Evaluating Endosomal Escape of Caspase-3-Containing Nanomaterials Using Split GFP

Area of Science:

• Biotechnology
• Nanomedicine
• Cell Biology

Background:

• Biologics require intracellular delivery for efficacy, often using nanocarriers that enter cells via endocytosis.
• Endosomal escape is a critical bottleneck, limiting therapeutic potential due to inefficient release from endosomes.
• Current methods for assessing cytosolic delivery are often inaccurate, hindering the development of effective delivery systems.

Purpose of the Study:

• To develop and validate a quantitative assay for assessing endosomal escape efficiency.
• To systematically investigate factors influencing nanocarrier endosomal escape, including material properties and surface modifications.
• To guide the development of optimized nanocarrier systems for enhanced intracellular protein delivery.

Main Methods:

• Utilized a split-complementation endosomal escape (SEE) assay to quantify cytosolic caspase-3 levels.
• Evaluated polymer nanogels and mesoporous silica nanoparticles as delivery vehicles.
• Investigated the impact of polymer composition, architecture, hydrophobicity, and surface functionalization on endosomal escape.

Main Results:

• Nanogel functionalization with cationic and pH-sensitive peptides significantly enhanced endosomal escape.
• Functionalized nanogels showed increased nanocarrier accumulation per endosome.
• Polymer structure had minimal impact on endosomal escape efficiency.

Conclusions:

• The SEE assay provides a robust framework for evaluating protein delivery vehicles and assessing endosomal escape.
• Surface functionalization of nanogels is a key strategy for improving cytosolic delivery of therapeutic proteins.
• This approach can be adapted for various protein cargos and delivery systems to optimize intracellular targeting.