Absolute quantitation of sub-micrometer particles in cells by flow cytometry

Area of Science:

• Nanotechnology
• Cell Biology
• Analytical Chemistry

Background:

• Accurate nanoparticle (NP) dose determination is crucial for pharmacological and toxicological studies.
• Lack of absolute quantitation in NP uptake studies hinders inter-study comparisons.
• Existing methods for NP quantitation are often complex or lack standardization.

Purpose of the Study:

• To develop and validate a flow cytometry (FC) based method for absolute quantitation of fluorescent NP uptake in cells.
• To establish a routine for rapid quantitative counting of endocytosed NPs.
• To demonstrate the utility of the method by measuring the uptake efficiency of specific NPs.

Main Methods:

• Utilized a standard non-scanning flow cytometer to analyze fluorescent NPs.
• Developed a method to obtain mean fluorescence intensity per particle and particle concentration (NPs/μL).
• Validated the FC method against confocal laser scanning microscopy for quantitative counting of endocytosed NPs in HeLa cells.

Main Results:

• Successfully developed a rapid quantitative counting routine for endocytosed NPs using FC.
• Validated the accuracy of the FC method through comparison with confocal microscopy.
• Demonstrated very low cellular uptake efficiency (approx. 10^-3%) for negatively charged poly(methyl methacrylate) NPs.

Conclusions:

• The presented FC approach enables absolute quantitation of NP uptake in cells.
• This method facilitates standardized and comparable NP uptake studies in pharmacology and toxicology.
• The findings highlight the low cellular uptake of specific negatively charged NPs, informing future material design and risk assessment.