Engineered exosomes deliver structurally optimized toad BAX to reactivate mitochondrial apoptosis in colorectal cancer

To evaluate the therapeutic potential of toad BAX from Xenopus laevis in colorectal cancer (CRC) and its delivery via engineered exosomes.

Functional Activity Evaluation: Assessed the activity of toad BAX in human CRC cell lines and mouse models.
Binding Quantification: Measured direct binding of toad BAX to human BCL-2 using microscale thermophoresis.
Mechanism Elucidation: Investigated the mechanism of action through JC-10 staining, subcellular fractionation, and liposome permeabilization assays.
Mutagenesis: Created a triple mutant of BAX to enhance binding affinity to BCL-2.
Exosome Packaging: Packaged optimized BAX into engineered exosomes for targeted delivery and assessed anti-tumor efficacy.

Toad BAX binds to human BCL-2 with a dissociation constant (Kd) of 12.7 ± 1.9 µM.
Toad BAX triggers mitochondrial outer membrane permeabilization, cytochrome c release, and caspase activation.
The triple mutant of BAX showed enhanced BCL-2 affinity and superior anti-tumor activity compared to wild-type BAX.
Exosome-mediated delivery of optimized BAX effectively targeted CRC cells and inhibited tumor growth in various models.

The study is preclinical and requires further validation in clinical settings.
Potential off-target effects and long-term safety of exosome-mediated delivery need to be assessed.