Integrated multiplatform method for in vitro quantitative assessment of cellular uptake for fluorescent polymer...

Area of Science:

• Biotechnology
• Nanomedicine
• Cell Biology

Background:

• Accurate quantification of nanoparticle (NP) cellular uptake is essential for developing effective drug delivery systems.
• Current methods lack the precision to determine the exact number of NPs internalized per cell.
• Novel experimental techniques are needed to overcome these limitations.

Purpose of the Study:

• To develop and validate a novel, quantitative method for assessing fluorescent NP uptake in cells.
• To determine the absolute number of NPs internalized by specific cell types.
• To investigate the influence of NP characteristics and exposure conditions on cellular internalization.

Main Methods:

• Combined flow cytometry and plate fluorimetry with cell counting for quantitative NP uptake analysis.
• Utilized biocompatible fluorescent NPs (20-300 nm, Rhodamine B dye) synthesized via emulsion free-radical polymerization.
• Calibrated microplate reader with serial NP dilutions to establish linearity between fluorescence and NP number.

Main Results:

• Established a linear relationship between average flow cytometry fluorescence and plate fluorimetry measurements.
• Quantified the absolute number of polymer NPs internalized by 4T1 mouse mammary tumor cells (10^3-10^4 NPs/cell).
• Observed intracellular NP concentrations up to 15 times higher than external concentrations.

Conclusions:

• The developed method provides a robust and quantitative evaluation of NP internalization.
• Cellular uptake is dependent on NP dimensions, surface charge, and exposure concentration.
• This technique advances the design and optimization of nanoparticle-based drug delivery systems.