Quantitative microscopy-based measurements of circulating nanoparticle concentration using microliter blood volumes

Abstract

Nanoparticles (NPs) are potential drug delivery vehicles for treatment of a broad range of diseases. Intravenous (IV) administration, the most common form of delivery, is relatively non-invasive and provides (in theory) access throughout the circulatory system. However, in practice, many IV injected NPs are quickly eliminated by specialized phagocytes in the liver and spleen. Consequently, new materials have been developed with the capacity to significantly extend the circulating half-life of IV administered NPs. Unfortunately, current procedures for measuring circulation half-lives are often expensive, time consuming, and can require large blood volumes that are not compatible with mouse models of disease. Here we describe a simple and reliable procedure for measuring circulation half-life utilizing quantitative microscopy. This method requires only 2μL of blood and minimal sample preparation, yet provides robust quantitative results.

Keywords: Circulation half-life; Nanoparticles; Quantitative microscopy.

Figure 1.. Reference NP corrects for heterogeneity within and between samples

(A) Representative images of Dil (red) and DiO (green) NPs demonstrating co-localization (overlay). (B) Independent regions within one sample showing varying intensity of Dil (top) and corresponding DiO-NP (bottom) images. Quantification of mean intensity for: (C) single sample of Dil-NP at 50 pg/mL or (D) 5 simultaneously collected samples from a single mouse before (black circles) and after referencing (gray squares); lines connect identical images. Values are normalized to the mean of their respective group (referenced or unreferenced). Scale bars represent 100 μm.

Figure 2.. Quantitative microscopy recapitulates expected concentration in vivo

Unreferenced (A) and referenced (B) standard of Dil-NPs. Each data point represents an individual image; line represents linear fit. (C) Quantification of circulating NP concentration 2 min after injection; each gray dot represents an individual image (10× per mouse). Theoretical values (dashed line) were calculated by amount of NP administered divided by mouse blood volume (from animal mass).

Figure 3.. Circulation half-life measured by quantitative microscopy

(A) Percentage of NPs remaining 4.5 h after injection determined by referenced microscopy or microplate. Dashed lines connect samples from the same animal. Solid black line depicts mean for 6 animals. (B, C) Circulation half-life measured by referenced microscopy (gray circles) displayed on a linear (B) and log/log plot (C). Line through data shows fit to a bi-exponential curve. Black X depicts value measured for microplate from panel A. All error bars represent standard deviation.