend the symposium, click here.

neuro-x-ad-flatThe symposium will begin at 8 a.m. with continental breakfast. Talks will begin at 9 a.m. and continue through 12:15 p.m. Speakers include: Alfredo Quiñones-Hinojosa, MD, FAANS, Professor of Neurological Surgery and Oncology Neuroscience and Cellular and Molecular Medicine; Jordan J. Green, PhD, Associate Professor of Biomedical Engineering, Ophthalmology, Neurosurgery, and Materials Science & Engineering; Ahmet Hoke MD, PhD, FRCPC, Professor, Neurology and Neuroscience; Patricia H. Janak, Professor, Department of Psychological and Brain Sciences/Department of Neuroscience in the Krieger School of Arts and Sciences; Piotr Walczak, MD, PhD, Associate Professor, Department of Radiology and Radiological Science; and Martin G. Pomper, MD, PhD, the William R. Brody Professor of Radiology and Radiological Science. This year’s symposium chairs are INBT director Peter Searson, Reynolds Professor, Materials Science and Engineering, and Dwight Bergles, Professor, the Solomon H. Snyder Department of Neuroscience, Department of Otolaryngology, Head & Neck Surgery.

The poster session will begin at 1:15 p.m. and conclude at 3:30 p.m. with poster prize presentations. Speaker talk titles, poster prizes and other details will be announced in the next few weeks. Don’t miss your chance to participate in one of Johns Hopkins largest, most popular and most well attended symposiums. Plan now to attend and present.

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

 

 

 

Podcast: Artificial blood vessel visualizes cancer cell journey

Researchers from Johns Hopkins Institute for NanoBioTechnology are visualizing many of the steps involved in how cancer cells break free from tumors and travel through the blood stream, potentially on their way to distant organs.  Using an artificial blood vessel developed in the laboratory of Peter Searson, INBT director and professor of materials science and engineering, scientists are looking more closely into the complex journey of the cancer cell.

Figure 1. 3D projection of a confocal z-stack shows human umbilical vein endothelial cells (HUVECs) forming a functional vessel immunofluorescently stained for PECAM-1 (green) and nuclei (blue).

Figure 1. 3D projection of a confocal z-stack shows human umbilical vein endothelial cells (HUVECs) forming a functional vessel immunofluorescently stained for PECAM-1 (green) and nuclei (blue). (Wong/Searson Lab)

INBT’s science writer, Mary Spiro, interviewed device developer Andrew Wong, a doctoral student Searson’s  lab, for the NanoByte Podcast. Wong is an INBT training grant student. Listen to NANOBYTE #101 at this link.

Wong describes the transparent device, which is made up of a cylindrical channel lined with human endothelial cells and housed within a gel made of collagen, the body’s structural protein that supports living tissues. A small clump of metastatic breast cancer cells is seeded in the gel near the vessel while a nutrient rich fluid was pumped through the channel to simulate blood flow. By adding fluorescent tags the breast cancer cells, the researchers were able to track the cells’ paths over multiple days under a microscope.

VIDEO: Watch how a cancer cell approaches the artificial blood vessel, balls up and then forces its way through the endothelial cells and into the streaming fluids within the channel of the device. (Video by Searson Lab)

The lab-made device allows researchers to visualize how “a single cancer cell degrades the matrix and creates a tunnel that allows it to travel to the vessel wall,” says Wong. “The cell then balls up, and after a few days, exerts a force that disrupts the endothelial cells. It is then swept away by the flow. “

Wong said his next goal will be to use the artificial blood vessel to investigate different cancer treatment strategies, such as chemotherapeutic drugs, to find ways to improve the targeting of drug-resistant tumors.

Results of their experiments with this device were published in the journal Cancer Research in September.

Andrew Wong (left) and Peter Searson. (Photo by Will Kirk/Homewood Photography)

Andrew Wong (left) and Peter Searson. (Photo by Will Kirk/Homewood Photography)

Check out this gallery of images from the Searson Lab. The captions are as follows:
Figure 1. 3D projection of a confocal z-stack shows human umbilical vein endothelial cells (HUVECs) forming a functional vessel immunofluorescently stained for PECAM-1 (green) and nuclei (blue).
Figure 2. 3D projection of a confocal z-stack shows human umbilical vein endothelial cells (HUVECs) forming a vessel with dual-labeled MDA-MB-231 breast cancer cells on the periphery.
Figure 3. Phase-contrast and fluorescence overlays depicting a functional vessel comprised of human umbilical vein endothelial cells (HUVECs) with dual-labeled MDA-MB-231 breast cancer cells on the periphery (green in the nucleus, red in the cytoplasm).

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

 

What’s mechanics got to do with tissue development?

A recent study at Harvard, published in the journal Science, found that mechanical factors play a significant role in tissue development. Learning these factors that contribute to the natural formation of tissues will not only improve our understanding of tissues, it will also improve our ability to engineer tissues in the future and improve our ability to discern developmental problems.

Intestinal villi small http://goo.gl/DlKA7p

Intestinal villi small http://goo.gl/DlKA7p

The walls lining the intestines are not smooth. They are covered with many tiny, finger-like protrusions, or villi, yielding a high surface area for high nutrient absorption. These villi are present in many different animals including humans, chickens, and mice. This study follows the chick’s gut from earlier embryonic stages through the gut formation.

In the beginning of gut formation, the intestine is a smooth, cylindrical tube. As the embryo matures, a outer layer of smooth muscle binds the inner regions. The inner region continues to expand, but the outer region restricts it causing the inner tube to buckle and bend back over on itself. As the embryo continues to grow, the outer layer is enhanced and strengthened, causing the inners layers to make smaller and tighter folds, eventually yielding the villi. This paper shows that without the outer muscle layer, the inner layer will continue to grow, but rather than forming villi, it just ends up with a larger circumference.

