Fluorescence Microscopy Imaging Calibration for Quantifying Nanocarrier Binding to Cells During Shear Flow Exposure

Abstract

Targeted drug delivery is a fast growing industry in healthcare and technologies are being developed for applications utilizing nanocarriers as vehicles for drug transport. As the size scale of these particles becomes further reduced, advanced fluorescence microscopy and image analysis techniques become increasingly important for facilitating our understanding of nanocarrier binding and avidity, thereby establishing the basis for nanocarrier design optimization. While there is a significant body of published work using nanocarriers in vitro and in vivo, the advent of smaller particles that have typically been studied (~500 nm) limits the ability to attain quantitative measurements of nanocarrier binding dynamics since image acquisition and analysis methods are restricted by microscopy pixel size. This work demonstrates the use of a novel calibration technique based on radioisotope counting and fluorescence imaging for enabling quantitative determination of nanocarrier binding dynamics. The technique is then applied to assess the temporal profile of endothelial cell binding of two antibody targeted nanocarrier types in the presence of fluid shear stress. Results are provided for binding of nanoparticles smaller than a microscopy image pixel.

Keywords: Binding; Calibration; Fluorescence Microscopy; Image Analysis; Nanocarrier; Nanogel; Quantitative Binding; Shear Stress; Targeted Drug Delivery.

Figure 1

DLS charts for (a) NGs and (b) PS beads.

Figure 2

PS beads binding at 0.1 Pa and 0.5 Pa over the 3 hour experiment. Seeding concentration of PS beads: 1.4E11 particles/mL.

Figure 3

NG binding at 0.1 Pa and 0.5 Pa over the 4 hour experiment. Seeding concentration of NGs: 7.6E10 particles/mL.

Figure 4

PS bead binding over 3 hours for high and low NC concentrations at (a) 0.1 Pa shear stress and (b) 0.5 Pa shear stress.

Figure 5

NG binding over 4 hours comparing TNF-α activated cell conditions to quiescent cell conditions at (a) 0.1 Pa shear stress and (b) 0.5 Pa shear stress.

Figure 6

NG binding curves comparing specific (R6.5 conjugation) to non-specific (IgG conjugation) binding under quiescent cell conditions at 0.5 Pa.

Figure 7

A NP can reside in any position relative to pixel arrangement (only 4 pixels shown for representational purposes) and being in the size range of 1 pixel, can confound results. Pixels will light up (give a signal, represented as yellow in figure) even if a portion of the NP falls within their “borders” (pixel sizes depend on objectives; in current work, size was calculated to be ~ 161.2 nm for a 40× oil immersion lens used in an Olympus IX 70 inverted microscope).

Figure 8

Bland-Altman analysis to validate FL counts and RL measure made with PS bead experiments.

Figure 9

Fluorescence images of NGs over HUVECs at 15 minutes (left), 60 minutes (center) and 240 minutes (right).