Synthetic solutions for ethical and effective drug testing

Improving human quality of life with drug treatments is a complicated issue. Drug certification, including drug safety and reliability, entails a long series of tests and government approvals before the drug is available for anyone to use.

Testing drugs is challenged by ethical and biological concerns. Testing new drugs on humans is usually part of a clinical trial and occurs near the end of a drug's path to public use. Before that point, a large amount of testing has been conducted on animals. There is a growing call to phase out animal testing, partly because of the differences in animal and human biology. Simply put, just because something works for mice doesn't mean it will work for a human being.

A group of academic researchers across institutions have joined forces with Virginia Tech's Jeff Schultz to find a solution that could give human-oriented results with synthetic tools. Their approach requires no human subjects or animals. Instead, it uses new technologies to create testing environments that are highly customizable. Drugs can be tested with cells, not creatures.

Funded by a $1.8 million grant from the National Institutes of Health (NIH), the team includes:

• Amrinder Nain, professor of mechanical engineering at Virginia Tech
• Rafael Davalos, Margaret P. and John H. Weitnauer Jr. Chaired Professor at Georgia Tech
• Seemantini Nadkarni, associate professor, Harvard Medical School and Massachusetts General Hospital
• Jeff Schultz, co-founder of 3D-printed microfluidics company Phase Inc., who also earned three degrees from the Virginia Tech Department of Material Science and Engineering

Breaking into the brain

Physiological barriers are common in the body, and one such barrier, called the blood-brain barrier, is made of a network of blood vessels and tissues. Its function is to allow helpful substances such as water and oxygen to enter the brain, but keep out harmful substances that could lead to disease or tumors. Recreating this intricate environment for drug testing has been challenging, and it is not uncommon for clinical trials to fail when they move from the lab.

Therapeutics fail in clinical trials because they can't cross the blood-brain barrier. The reality is that the devices that have been created in a lab don't work and they allow too much to pass through. This gives false information that molecules can get through, and when you get into a clinical trial, the drugs fail because the human brain conditions haven't been properly duplicated."

Rafael Davalos, Georgia Tech

The team is approaching the problem with Phase's proprietary 3D-printing method, creating microfluidics at previously unattainable resolutions that also are highly reproducible and scalable. Microfluidics are remarkably small devices where cells and fluids can be manipulated to create an "organ on a chip" that mimics the behavior and function of human organs. While this project is focused on the blood-brain barrier, the core technology has wide-ranging applications for other organs such as the liver, lungs, and skin.

Schultz has spent his career inventing and scaling 3D-printing techniques for both startup companies and international conglomerates. Building on the strength of that experience, he turned his attention to apply the flexibility of 3D printing to the biomedical world.

"We're building something that more realistically mimics the geometry of the body compared to other microfluidics," said Schultz. "Harnessing the design freedom of 3D printing allows us to create devices that have the same curvature, size of veins, and functionality of the human body. We can put in valves similar to the heart that are accustomed to pulsating mechanical stresses. This gives us the opportunity to see results that are closer to real life than if the cells were laying flat in a dish, and is done in other conventional microfluidic devices, but has yet to be applied to the blood brain barrier."

Synthetic devices and living cells

Schultz and Davalos have already collaborated on new methods for 3D printing medical devices using materials that had been problematic in drug trials up to that point. In phase one of this project, they devised a way to 3D-print polydimethylsiloxane (PDMS), a silicone polymer that could be used to mimic the blood-brain barrier. That project received $173,000 from the NIH.

"The challenge we set out to solve was with the materials," said Schultz. "There were no materials you could 3D-print for microfluidics that were widely accepted as safe for cells. PDMS was used for over two decades but wasn't 3D printable. We set out to develop a technology to 3D-print that material, which the NIH funded us to do in phase one of the project."

The material needed to be safe for cells so that cells could grow on the platform and provide conditions for testing the viability of various drugs. To make an artificial blood-brain barrier, the blood and tissue cells that form the barrier in a living body were grown on the 3D-printed piece, hence the "organ on a chip." The advantage of 3D printing is that the framework creates different pathways and architectures, which might lead to customizing the synthetic blood-brain barrier to match the patient's own.

After seeing success in the first phase, Schultz and Davalos saw possibilities in expanding the project. Amrinder Nain had expertise and tools ready for the task and had previously collaborated with Davalos.

Davalos' team has developed other organ-on-a-chip platforms to test the behavior of biological processes at the small scale. Philip Graybill, a recent Virginia Tech graduate on Davalos's team focused on developing such microfluidic models of the blood-brain barrier as well as how single cells respond to electromechanical cues using Nain's nanofiber platform. Through their collaboration, Graybill recognized a chance to fold one technology into another to build a more accurate model of what occurs in the brain. 

Making a mesh

Nain's specialty is research with nanofiber membranes that perform in much the same way as living tissues, created using a lattice of spun fibers that crisscross one another at the nanoscale. Those membranes became the key for the next evolution of the device and helped the team pick up a second round of NIH funding. Davalos and Nain's group recently published the first-ever ultra-thin and ultra-porous blood-brain barrier (BBB) that is roughly 70 percent thinner than other existing methods to study it. 

"What's really nice about using Amrinder [Nain] 's fiber network is that it's so thin, you can have cells on either side that can communicate," said Davalos. "This creates tight junctions between cells to prevent therapeutics from passing through."

This is precisely the level of control that is required to match findings from clinical trials. With this development, the team has given future researchers a reliable and faster turn-around tool for drug testing in physiologically relevant environments while minimizing animal models.

"In the blood-brain barrier, there is a physical membrane," said Schultz. "Amrinder [Nain]'s nanofiber membrane mimics the thickness and porosity of the mechanism in a real brain better than most mimics used in similar devices. When we proposed phase two to the NIH, we proposed using those membranes integrated into our previous PDMS microfluidic device."

To create the organ-on-a-chip approach, every team member used their specialties. The process generally goes like this: 

• Nain's team produces the ultra-thin and nanoporous membrane mimics.
• Schultz's team receives the membrane, constructs a design including it, then uses a system developed by Nadkarni's Harvard team to test the behavior of the material.
• The finished pieces are sent to Davalos's team to outfit them with cells and conduct the biological tests.

"Organ-on-a-chip technologies are now projected to be standard lab protocols in the 21st century," said Nain. "Our technological breakthroughs have enabled the thinnest BBB in the market. In future design iterations, we expect to meet the dimensions and architectures present in the human body to achieve physiological outputs in a lab setting. When realized, this will transform how we test drugs and study bioengineering and biophysics."