October 11, 2014
MIT engineers have devised a way to rapidly test hundreds of different drug-delivery vehicles in living animals, making it easier to discover promising new ways to deliver a class of drugs called biologics, which includes antibodies, peptides, RNA, and DNA, to human patients.
In a study appearing in the journal Integrative Biology, the researchers used this technology to identify materials that can efficiently deliver RNA to zebrafish and also to rodents.
This type of high-speed screen could help overcome one of the major bottlenecks in developing disease treatments based on biologics: how to find safe and effective ways to deliver them.
“Biologics is the fastest growing field in biotech, because it gives you the ability to do highly predictive designs with unique targeting capabilities,” says senior author Mehmet Fatih Yanik, an associate professor of electrical engineering and computer science and biological engineering. “However, delivery of biologics to diseased tissues is challenging, because they are significantly larger and more complex than conventional drugs.
Automating large-scale studies
Zebrafish are commonly used to model human diseases, in part because their larvae are transparent, making it easy to see the effects of genetic mutations or drugs.
In 2010, Yanik’s team developed a technology for rapidly moving zebrafish larvae to an imaging platform, orienting them correctly, and imaging them. This kind of automated system makes it possible to do large-scale studies because analyzing each larva takes less than 20 seconds, compared with the several minutes it would take for a scientist to evaluate the larvae by hand.
For this new study, Yanik’s team developed a new technology to inject RNA carried by nanoparticles called lipidoids. These fatty molecules have shown promise as delivery vehicles for RNA interference, a process that allows disease-causing genes to be turned off with small strands of RNA.
Yanik’s group tested about 100 lipidoids that had not performed well in tests of RNA delivery in cells grown in a lab dish. They designed each lipidoid to carry RNA expressing a fluorescent protein, allowing them to easily track RNA delivery, and injected the lipidoids into the spinal fluid of the zebrafish.
To automate that process, the zebrafish were oriented either laterally or dorsally once they arrived on the viewing platform. Once the larvae were properly aligned, they were immobilized by a hydrogel. Then, the lipidoid-RNA complex was automatically injected, guided by a computer vision algorithm. The system can be adapted to target any organ, and the process takes about 14 seconds per fish.
A few hours after injection, the researchers imaged the zebrafish to see if they displayed any fluorescent protein in the brain, indicating whether the RNA successfully entered the brain tissue, was taken up by the cells, and expressed the desired protein.
The researchers found that several lipidoids that had not performed well in cultured cells did deliver RNA efficiently in the zebrafish model. They next tested six randomly selected best- and worst-performing lipidoids in rats and found that the correlation between performance in rats and in zebrafish was 97 percent, suggesting that zebrafish are a good model for predicting drug-delivery success in mammals.
The idea is to identify useful drug delivery nanoparticles using this miniaturized system.
The researchers are now using what they learned about the most successful lipidoids identified in this study to try to design even better possibilities. “If we can pick up certain design features from the screens, it can guide us to design larger combinatorial libraries based on these leads,” Yanik says.
Yanik’s lab is currently using this technology to find delivery vehicles that can carry biologics across the blood-brain barrier — a very selective barrier that makes it difficult for drugs or other large molecules to enter the brain through the bloodstream.
The research was funded by the National Institutes of Health, the Packard Award in Science and Engineering, Sanofi Pharmaceuticals, Foxconn Technology Group, and the Hertz Foundation.
Abstract of Organ-targeted high-throughput in vivo biologics screen identifies materials for RNA delivery
Therapies based on biologics involving delivery of proteins, DNA, and RNA are currently among the most promising approaches. However, although large combinatorial libraries of biologics and delivery vehicles can be readily synthesized, there are currently no means to rapidly characterize them in vivo using animal models. Here, we demonstrate high-throughput in vivo screening of biologics and delivery vehicles by automated delivery into target tissues of small vertebrates with developed organs. Individual zebrafish larvae are automatically oriented and immobilized within hydrogel droplets in an array format using a microfluidic system, and delivery vehicles are automatically microinjected to target organs with high repeatability and precision. We screened a library of lipid-like delivery vehicles for their ability to facilitate the expression of protein-encoding RNAs in the central nervous system. We discovered delivery vehicles that are effective in both larval zebrafish and rats. Our results showed that the in vivo zebrafish model can be significantly more predictive of both false positives and false negatives in mammals than in vitro mammalian cell culture assays. Our screening results also suggest certain structure–activity relationships, which can potentially be applied to design novel delivery vehicles.