High-throughput quantitative microscopy-based half-life measurements of intravenously injected agents

Abstract

Accurate analysis of blood concentration and circulation half-life is an important consideration for any intravenously administered agent in preclinical development or for therapeutic application. However, the currently available tools to measure these parameters are laborious, expensive, and inefficient for handling multiple samples from complex multivariable experiments. Here we describe a robust high-throughput quantitative microscopy-based method to measure the blood concentration and circulation half-life of any fluorescently labeled agent using only a small (2 µL) amount of blood volume, enabling additional end-point measurements to be assessed in the same subject. To validate this method, we demonstrate its use to measure the circulation half-life in mice of two types of fluorescently labeled polymeric nanoparticles of different sizes and surface chemistries and of a much smaller fluorescently labeled monoclonal antibody. Furthermore, we demonstrate the improved accuracy of this method compared to previously described methods.

Keywords: circulation half-life; drug delivery; nanoparticle; quantitative microscopy.

Fig. 1.

Schematic illustrating the workflow for half-life measurements and end-point analyses using high-throughput quantitative microscopy.

Fig. 2.

Tail-vein and RO injections of polymeric nanoparticles yield identical measurements of half-life. (A) Schematic of polymeric nanoparticles composed of a PLA-PEG copolymer encapsulating fluorescent dye and routes of IV administration. (B) Representative fluorescence images of dye-loaded nanoparticles at different concentrations in blood. (C) Circulation half-life measurements after IV administration of fluorescent polymeric nanoparticles using quantitative microscopy (n = 3 mice per group per time point). Error bars represent SEM. Representative end-point (D) flow cytometry histograms from homogenized livers, (E) mean fluorescence intensity (MFI) values for homogenized organs (n = 3 to 4 mice per group per time point; error bars represent SEM), and (F) IVIS analyses of NP uptake in various tissues. (Scale bars, 100 μm.)

Fig. 3.

Mouse antibodies remain in circulation for an extended time. (A) Schematic of a fluorescently labeled mouse anti-human CD45 antibody. (B) Representative phase-contrast and fluorescence images of blood with or without fluorescently labeled antibodies at different concentrations in blood. (C) Circulation half-life measurements after IV administration of fluorescently labeled antibodies using quantitative microscopy (n = 3 mice per group per time point). Error bars represent SEM. Representative end-point (D) flow cytometry histograms from homogenized livers, (E) MFI values for homogenized organs (n = 3 to 4 mice per group per time point; error bars represent SEM), and (F) IVIS analyses of NP uptake in various tissues. (Scale bars, 100 μm.)

Fig. 4.

Nanoparticle size, surface chemistry, and multiplexed administration dictate circulation half-life and biodistribution. (A) Schematic of PLGA and PLA-PEG polymeric nanoparticles encapsulating fluorescent dye. Circulation half-life measurements following IV administration of (B) DiD-loaded PLGA nanoparticles and (C) DiD-loaded PLA-PEG nanoparticles in male (light blue) and female (light red) mice (n = 3 mice per group per time point), and in a multiplexed experiment (dark blue) where mice were simultaneously administered DiI-loaded PLA-PEG and DiD-loaded PLGA NPs using quantitative microscopy (n = 3 mice per group per time point). Error bars represent SEM.

Fig. 5.

A comparison of nanoparticle concentration using previous methods shows similar concentration with reduced deviation. (A) Circulation half-life measurement at a single time point (4 h) after IV administration of PLA-PEG nanoparticles using the traditional method to dissolve nanoparticles, the initial improved protocol moving to quantitative microscopy, and the high-throughput method described here (n = 6 images per group). Error bars represent SD. Images obtained through these techniques demonstrate the variation between regions imaged using a microscope slide (B and C) and the consistency obtained using a glass-bottom plate (D). (Scale bars, 100 µm.)