Peptide-guided nanoparticles deliver mRNA to neurons

Penn engineers have modified lipid nanoparticles (LNPs) – the revolutionary technology behind mRNA COVID-19 vaccines – to not only cross the blood-brain barrier (BBB), but also target specific types of cells, including neurons. This breakthrough marks an important step toward potential next-generation treatments for neurological diseases such as Alzheimer’s and Parkinson’s.

In a new article in Nano lettersThe researchers demonstrate how peptides (short chains of amino acids) can serve as precise targeting molecules, allowing LNPs to deliver mRNA specifically to endothelial cells that line blood vessels in the brain, as well as to neurons.

This represents an important advance in delivering mRNA to cell types that would be essential for treating neurodegenerative diseases; such treatments will need to ensure that the mRNA arrives at the right place. Previous work by the same researchers proved that LNPs can cross the BBB and deliver mRNA to the brain, but did not attempt to control which cells the LNPs target.

“Our first paper was a validation lipid nanoparticle design,” says Michael J. Mitchell, associate professor of bioengineering (BE) and senior author of the paper.

“It was like showing that we could send a package from Pennsylvania to California, but we had no idea where it would end up in California. Now, thanks to peptides, we can send the package to specific destinations with common features, like every house with a red mailbox.

The challenge of accessing the brain

Crossing the BBB is difficult because the structure has evolved to prevent entry of virtually all dangerous or foreign molecules, including most drugs; The mRNA molecules are too large to penetrate the barrier, as are most pharmaceuticals. The BBB also actively expels materials it deems unsafe.

“You can inject treatment directly into the brain or spine, but these are highly invasive procedures,” says Emily Han, a doctoral student at the Mitchell Lab and first author of the paper.

Because the BBB allows fat-soluble molecules (like alcohol and THC, which is why these substances affect the brain), to pass through, some formulations of LNP, which are partly made up of the same family of fatty compounds found in everyday oils, can sneak into the brain.

Peptides vs Antibodies

Until now, most research into targeting specific organs with LNPs has focused on combining them with antibodies, large proteins that function as biological tags. “When you put antibodies on LNPs, they can become unstable and larger, making it very difficult to pass through the barrier,” says Han.

Unlike antibodies, which can be several hundred amino acids long, peptides are only a few dozen amino acids long. Their smaller size means they are not only easier to place in large numbers on LNPs, but also cheaper to manufacture. Peptides are also much less likely than antibodies to aggregate during LNP formulation or cause unintended immune responses.

The choice to use peptides began with Han’s unexpected encounter with a bat that flew into his room, potentially exposing him to rabies. While researching the vaccines she received against the disease, Han learned that one of the ways the rabies virus crosses the BBB is through the rabies virus glycoprotein.

“I then came across one of our most promising targeting peptides,” says Han, a molecule known as RVG29, a 29 amino acid segment of this protein.

Test the concept

To confirm that the peptides worked as expected, the researchers first needed to verify that they adhered to the LNPs. “Our LNPs are a complex mixture of nucleic acids, lipids and peptides,” explains Han. “We had to optimize the quantification methods to distinguish the peptides from all these other signals.”

Once they knew that the peptides had adhered to the LNPs, the researchers then had to determine whether the peptide-functionalized LNPs (pLNPs) actually reached their intended targets in animal models.

“It’s really difficult to set up,” says Han, “because in the brain there are so many different cell types and a lot of fat that can interfere with the measurements.”

Over more than six months, Han painstakingly developed a protocol to carefully disassemble brain tissue, almost like a mechanic would disassemble an engine.

Future Directions

Next, the team aims to determine what fraction of neurons should be treated with pLNPs to significantly relieve symptoms or potentially cure neurological diseases. “Going back to the same analogy, should we send them to every house with a red mailbox, or just 10% of them? Would 10% of neurons be enough?” Mitchell asks.

Answering this question will guide the development of even more effective delivery strategies, bringing the promise of mRNA-based treatments for Alzheimer’s, Parkinson’s and other brain diseases closer to reality.