This study goes on to show that across different animals (xenopus, chick, and mouse), while the time scales and intermediate steps may vary, the constraints from the outer loop cause the buckling of the inner layer into the villi.

This research establishes that in natural formation of specific tissues—and consequently engineered tissues—mechanical factors must not be ignored.

Villification: How the Gut Gets Its Villi 

Charli Dawidczyk is a PhD candidate in Materials Science and Engineering working in Peter Searson’s research group.

 

Unlocking the mysteries of the blood-brain barrier

It might astonish you to know that, although we use our brains all the time, science knows very little about how they actually work. That is why recently, President Barack Obama announced a $100 million initiative to map the human brain.

“We can identify galaxies light-years away; we can study particles smaller than an atom; but we still haven’t unlocked the mysteries of the three pounds of matter that sits between our ears,” Obama said in a press conference on the announcement April 2.

The blood-brain barrier involves functional interactions between endothelial cells that form brain capillaries, astrocytes, and pericytes in a complex microenvironment. (Illustration by Martin Rietveld)

The blood-brain barrier involves functional interactions between endothelial cells
that form brain capillaries, astrocytes, and pericytes in a complex microenvironment. (Illustration by Martin Rietveld)

Obama’s Brain Research Through Advancing Innovative Neurotechnologies (BRAIN) project will seek to discover what occurs between the 100 billion cells firing inside the brain with the goalof helping to prevent and even cure neurological diseases, such as Alzheimer’s or Parkinson’s, that affect as many as 100 million Americans.

Johns Hopkins University is at the forefront of brain science research. The Brain Science Institute (BSi) at the Johns Hopkins School of Medicine was launched to develop new multidisciplinary research teams; create cutting edge-research cores for use by all brain researchers at Hopkins; and foster translation of discoveries to treatments of brain diseases, in part, by improving our ability to partner with industry and biotechnology.

In 2012, Peter Searson, professor of materials science and engineering and director of Johns Hopkins Institute for NanoBioTechnology (INBT), joined forces with Jeffrey Rothstein MD, PhD, director of the BSi, to create the Blood-Brain Barrier Working Group. This group brings together researchers with diverse interests and expertise to address key problems associated with drug delivery, to discover the role of the blood-brain barrier (BBB) in disease, and to elucidate the structure and function of the BBB.

“The blood-brain barrier is a dynamic interface that separates the brain from the circulatory system and protects the central nervous system from potentially harmful chemicals while, at the same time, regulating transport of essential molecules and maintaining a stable environment,” Searson said. “It is formed from highly specialized endothelial cells that line the brain capillaries, which transduce signals in two directions: from the vascular system and from the brain. The structure and function of the BBB is dependent upon the complex interplay between different cell types, specifically the endothelial cells, astrocytes and pericytes, within the extracellular matrix of the brain and with the blood flow in the capillaries.”

Although the BBB serves the important purpose of tightly regulating the environment of the brain and preventing sudden changes, which the brain cannot tolerate, Searson said, “this interface also blocks the passage of drug molecules to treat disease, neurodegenerative disorders, inflammation or stroke. Unfortunately, animal models are insufficient for use in under-standing how the human blood-brain barrier functions or responds to drugs. In addition, little is known about how disease, inflammation or stroke disrupts or damages the blood-brain barrier.”

With this in mind, the BBB working group has two primary goals, Searson explained. “Our long-term goal is to build an artificial microvessel that will be the first platform that recapitulates a brain capillary in its local microenvironment. This will enable fundamental studies as well as drug discovery and the development of methods to cross the blood-brain barrier,” Searson said.

The second goal is to understand how the blood-brain barrier can be damaged or disrupted so that strategies can be developed to repair it. Injury and disease can disrupt the normal structure and function of the blood brain barrier.

Currently the BBB Working Group has 40 researchers from disciplines as diverse as anesthesiology, materials science and engineering, pharmacology and oncology. Three postdoctoral fellows and 12 pre-doctoral students are also involved. The group meets monthly and hosts expert speakers on various topics. The working group website also lists current funding opportunities to which members can apply and conferences and workshops of interest.

Membership in the working group is open to any student, faculty member or staff at Johns Hopkins University in any discipline.

Visit the Blood-Brain Barrier Working Group website here.

This article was written by Mary Spiro and appeared in the 2013 issue of Nano-Bio Magazine.

Podcast: Nanotech method to study cell detachment could lead to improved cancer therapies

Peter Searson

Peter Searson

Cancer spreads from organ to organ when cells break free from one site and travel to another. Understanding this process, known as metastasis, is critical for developing ways to prevent the spread and growth of cancer cells. Peter Searson, Reynolds Professor of Materials Science and Engineering in the Whiting School of Engineering and director of the Institute for NanoBioTechnology, led a team of engineers who have developed a method to specifically measure detachment in individual cells.

The method, which uses lab-on-a-chip technology, allows researchers to observe and record the exact point when a cell responds to electrochemical cues in its environment and releases from the surface upon which it is growing. Better knowledge of the biochemistry of cell detachment could point the way to better cancer therapies. In this “Great Ideas” podcast, Elizabeth Tracey, communications associate for the School of Medicine, interviews Searson about this current research.

“…We know that processes like cell detachment are important in cancer metastasis, where cells become detached from tumors…” Peter Searson

Click here to listen:  Great Ideas Podcast: Peter Searson

Related links:

You can watch a video and read more about Searson’s method of studying cell detachment here.

Peter Searson’s INBT profile page.

This podcast was originally posted to the Johns Hopkins University “Great Ideas” web page. To view the original posting, click here.