New ‘Trojan horse’ drug delivery system uses protein-based microdroplets

Scientists at Nanyang Technological University in Singapore (NTU Singapore) have developed a new method of delivering drugs into human cells using large biological molecules, first enclosing them in a microdroplet at protein base.

This discovery promises to be faster, safer, more effective and better suited to gene therapy, cancer treatment and vaccine delivery, including mRNA-based vaccines such as those currently used for Covid-19 vaccinations by Pfizer and Moderna.

These microdroplets, made up of small proteins called peptides, can contain large biomacromolecules that carry drugs inside. In doing so, they allow these biological molecules to enter cells, which molecules cannot do on their own.

Biomacromolecules are large biological molecules such as nucleic acids (DNA, mRNA), proteins and carbohydrates. They are of great research interest as drug carriers because they can transport a large amount of drugs, are non-toxic, able to target specific sites, and do not trigger the body’s immune response. This makes them preferable and advantageous over the synthetic supports currently used in the market.

However, their large size and inability to cross the cell membrane have prevented them from being widely used clinically.

Now the NTU research team, led by Professor Ali Miserez from the School of Materials Science and Engineering and the School of Biological Sciences, has shown in laboratory experiments that their method of enclosing first drug-carrying biomacromolecules in protein-based microdroplets allows them to reliably and efficiently enter cells, overcoming the primary challenge of cell entry.

“Biomacromolecules are promising therapeutic prospects for the treatment of various diseases because they have high potency, specificity and are very safe,” Professor Miserez said. “Despite this broad potential, biomacromolecules suffer from a major drawback: they are impermeable to the cell membrane and therefore cannot enter the cell on their own. They need help, which is where our platform gets ready.”

The results were published in the scientific journal natural chemistry in February. The study was funded by a Tier 3 grant from the Department of Education.

The research team has filed two patents based on their published study and is working to commercialize their drug delivery platform method through NTUitive, the company’s innovation and enterprise company. ‘University.

Development of the team’s new drug delivery system is aligned with NTU’s commitment to innovation in its recently announced 2025 strategic plan, which aims to translate research into products and outcomes that improve quality of life. .

A new delivery system bypasses the cell membrane

Researchers synthesized a peptide derived from squid beak to form the microdroplet due to its biological origin, high efficiency in molecule storage and low toxicity. They were then able to trap biomacromolecules inside through a process called liquid-liquid phase separation (LLPS).

This LLPS process, similar to how oil and water can mix while easily separating into two separate liquids, forms what is called a coacervate.

This coacervate is able to fuse into the cell membrane, although the exact reason for this is currently unknown. “Presumably the liquid-like properties of coacervates obtained via the liquid-liquid phase separation process are critical in their ability to cross the cell membrane, although the precise mechanism of entry is still unclear and is currently unclear. ‘study,’ said the paper’s first author, Yue Sun, a doctoral student at NTU.

Basically, this discovery allows biomacromolecules to avoid endocytosis – the process by which cells allow foreign substances to enter by surrounding them with a protective membrane.

Traditional methods of drug delivery cannot cross the cell membrane without first being taken up by the cell and enveloped in a “bubble” of cell membrane, or endosome. Therefore, these types of drug packages must also be coded with additional instructions to “escape” the endosome in order for them to efficiently deliver drugs into the cell.

The team’s coacervates are able to smoothly cross the cell membrane without triggering endocytosis. Once inside the cell, the carrier droplets disintegrate and release the biomacromolecules to do their job of treating various diseases, including cancer and metabolic diseases.

“You can think of these droplets as molecular ‘Trojan horses’: they trick cells into letting them in, and once inside they deliver the biomacromolecular ‘soldiers’ that target disease,” said Professor Miserez. .

Bypassing endocytosis is crucial because it reduces the effectiveness of drugs. Since the peptide microdroplets developed by the NTU team can bypass this process to enter the cell unimpeded, their drug cargo can work at full strength, Professor Miserez said.

A delivery system for a variety of drugs

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

The protein-based cargo not only managed to enter and be released into the cells, but also maintained its bioactivity and efficacy. The team found that the cell-entry process had a 99% success rate compared to the 50-70% of currently commercially available synthetic media.

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 distribution systems must be created separately for different types of drugs.

“Using our peptide droplets as a drug delivery system eliminates the need to manufacture drug carriers that must escape from the endosome to deliver their cargo,” said Professor Miserez. “Additionally, the disadvantage of these drug carriers is that they must also be tailored to the particular drug being used or administered. Such manufacturing methods can be complex, time-consuming, and often contain organic solvents that reduce bioactivity and the effectiveness of the drug shipment.”

“However, our peptide droplets can function as a universal delivery system without requiring individual adjustment. A delivery system for a range of proteins of different sizes, from large to small, and which can transport both proteins positively and negatively charged. , is very appealing,” Professor Miserez added.

This discovery may lead to better targeted drug delivery systems that are cheaper, safer and more effective.

The potential future of drug delivery

The researchers were also able to deliver mRNA molecules into cells with this method. This opens up 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 various diseases such as cancer. and metabolic and infectious diseases,” Professor Miserez said. .

Independently commenting on the study, Dr Mahmood Ahmed, Chief Scientific Officer of artificial intelligence drug development company Biotech Talo Labs, said: “The exciting findings reported by NTU scientists are paving the way to fill some key gaps in delivering a range of therapeutic modalities to the site of action.The data reported here demonstrates the potential of these biocompatible coacervates to cross cell membranes and deliver diverse sets of large molecular entities, with the ability to tune and control payload release. Continued development of this discovery will further strengthen the translational utility of these unique coacervates and establish a transformational delivery technology platform.”

The team has filed two patents related to the study. The first patent is the team’s method for preparing microdroplet peptides to function as drug carriers. The other patent relates to the method of allowing microdroplets to enter the cells and then disintegrate once inside the cells so as to release the packaged drugs.

While real-world applications remain at least five years away, the scientists say their research has attracted initial interest from pharmaceutical and drug-development companies. The team is looking to deepen their research by starting animal studies this year.

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