Live Cell Imaging by Förster Resonance Energy Transfer Fluorescence to Study Trafficking of PLGA Nanoparticles and the Release of a Loaded Peptide in Dendritic Cells

Abstract

Our previous study demonstrated that a selected β-lactoglobulin-derived peptide (BLG-Pep) loaded in poly(lactic-co-glycolic acid) (PLGA) nanoparticles protected mice against cow's milk allergy development. However, the mechanism(s) responsible for the interaction of the peptide-loaded PLGA nanoparticles with dendritic cells (DCs) and their intracellular fate was/were elusive. Förster resonance energy transfer (FRET), a distance-dependent non-radioactive energy transfer process mediated from a donor to an acceptor fluorochrome, was used to investigate these processes. The ratio of the donor (Cyanine-3)-conjugated peptide and acceptor (Cyanine-5) labeled PLGA nanocarrier was fine-tuned for optimal (87%) FRET efficiency. The colloidal stability and FRET emission of prepared NPs were maintained upon 144 h incubation in PBS buffer and 6 h incubation in biorelevant simulated gastric fluid at 37 °C. A total of 73% of Pep-Cy3 NP was internalized by DCs as quantified using flow cytometry and confirmed using confocal fluorescence microscopy. By real-time monitoring of the change in the FRET signal of the internalized peptide-loaded nanoparticles, we observed prolonged retention (for 96 h) of the nanoparticles-encapsulated peptide as compared to 24 h retention of the free peptide in the DCs. The prolonged retention and intracellular antigen release of the BLG-Pep loaded in PLGA nanoparticles in murine DCs might facilitate antigen-specific tolerance induction.

Keywords: Förster resonance energy transfer; cyanine-3; cyanine-5; dendritic cells; peptide delivery; poly(lactic-co-glycolic acid) nanoparticles.

Figure 1

Structures of parent β-lactoglobulin-derived peptide (A) and Pep-Cy3 (B), UPLC chromatograms of Pep-Cy3 (C) used for determination of purity using UV detection at 210 nm (blue curve) and fluorescence detection at λ_ex/em = 555/570 nm (red curve) and (D) molecular mass of the synthesized Pep-Cy3 by mass spectrometry (Theoretical mass = 2343.3 g/mol; mass found (M2+) = 1172.6 and (M3+) = 782.1; and calculated mass = 2343.2 g/mol or 2343.3 g/mol). Cy5 was conjugated to PLGA-COOH by using carbodiimide/NHS chemistry as described previously [29] (Figure 2A). Gel permeation chromatography (GPC) of the synthesized PLGA-Cy5 showed overlapping peaks at the same retention time when using the refractive index (RI) and UV detection at 650 nm (Figure 2B,C, respectively), while no free dye was detected (Figure 2C), which demonstrates the successful conjugation of the acceptor dye Cy5 to PLGA-COOH.

Figure 2

Synthesis and characterization of PLGA-Cy5. (A) Synthesis route of PLGA-Cy5 by conjugation of Cy5 amine to PLGA-COOH using carbodiimide/NHS chemistry. GPC analysis of PLGA-Cy5 conjugate (blue) and free Cy5 (red) by using refractive index (RI) detection (B) and UV detection at 650 nm (C).

Figure 3

Fluorescence spectra in PBS (pH 7.4) at λ_ex = 555 nm: 0.5 mg/mL 9% PLGA-Cy5 NP (containing 9 wt% labeled polymer), 1% Pep-Cy3 NP (containing 1 wt% feed Pep-Cy3), and FRET NP (containing 1 wt% feed Pep-Cy3 and 1.5–9 wt% PLGA-Cy5).

Figure 4

Fluorescence intensity of 0.2 mg/mL 1 % Pep-Cy3 NP, 9% PLGA-Cy5 NP and FRET NP 0.8:1 (1 wt% Pep-Cy3:9 wt% PLGA-Cy5) incubated in PBS (pH 7.4) for 144 h at 37 °C. From left to right: fluorescence intensity of (A) Cy3 (λ_ex/em = 555/570 nm), (B) Cy5 (λ_ex/em = 646/662 nm), and (C) FRET (λ_ex/em = 555/662 nm).

Figure 5

Fluorescence intensity of 0.2 mg/mL 1% Pep-Cy3 NP, 9% PLGA-Cy5 NP, and FRET NP 0.8:1 (1 wt% Pep-Cy3:9 wt% PLGA-Cy5), incubated for 6 h at 37 °C in FaSSGF (pH 1.6, with pepsin) (A–C) and in FaSSGF (pH 1.6, without pepsin) (D–F). From left to right: fluorescence intensity of Cy3 (λ_ex/em = 555/570 nm), Cy5 (λ_ex/em = 646/662 nm), and FRET (λ_ex/em = 555/662 nm).

Figure 6

Flow cytometry data of human immature moDC after 2.5 h incubation with 0.06 mg/mL 2% Pep-Cy3 NP at 37 °C. Percentages of viable cells (A), CD11c+CD14- human moDC viable cells (B), and cellular uptake (Cy3-positive CD11c+CD14- cells) (C) were gated using medium-treated human immature moDC as control in the flow cytometry analysis. Data are presented as mean ± SEM, n = 3. Human immature moDC were derived and cultured from three different donors.

Figure 7

Representative fluorescent microscopy images of cellular uptake of 2% Pep-Cy3 NP (0.06 mg/mL) by day 7 human moDC: medium-treated control (A) and internalized 2% Pep-Cy3 NP after (B) 2.5 and (C) 18.5 h of incubation at 37 °C; bars indicate 20 μm.

Figure 8

(A) Experimental scheme and (B) cellular images of internalized fluorescent nanoparticles and free Pep-Cy3 by murine DC 2.4. Cells were washed and live-imaged over 144 h after 2.5 h of incubation of DC 2.4 cells with medium, 9% PLGA-Cy5 NP, 1% Pep-Cy3 NP, FRET NP 0.8:1 (1% Pep-Cy3:9% PLGA-Cy5), or 1 µg/mL free Pep-Cy3. Shown are representative merged confocal fluorescence microscopy images (B) of nuclei (Hoechst, blue, λ_ex = 405 ± 5 nm), Cy3 (orange, λ_ex = 561 ± 2 nm), Cy5 (red, λ_ex = 640 + 4/−5 nm), and FRET (λ_ex = 561 ± 2 nm) taken 2, 24, 96, and 144 h after the washing step; bars indicate 10 μm. (C) Fluorescence from the FRET NP 0.8:1 and free Pep-Cy3 treated DC 2.4 cells in confocal image of Cy3 alone and merged confocal images of Cy3 (orange), nuclei (Hoechst, blue), and bright field (BF, confocal path) 2 (left panel) and 24 h (right panel) after the washing step, respectively.

Figure 9

Intracellular localization of FRET NP after internalization by DC 2.4 cells. According to the scheme of Figure 8A, DC 2.4 cells were incubated with FRET NP 0.8:1 (1% Pep-Cy3:9% PLGA-Cy5) for 2.5 h and subsequently washed. Shown are representative confocal fluorescence microscopy images of DC 2.4 cultured with FRET NP (A) 2, (B) 24, (C) 96, and (D) 144 h after the washing step; bars indicate 10 μm. Colocalizations of Cy3 (orange, λ_ex = 561 ± 2 nm), Cy5 (red, λ_ex = 640 + 4/−5 nm), and FRET (purple, λ_ex = 561 ± 2 nm) fluorescence are indicated with the yellow arrows and numbers.