Abstract
Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed. Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStream(X) system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.
Highlights
Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells[1,2,3,4,5]
Studies have shown that biodegradable nanoparticles based on poly(lactic-co-glycolic acid (PLGA) or polyanhydrides can be used to deliver encapsulated antigens or drugs to target cells
Conventional microscopy and flow cytometry have been used for quantifying particle uptake; their respective limitations with high throughput and resolution call for alternative approaches to study internalization
Summary
1. Fabricate 1% FITC-loaded nanoparticles as described previously[11]. Particles are fabricated by polyanhydride anti-solvent nanoencapsulation, in which the polymer is dissolved in methylene chloride (4 °C at a concentration of 25 mg/mL) and precipitated in pentane (-30 °C at a ratio 1:200 methylene chloride: pentane). After evaporating any residual solvent, weigh the dried polyanhydride nanoparticles using sterilized weigh paper. 2. Add 5 mg of nanoparticles to 0.5 mL of cold phosphate buffered saline (PBS, calcium and magnesium free, pH 7.4) in a 1.5 mL microcentrifuge tube and keep it on ice until the nanoparticles are added to the RAW 264.7 cells. 3. Sonicate the nanoparticle suspension (while keeping on ice) using an ultrasonic liquid processor fitted with a microtip for approximately 25 s at 4 to 6 joules
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