Structure and Dynamics of Thermosensitive pDNA Polyplexes Studied by Time-Resolved Fluorescence Spectroscopy

Area of Science:

• Polymer Chemistry
• Biomaterials Science
• Gene Delivery Systems

Background:

• Stimuli-responsive polymers are promising for nucleic acid delivery.
• Understanding polymer structure-property relationships is crucial for optimizing polyplex formation and stability.
• Thermosensitive polymers offer tunable characteristics for controlled release applications.

Purpose of the Study:

• To investigate the structure and dynamics of thermosensitive polyplexes using time-resolved fluorescence spectroscopy.
• To synthesize and characterize novel heterofunctional polymers for nucleic acid delivery.
• To elucidate the influence of polymer architecture and environmental factors on polyplex properties.

Main Methods:

• Synthesis of linear triblock copolymers (NPD) using a novel heterofunctional PEG macroinitiator (ATRP/RAFT).
• Labeling of polymer chain-ends (Cy3) and plasmid DNA (pDNA) (FITC) for FRET studies.
• Time-resolved fluorescence resonance energy transfer (FRET) to track polyplex structure, dynamics, and polymer exchange at different temperatures and N/P ratios.

Main Results:

• Successful synthesis of thermosensitive (PNIPAM) and cationic (PDMAEMA) triblock copolymers (NPD).
• FRET confirmed stable polyplex formation at both 4 and 37 °C, indicating thermosensitive block integration did not compromise structure.
• NPD and PD polyplexes exhibited less dense cores and dynamic behavior with polymer exchange between core and shell, unlike static homopolymer polyplexes.

Conclusions:

• Thermosensitive triblock copolymers form dynamic, less dense pDNA polyplexes with tunable properties.
• The dynamic nature of these polyplexes facilitates polymer exchange, potentially improving pDNA release and transfection efficiency.
• This research provides fundamental insights for designing advanced polymer-based gene delivery systems.