Molecular syringe injects drugs into cells

bacteria injectors

Electron micrograph of Photorhabdus virulence cassettes. © Joseph Kreitz, Broad Institute of MIT and Harvard/ McGovern Institute for Brain Research at MIT

Even the best therapeutics can only work if they get to the right place in the body. Researchers have now developed an innovative system for this: they have modified a molecular syringe made of bacteria in such a way that it can be loaded with various cargo proteins and specifically binds to selected target structures. The technique has shown promise in cell cultures with human cells and in experiments with living mice. If further studies are also successful, the nano-syringe could open up new therapy options in the future, for example in the fight against cancer or in gene therapies.

Bacteria have evolved a variety of tactics to deliver proteins into the cells of their hosts. Bacteria of the genus Photorhabdus use a so-called extracellular contractile injection system (eCIS). These are a type of nano-syringes loaded with a toxin that the bacteria release into their environment. When the small poison transporters hit their target – in the case of Photorhabdus this is the cells of insects – they bind to certain surface structures and inject their cargo into the cell. The affected cells then die. Since Photorhabdus kills insects effectively in this way, the bacterium is used in agriculture as a natural pesticide.

Cell injection system

A further development of the system could now possibly revolutionize medicine. "The delivery of therapeutic molecules is a major bottleneck in medicine, and we need a wide range of ways to deliver these powerful new therapies to the right cells in the body," says Feng Zhang of the Broad Institute at MIT and Harvard in Cambridge. Photorhabdus' injection system offered him, first author Joseph Kreitz and their team a promising basis. "By learning how nature transports proteins, we were able to develop a new platform that can help bridge this gap," Zhang said.

On the way to this goal, the research team first analyzed the bacterial injection system in detail - and came across a useful property: the approximately 100 nanometer long, syringe-like constructs have a modular structure. In the so-called Photorhabdus virulence cassette (PVC), a tail fiber is responsible for recognizing and binding to specific structures on the target cell. The protein load is independent of the exact structure of the tail fiber and is contained in a tube within a sheath. After the machine docks with a cell, this tube contracts, mechanically forcing its charge inside the cell.

Killed cancer cells in cell culture

With the help of the adaptive AI system AlphaFold, which can predict the three-dimensional structure of proteins based on the amino acid sequence, the research team developed modified tail fibers that recognize certain structures on human cells instead of insect cells. "This is a nice example of how protein engineering can change the biological activity of a natural system," says Kreitz. "Our work confirms that protein engineering is a useful tool in bioengineering and in the development of new therapeutic systems."

As a proof of concept, the researchers designed the tail fibers to recognize a specific receptor that is typical of human cancer cells. In addition, they showed that they can load the molecular syringe with different types of proteins - including toxins against tumor cells and the DNA-cutting enzyme Cas9, which is used in gene editing. When they added nano-syringes programmed for cancer cells, loaded with a chemotherapy drug, to a cell culture containing human cancer cells, they reliably killed almost all of the cancer cells without affecting other cells. In tests with other targets and payloads, the system was not quite as efficient, but also showed promise.

Successful in the living organism

To test whether eCIS can also be used in living organisms, the team injected suitably programmed molecular syringes directly into the brains of living mice. In this case, the tail fibers were tailored to the mice's brain cells and contained a green fluorescent protein as cargo. And sure enough, a green glow in the mice's brains indicated that the transfer was successful. Follow-up examinations a day later revealed that the empty syringes were still attached to the outside of some mouse brain cells. After a week, however, such residues were no longer detectable. The researchers therefore assume that the nano-machines will be broken down by the body within a short time.

The team did not find any immune reactions to the foreign bodies. "However, they injected the PVCs directly into the brain, and a rather low immune response is expected there," points out Stefan Raunser from the Max Planck Institute for Molecular Physiology in Dortmund, who was not involved in the study. "I can imagine that the PVCs trigger an immune response if they are administered into the blood, for example."

More challenges

There are other potential obstacles that need to be overcome before the system can possibly be used therapeutically. "The disadvantage of the size of the PVC protein is that it makes it more difficult to penetrate dense tissue, such as in solid tumors," explains Raunser. In addition, the receptors on the cell surface of tumor cells cannot always be clearly distinguished from those of healthy body cells. Depending on the type of cancer, it could be difficult to find target structures that are specific to the tumor cells. "This problem is not solved with this method and one has to reckon with the fact that there are many off-targets as a result," says Raunser.

Kreitz assumes that the eCIS system can be significantly further developed in the future, for example to transport other loads such as DNA or RNA in addition to proteins and to bind them even more precisely to target cells. "We and others have shown that these types of systems are incredibly diverse in the biosphere, but they're not very well characterized," he says. "We believe that these types of systems play very important roles in biology that have yet to be explored."

Source: Joseph Kreitz (Broad Institute of MIT and Harvard, Cambridge, USA) et al., Nature, doi: 10.1038/s41586-023-05870-7

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