Protein Encapsulation: A Nanocarrier Approach to the Fluorescence Imaging of an Enzyme-Based Biomarker

Abstract

Here, we report a new pentafluoropropanamido rhodamine fluorescent probe (ACS-HNE) that allows for the selective detection of neutrophil elastase (NE). ACS-HNE displayed high sensitivity, with a low limit of detection (<5.3 nM), and excellent selectivity toward elastase over other relevant biological analytes and enzymes. The comparatively poor solubility and cell permeability of neat ACS-HNE was improved by creating an ACS-HNE-albumin complex; this approach allowed for improvements in the in situ visualization of elastase activity in RAW 264.7 cells relative to ACS-HNE alone. The present study thus serves to demonstrate a simple universal strategy that may be used to overcome cell impermeability and solubility limitations, and to prepare probes suitable for the cellular imaging of enzymatic activity in vitro.

Keywords: BSA-based nanocarrier; cell imaging; elastase detection; fluorescence imaging; nanocarrier-based enzyme detection.

Scheme 1

Development of a rhodamine fluorescent probe for the detection of neutrophil elastase (NE).

Figure 1

Fluorescence emission spectra of ACS-HNE (5 μM) over time (24 h) in buffered elastase enzyme (2 μM) solution (in PBS buffer, pH = 7.40); λ_ex = 496 nm.

Figure 2

Widefield fluorescence micrographs of RAW 264.7 cells incubated with ACS-HNE (20 μM) before (–) or after (+) addition of human NE (HNE, 4 ng μL⁻¹, 100 μL); Excitation and emission wavelengths for ACS-HNE are 460–490 nm and 500–550 nm, respectively. The insert shows magnified sections of the corresponding fluorescence micrograph.

Figure 3

Widefield fluorescence micrographs of RAW 264.7 cells incubated with ACS-HNE/BSA (20/100 μM) before (–) or after (+) addition of human NE (HNE, 4 ng μL⁻¹, 100 μL); Excitation and emission wavelengths for ACS-HNE are 460–490 nm and 500–550 nm, respectively. The inset shows magnified sections of the corresponding fluorescence micrograph.