'Trojan Horse' drug delivery system developed

Scientists from Nanyang Technological University, Singapore (NTU Singapore) have created a novel method of delivering drugs into human cells using large biological molecules, by first encasing them in a protein-based microdroplet. These microdroplets, made up of small proteins named peptides, can encase large biomacromolecules that carry drugs inside them, thus allowing these biological molecules to enter cells — something the molecules cannot do by themselves.

Biomacromolecules are large biological molecules such as nucleic acids (DNA, mRNA), proteins and carbohydrates. They are of great research interest as drug carriers, as they can carry a large amount of drugs, are non-toxic, are able to target specific sites and do not trigger the body’s immune response. This makes them preferable and advantageous over synthetic carriers currently used in the market; however, their large size and inability to pass through the cell membrane have held them back from widespread clinical use.

“Biomacromolecules are promising therapeutic prospects for the treatment of various diseases as they have high potency, specificity and are very safe,” said research leader Professor Ali Miserez. “Despite this broad potential, biomacromolecules suffer from a major drawback: they are impermeable to the cell membrane and thus cannot penetrate the cell by themselves. They need help, which is where our platform comes into place.”

The NTU researchers have now shown in lab experiments that their method of first encasing drug-carrying biomacromolecules in protein-based microdroplets lets them reliably and effectively enter cells. Their findings have been published in the journal Nature Chemistry.

The researchers synthesised a peptide derived from squid beak to form the microdroplet due to its biological origin, high efficiency in storing molecules and low toxicity. They were then able to entrap biomacromolecules inside it through a process called liquid-liquid phase separation (LLPS).

This LLPS process, similar to how oil and water can mix together yet easily separate into two distinct liquids, forms what is known as a coacervate. This coacervate is able to merge into the cell membrane, although the exact reason why is currently unknown.

“Presumably, the liquid-like properties of coacervates achieved via the liquid-liquid phase separation process is critical in their ability to cross the cell membrane, although the precise entry mechanism is still unclear and currently under investigation,” said NTU PhD student Yue Sun, first author on the study.

Traditional drug delivery methods cannot cross into the cell membrane without first being caught by the cell and wrapped within a ‘bubble’ of cell membrane, or endosome; therefore, these types of drug packages must also be encoded with further instructions to ‘escape’ the endosome in order for them to efficiently release the drugs within the cell. The team’s coacervates are able to smoothly cross the cell membrane without triggering the bubble.

Once inside the cell, the carrier droplets disintegrate and release the biomacromolecules to do their job of treating various illnesses, including cancer and metabolic diseases, with their drug cargo operating at full strength. Prof Miserez said, “You can think about these droplets as molecular ‘Trojan Horses’: they trick the cells into letting them enter, and once inside they deliver the biomacromolecular ‘soldiers’ that target the disease.”

In lab experiments, the team was able to successfully deliver fluorescent proteins, which are commonly used to demonstrate the efficiency of drug carriers, as well as the protein drug saporin through this method. By themselves, these proteins cannot enter the cell.

The protein-based cargo was not only able to successfully enter and be released into the cells, but also maintained its bioactivity and efficacy. The team discovered that the cell entry process had a 99% success rate compared to the 50–70% of current commercially available synthetic carriers.

The research team demonstrated that a wide range of biomacromolecules can be loaded into their microdroplets, from small peptides to enzymes to mRNAs. This makes it viable as a universal drug delivery system. All current delivery systems have to be created separately for different types of drugs.

“Using our peptide droplets as a drug delivery system does away with the need to fabricate drug carriers that have to escape the endosome in order to deliver their cargo,” Prof Miserez said. “Furthermore, the drawback of such drug carriers is that they also have to be tailored to the particular drug that is being used or delivered. Such fabrication methods can be complex, time-consuming and often contain organic solvents that reduce the bioactivity and efficiency of the drug cargo.

“However, our peptide droplets can work as a universal delivery system without the need for individual adjustment. One delivery system for a whole range of proteins of various sizes, from big to small, and that can carry both positively and negatively charged proteins, is very appealing.”

This discovery can lead to better targeted drug delivery systems that are cheaper, safer and more effective. The researchers were also able to deliver mRNA molecules into cells with this method, opening the possibility of using mRNA in gene therapy — a possible treatment for serious diseases such as cancer, genetic disorders or infectious diseases.

“The versatility of drug delivery and subsequent release allows these coacervates to deliver a single or a combination of macromolecular drugs, making this delivery platform very promising for the treatment of a variety of illnesses such as cancer, and metabolic and infectious diseases,” Prof Miserez said.

The researchers have filed two patents related to the study — one for their method of preparing the peptides into microdroplets for them to function as drug carriers, the other for their method allowing the microdroplets to enter cells and then disintegrate once inside the cells so as to release the packaged drugs — and are now looking to further their research by beginning animal studies this year. While real-world applications remain at least five years away, the scientists say their work has garnered initial interest from pharmaceutical and drug development companies.

Image credit: ©stock.adobe.com/au/epic_images

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