Visualization of Apoptosis in Three-Dimensional Cell Aggregates Based on Molecular Beacon Imaging

Area of Science:

• Biotechnology
• Cell Biology
• Nanomedicine

Background:

• Visualizing apoptosis in 3D cell aggregates is challenging.
• Molecular beacons (MB) offer potential for detecting specific mRNA targets like caspase-3 for apoptosis.
• Enhancing MB delivery into cells is crucial for effective imaging.

Purpose of the Study:

• To develop and validate a method for visualizing cell apoptosis in 3D cell aggregates using molecular beacons (MB).
• To investigate the distribution and localization of apoptosis markers within 3D cell aggregates.
• To assess the efficacy of cationized gelatin nanospheres (cGNS) for MB delivery and imaging.

Main Methods:

• Incorporation of caspase-3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MB into cationized gelatin nanospheres (cGNS).
• Preparation of dual-color imaging cGNS co-incorporating both MB types (cGNS_{dual MB}).
• Incubation of MB-labeled cGNS with mouse mesenchymal stem cells and formation of 3D cell aggregates for imaging.

Main Results:

• cGNS effectively delivered MB into cells for both 2D cultures and 3D aggregates, enabling homogenous fluorescence detection.
• Dual-color imaging accurately detected Fas-mediated apoptosis in 3D aggregates.
• Apoptosis marker distribution varied based on the inducer, with caspase-3 MB localized to the aggregate surface for Fas-mediated apoptosis and interior for camptothecin-induced apoptosis.

Conclusions:

• Cationized gelatin nanospheres (cGNS) loaded with molecular beacons (MB) provide a promising system for visualizing cell apoptosis in 3D cell aggregates.
• This technique allows for non-destructive, detailed imaging of apoptotic processes within complex 3D cellular structures.
• The localization patterns observed suggest MB fluorescence can reflect the penetration dynamics of different apoptosis inducers.