A novel method to enable intracellular delivery

Medical research has made incredible strides in recent decades, with scientists uncovering new insights at the cellular level. This deeper understanding has led to smaller, more personalised treatments for conditions once considered untreatable. With increased knowledge of genetic conditions and cellular responses, a significant amount of research is now focussed on effectively altering them at a fundamental level.

For advanced treatments to work, particularly those performed at the cellular level, therapeutic agents must be delivered directly into the target cells. However, cells have membranes that block unwanted substances, presenting a significant hurdle for effective treatment delivery. There are two main approaches to overcome this. The first is to use carriers to help bring the agents through the cell membranes, while the second is to create temporary holes (known as transient nanopores) in the membrane to allow entry.

“Previous research tells us that the fast deformation of cells and the recovery of cells are key to enabling high efficiency for intracellular delivery,” said Associate Professor Ai Ye from the Singapore University of Technology and Design (SUTD).

However, existing methods using both approaches, such as electroporation and lipofection, can either create irreversible cell damage or cause high toxicity. Without a suitable method of facilitating the delivery of therapeutic agents into target cells, new therapeutic techniques such as CAR-T therapy and gene editing have mostly been restricted in their applications.

Tapping into their expertise on single cell level deformation using microfluidic technology, Assoc Prof Ai and his team explored the use of stainless steel filters and viscoelastic fluids to mechanically create transient nanopores. They proposed a novel technique that could open new doors in research on intracellular delivery in the paper, “Enhanced intracellular delivery via stainless steel filters and viscoelastic fluids: A high-efficiency alternative to conventional transfection”.

Assoc Prof Ai initially planned to use micro-constrictions in microfluidic chips and viscoelastic fluids to enable intracellular delivery—a technique he had used in previous research. After consulting other research groups and companies in the Accelerating Research & Innovation for SUTD Entrepreneurs (ARISE) programme, he realised he needed to find a replacement for the microfluidic chip to improve the throughput.

This led to the idea of using stainless steel filters in place of silicon-based wafers, which are used in traditional microfluidic devices. Stainless steel filters have apertures larger than the average cell diameter, which helps to prevent potential clogging and, thus, minimises the possibility of cell death.

In the study, the stainless steel filters served as a template for ensuring consistently-sized apertures, upon which the viscoelastic fluids exerted force to deform and create transient nanopores on the cells mechanically. A test of the team’s prototype showed that this method can achieve delivery efficiencies as high as 94.7 percent, while also ensuring that the cells are still mostly viable.

Assoc Prof Ai explained that these could be achieved due to the speed of the deformation, which was in the range of a few microseconds. “Such a small deformation time enables fast generation of nanopores on cell membranes and fast intracellular-extracellular volume exchange,” he said. “Therefore, the damage to cells is reduced, especially when it is compared to electroporation.”

While the results are promising, Assoc Prof Ai believes that more work needs to be done. He said “We need to test more cells to prove the universality of this method, which still needs a lot more effort.”

In addition, his team plans to build a standalone prototype that will be more accessible to commercial laboratories that may not have microfabrication capabilities. Overall, Assoc Prof Ai is optimistic about his team’s technique's potential to advance cellular engineering research.

“We hope this technology can lower the requirements for researchers to participate in the improvement of CAR-T therapy and gene editing,” he mused. “We believe that this technology has high universality and uniformity, which can simplify the cell engineering process.”

 

Acknowledgements

This work was supported by the SUTD Growth Plan Grant for Healthcare (PIE-SGP-HC-2022-02) and the SUTD Gap Funding (GAP-035) awarded to Y.A.

 

Reference

Enhanced intracellular delivery via stainless steel filters and viscoelastic fluids: A high-efficiency alternative to conventional transfection. Analytical Chemistry, https://doi.org/10.1021/acs.analchem.5c02251

 

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