Quantification of nanoparticle endocytosis based on double fluorescent pH-sensitive nanoparticles

Area of Science:

• Biomaterials Science
• Nanotechnology
• Cell Biology

Background:

• Amorphous silica nanoparticles offer inertness and stability for biomedical applications.
• Investigating nanoparticle uptake and biological activity is critical for nanomedicine development.
• Assessing the safety and cellular interactions of silica nanoparticles is essential before clinical use.

Purpose of the Study:

• To adapt a method for quantifying nanoparticle endocytosis using pH-sensitive, dual-fluorescent particles.
• To evaluate the endocytosis of 60 nm silica nanoparticles in RAW 264.7 macrophages.
• To compare the cellular uptake and biological impact of nanoparticles versus submicronic particles.

Main Methods:

• Development of silica nanoparticles incorporating FITC (green fluorescence at physiological pH) and pHrodo™ (red fluorescence increases with acidification).
• Utilizing time-lapse microscopy and fluorimetry to quantify nanoparticle endocytosis by macrophages.
• Assessing cytotoxicity, pro-inflammatory response, and oxidative stress induced by the particles.

Main Results:

• The dual-fluorescent method successfully distinguished between extracellular and intracellular nanoparticles based on pH-dependent fluorescence.
• 60 nm silica nanoparticles exhibited higher uptake and reactivity in RAW 264.7 macrophages compared to 130 nm submicronic particles.
• The developed method for endocytosis quantification was validated.

Conclusions:

• The pH-sensitive dual-fluorescent nanoparticle system is effective for quantifying cellular endocytosis.
• Nanoparticle size influences cellular uptake and biological reactivity, with smaller particles showing greater interaction.
• This validated method advances the safety evaluation of silica nanoparticles for nanomedicine.