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Drug Delivery

Labyrinthine lipid nanoparticles may boost RNA delivery

Lipids with complex interiors facilitate the escape of their cargo into the cytoplasm

by Shi En Kim
June 28, 2023 | A version of this story appeared in Volume 101, Issue 21


A composite of an cryo-electron micrograph and a schematic of an endosomal escape event during RNA drug delivery by a lipid nanoparticle. The lipid nanoparticle, delineated in red, fuses with the bilayer membrane of the endosome to open up a pore in the endosome and enable the RNA cargo to escape. The depicted lipid nanoparticle has a mixed internal geometry of cubic and hexagonal phases. The escaping RNA strand is depicted as a squiggly grey line.
Credit: Lining Zheng and Alex D. Jerez, UIUC
A lipid nanoparticle, delineated in red, fuses with the bilayer membrane of an endosome to allow the RNA strand (squiggly grey line) to escape its nested enclosure. This lipid has a mixed internal geometry of cubic and hexagonal phases, which are captured using cryo-electron microscopy.

The COVID-19 vaccine’s success was a validation of RNA therapeutics as much as it was for the lipid nanoparticle (LNP) technology that delivered it. But with the boom of RNA medicines extending beyond vaccines, there’s always room for improvement in their lipid carriers. A new study shows that tweaking the internal structures of these oleaginous capsules can make it easier for the RNA cargo to access the cellular cytoplasm and do its job (PNAS 2023, DOI: 10.1073/pnas.2301067120).

A complicated sequence of events follows once an RNA drug is injected into the body. After the lipid-packaged RNA arrives at a cell, it pushes through the cell membrane, inadvertently wrapping itself with part of the phospholipid bilayer in a process called endocytosis. The engulfed package sits inside an endosome in the cell. For the RNA strand to take effect as a drug, it needs to bust out of its endosome enclosure and reach the cytoplasm. But the cell may mobilize to chew up foreign substances before the RNA cargo gets free. Estimates for the rate of endosomal escape are as low as 1%.

The new study’s corresponding author, Cecilia Leal, likens the encapsulated LNP-RNA complex to a plastic shopping bag of apples, with the endosomal membrane resembling the bag itself—flimsy, yet tough. “Try to puncture a pore in a floppy plastic bag,” the University of Illinois Urbana-Champaign materials scientist says. “This is a harder problem than it looks.”

Fortunately, the accompanying LNPs themselves can facilitate its flight. The LNP needs to fuse with the endosomal membrane, commingling fats from both surfaces, to open up pores and spill its guts.

In traditional drugs, LNPs rely on a drop in the surrounding pH to become favorable toward fusing. In an acidic environment, the LNPs gain a positive charge and coalesce with the negatively charged endosomal walls. But there is a competing attraction to the RNA payload itself, which is also negatively charged.

Leal’s solution to make LNPs more fusion-prone is to make them less stable. To do so, her group mixed lipids with varying head and tail lengths. Lipids generally pack together to minimize their exposure to the overwhelmingly aqueous cellular environment. But when the participating lipids come in diverse molecular shapes and sizes, they will assemble into complex droplets with nanoscale undulations rather than blobs with smooth surfaces. Changing up the mixing ratios of these lipids, the researchers fashioned nanoparticle complexes with varying interiors, such as multilayer globules resembling an onion and nanospheres with pores arranged hexagonally. By changing how the lipids pack, the authors alter the LNP’s elastic energy cost of popping the endosome.

The researchers put their LNPs to the test in cultures of live cancer cells. Tagging the LNP carriers, their cargo, and endosomes with fluorescent dyes, the study authors observed that LNPs-RNA complexes with cubic pore arrangements—“cuboplexes,” the team calls them—outperformed other LNP geometries at releasing the RNAs into the cellular environs.

The study sheds light on the fundamentals of lipid delivery, an important “first step” towards the rational design of LNPs, says Gaurav Sahay of Oregon State University who wasn’t involved in the research. In the future, he would like to see whether the enhanced performance of the cuboplexes hold up inside the body or in a closer proxy environment. For example, he notes that liver-homing lipoproteins tend to cling to LNPs soon after they enter the body. He wonders whether they will modify the cuboplex structure and alter the delivery efficiency.

Leal acknowledges that her group hasn’t worked out all the mechanistic details of why the internal structure exerts an effect on endosomal elusion. In fact, the entire field still relies on a trial and error approach to design LNPs, and a more systematic study is long overdue. Leal has recently received a research grant to pursue the mechanics of LNP-endosome fusion further. “It’s literally a million-dollar question,” she says.


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