How mRNA Opens the Cell Door—the Janitor Holds the Key

The game is starting any minute. He sprints to the gym door, grabs the handle to throw it open, and—his heart sinks. The door is locked. He pounds on the door, but no one can hear him over the pep band’s performance of the fight song. He is hopeless, until a janitor appears. The janitor rifles through his key ring, finally producing the one that opens the door. He barrels into the gym, and is met with cheers from the crowd and sighs of relief from his teammates. What would they do without their captain, after all?

The COVID-19 vaccine has a star athlete that we’ve all heard about—mRNA. But it also has a janitor, and without it, the mRNA would miss the big game.

Like the team captain, mRNA is locked out. As it tries to enter an immune cell, the negative charges along its backbone repel the negative charges on the cell membrane, preventing it from getting inside. And anything jostling the door handle of the cell, so to speak, is seen as an invader and is promptly ripped to shreds by nucleases, enzymes that can cleave the bonds of genetic material. 

That’s why mRNA needs a janitor to come to its rescue, and this janitor’s embroidered jumpsuit reads “ionizable lipid nanoparticles.” The mRNA is encased inside these tiny vesicles made up of fat-like molecules called lipids. The ionizable lipid nanoparticles—which are about 1/50,000 the diameter of a human hair in size—can easily cross the cell membrane, allowing the mRNA inside them to sneak into the cell.

Here’s how it works: the ionizable lipid develops an electrical charge in response to the pH of its environment. At the physiological pH of the body, the lipid has a neutral charge, which helps the vesicle interact with the cell membrane without repulsion. Proteins on the cell surface bind to the particle and help the cell to engulf it. The vesicle is now trapped in a little bubble of pinched-off cell membrane, the interior of which is acidic. This change in pH causes the ionizable lipids to develop a positive charge, which somehow triggers the nanoparticle to escape from the cell membrane bubble and fall apart, releasing its mRNA contents. Scientists aren’t entirely sure of the mechanism, but it does the job.

Developing an ionizable lipid was critical for delivering mRNA into the cell, and it wasn’t an easy task. Pharmaceutical companies spent years optimizing the chemical structures of their lipids, watching them fail when tested in cells or humans, and going back to the drawing board. One major breakthrough came when Alnylam Pharmaceuticals developed an ionizable lipid called MC3 that worked in clinical trials. However, MC3 was used to carry siRNA, a different type of RNA. siRNA is a short RNA sequence that suppresses one gene that’s thought to be causing trouble, while mRNA encodes an entire protein. Due to its much larger size, mRNA was incompatible with MC3. However, the lipid provided a valuable starting point for the design of new ionizable lipids that could successfully encapsulate mRNA.

Various biological and synthetic molecules—including cholesterol, another type of lipid called phospholipids and a compound called polyethylene glycol—are often added to the ionizable lipids to reinforce the structure of the nanoparticle. Researchers are currently exploring different formulations to further tune the balance between the particle’s stability and degradability, maximizing the mRNA load the cell receives. For example, one study showed that incorporating cholesterol molecules with specific chemical modifications greatly enhances mRNA delivery by ionizable lipid nanoparticles.

Additionally, scientists are interested in developing nanoparticles that can deliver mRNA to specific types of cells. The administration route of the vaccine currently offers some control—for example, intramuscular injections like the COVID-19 vaccine naturally target immune cells. But to selectively access other classes of cells, scientists are designing particles that recognize surface proteins that are unique to one cell type. In one study, researchers decorated nanoparticles with biological molecules that bind to proteins presented on the surface of the endothelial cells that line the lung to direct mRNA delivery to the lungs. 

Ionizable lipid nanoparticles show great promise in facilitating the delivery of a wide range of mRNA-based therapeutics. But regardless of what the future holds, let’s promise to never underappreciate the janitor again. 

Other References:

• Cross, Ryan. Without these lipid shells, there would be no mRNA vaccines for COVID-19. C&En Magazine. https://cen.acs.org/pharmaceuticals/drug-delivery/Without-lipid-shells-mRNA-vaccines/99/i8
• Aldosari, Basmah; Alfagih, Iman; Almurshedi, Alanood.  Lipid nanoparticles as delivery systems for RNA-based vaccines. Pharmaceutics 2021, 13, 206. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913163/pdf/pharmaceutics-13-00206.pdf
• Let’s talk about lipid nanoparticles. Nat. Rev. 2021, 6, 99. https://www.nature.com/articles/s41578-021-00281-4