Amphipathic helical peptide-based fluorogenic probes for a marker-free analysis of exosomes based on membrane-curvature sensing

Abstract

With increasing knowledge about the diverse roles of exosomes in the biological process, much attention has been paid to develop analytical methods for detection and quantification of exosomes. Immunoassays based on the recognition of exosomal protein markers by antibodies were widely used. However, considering that exosomal protein composition varies with the cell type, the protein markers should be carefully selected for a sensitive and selective analysis of target exosomes. Herein, we developed a new class of exosome-binding fluorogenic probes based on membrane curvature (MC) sensing of amphipathic helical (AH) peptides for exosome analysis without the need to use protein markers on the exosomal membranes. The C-terminal region of apolipoprotein A-I labeled with Nile red (ApoC-NR) exhibited a significant fluorescence enhancement upon selective binding to the highly curved membranes of synthetic vesicles. Circular dichroism (CD) measurements involving 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-2-dioleoyl-sn-glycerol (DOG) vesicles suggested that ApoC-NR recognizes the lipid packing defects in the surface of highly curved membranes via the hydrophobic insertion of the α-helix structure of the ApoC unit. ApoC-NR exhibited a stronger binding affinity for exosome-sized vesicles and a higher MC selectivity compared to all other previously reported peptide probes. ApoC-NR can be used in a simple and rapid "mix and read" analysis of various kinds of exosomes derived from different cell types (limit of detection: -10⁵ particles/μL) without being influenced by the variation in the expression of the surface proteins of the exosomes, which stands in sharp contrast to immunoassays.

Fig. 1. Schematic illustration of the exosome analysis with the AH peptide-based fluorescent probe ApoC-NR (cf. Fig. S1†). A helical wheel representation of ApoC sequence is also shown, wherein the hydrophobic and the polar residues are orange and blue, respectively.

Fig. 2. Fluorescence spectra of ApoC-NR in the (a) absence and presence of (b) V₁₁₀, (c) V₃₅₀, and (d) V₆₅₀. Inset: Comparison of the fluorescence response for the vesicles between ApoC-NR and a free NR molecule. F and F_V110 denote the fluorescence intensity of the probe in the presence of synthetic vesicles and V₁₁₀, respectively. Excitation, 552 nm. Analysis, 613 nm (ApoC-NR); 633.5 nm (free NR).

Fig. 3. Titration curve for the binding of ApoC-NR to V₁₁₀. [ApoC-NR] = 2.0 μM. r and r₀ denote the fluorescence anisotropy of ApoC-NR in the presence of synthetic vesicles and absence of the vesicles, respectively. The obtained curve was analyzed with the fitting equation.

Fig. 4. CD spectra of ApoC-NR in the (a) absence and presence of (b) V₁₁₀, (c) V₃₅₀ and (d) V₆₅₀. [ApoC-NR] = 2.0 μM, [total lipid] = 100 μM.

Fig. 5. Dependence of the K_d values of ApoC-NR and MARCKS-ED-NBD on the size of the synthetic vesicles.

Fig. 6. Fluorescence response of ApoC-NR (2.0 μM) for Exo-K562 (0.082–8.2 × 10⁷ particles/μL). Inset: calibration curve of Exo-K562 based on the fluorescence response (F/F₀) of ApoC-NR. F and F₀ denote the fluorescence intensity of ApoC-NR at 620 nm in the presence and absence of Exo-K562, respectively. Excitation, 552 nm.