Evaluation of Complex Formation between Aminated Polyrotaxane and Proteins Using FRET

Area of Science:

• Biomaterials Science
• Cellular Biology
• Drug Delivery Systems

Background:

• Aminated polyrotaxane (amino-PRX) is an efficient intracellular carrier for biopharmaceuticals.
• The detailed intracellular behavior of amino-PRX and its complexes, especially for antibody delivery, requires further investigation.

Purpose of the Study:

• To visualize the intracellular distribution of amino-PRX carriers and their complexes using fluorescence.
• To examine the intracellular behavior of amino-PRX complexes and assess their potential for antibody delivery.

Main Methods:

• Preparation of fluorescently capped amino-PRX (DET-PRX-FAM) using α-cyclodextrin and polyethylene glycol.
• Utilizing Förster resonance energy transfer (FRET) to observe complex formation between the carrier and model proteins (phycocyanin, IgG).
• Confocal microscopy in HeLa cells to visualize carrier, drug, and FRET fluorescence.

Main Results:

• DET-PRX-FAM (2 kDa) showed lower cytotoxicity than the 20 kDa variant in HeLa cells.
• FRET-derived fluorescence confirmed complex formation between DET-PRX-FAM (2 kDa) and model proteins.
• Intracellular visualization of carrier, protein, and FRET signals demonstrated successful complex delivery and distribution.

Conclusions:

• Fluorescently capped amino-PRX enables visualization of intracellular carrier and complex dynamics.
• The study validates amino-PRX as a potential carrier for intracellular antibody delivery.
• This approach enhances understanding of carrier-mediated intracellular transport mechanisms.