Sequential intracellular delivery of genetic coding molecules using an acoustic electric microfluidic platform

Area of Science:

• Cell Biology
• Biotechnology
• Bioengineering

Background:

• Intracellular delivery of genetic molecules is crucial for cell biology and gene therapies.
• Current co-transfection methods face limitations in efficiency and precision due to cargo interactions.
• Next-generation cell engineering requires precise delivery of multiple genetic payloads.

Purpose of the Study:

• To develop a novel method for efficient sequential intracellular transfection of genetic molecules.
• To overcome the limitations of traditional co-transfection techniques.
• To advance cell and gene therapies reliant on multiplexed intracellular delivery.

Main Methods:

• Utilized acoustic microstreaming in a microfluidic platform (AESOP).
• Generated acoustic microstreaming vortices to trap cells and enable sequential molecule delivery without external pumps.
• Employed a combination of mechanical shearing and electric fields for transfection.

Main Results:

• Demonstrated up to a 7-fold increase in transfection efficiency compared to co-transfection.
• Sequential delivery via AESOP eliminated cargo competition, enhancing precision.
• Successfully transfected plasmid DNA and Cas9 ribonucleoprotein (RNP) sequentially.

Conclusions:

• AESOP provides an efficient and precise method for sequential intracellular transfection.
• This technology supports advancements in cancer immunotherapy, mRNA therapies, and CRISPR-Cas9 gene editing.
• The platform addresses key challenges in multiplexed intracellular delivery for therapeutic applications.