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
:1. Introduction
2. Results and Discussion
2.1. Preparation and Characterization of the Cy3-Labeled β-Lactoglobulin-Derived Peptide and Cy5-Labeled PLGA
2.2. Characteristics of Single- and Dual-Labeled PLGA Nanoparticles
2.3. Optimization of FRET Efficiency of the NPs Labeled with the Cy3 and Cy5
2.4. Colloidal Stability and Time-Dependent Fluorescence of Labeled PLGA NP in PBS and Biorelevant Fasted-State-Simulated Gastric Fluid
2.5. Cellular Uptake of FRET NPs by Human Immature moDC and Their Intracellular Trafficking in Murine Dendritic Cells DC 2.4
3. Materials and Methods
3.1. Materials
3.2. Solid-Phase Peptide Synthesis and Characterization
3.3. Conjugation of Cy5 to PLGA-COOH
3.4. Preparation of Nanoparticles
3.5. Characterization of the Nanoparticles
3.6. Peptide Encapsulation Efficiency
3.7. Fluorescence Characteristics of Labeled Nanoparticles
3.8. Fluorescence Intensity of Labeled PLGA NP in PBS or Fasted-State-Simulated Gastric Fluid
3.9. Culture of Human-Monocytes-Derived Dendritic Cells (moDC)
3.10. Culture of Murine Bone-Marrow-Derived Dendritic Cell Line DC 2.4
3.11. Internalization of Pep-Cy3/NP by Human moDC Determined by Fluorescence Microscopy and Flow Cytometry
3.12. Cellular Uptake and Intracellular Trafficking of Nanoparticles by Murine DC 2.4
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Formulation (n = 1) | Nanosight Analysis 1 | Zetasizer Nano S 2 | Zeta-Potential (mV) | EE 3 (%) | LC 4 (%) | Actual Molar Ratio Pep-Cy3: PLGA-Cy5 | Yield (%) | EPR 5 (%) | |
---|---|---|---|---|---|---|---|---|---|
Dh (nm) | Dh (nm) | PDI | |||||||
0.1% Pep-Cy3 NP * | - | 616 ± 24 | 0.15 ± 0.06 | −2.5 ± 0.1 | - | - | 63 | - | |
0.5% Pep-Cy3 NP * | - | 463 ± 19 | 0.22 ± 0.01 | −2.1 ± 0.2 | 31 | 0.16 | 82 | - | |
1% Pep-Cy3 NP * | - | 338 ± 10 | 0.15 ± 0.01 | −1.9 ± 0.4 | 31 | 0.31 | 73 | - | |
1.5% Pep-Cy3 NP * | - | 526 ± 10 | 0.20 ± 0.04 | −1.8 ± 0.1 | 44 | 0.66 | 62 | - | |
2% Pep-Cy3 NP * | - | 574 ± 22 | 0.15 ± 0.02 | −2.1 ± 0.2 | 51 | 1.02 | 77 | - | |
Empty PLGA NP | 291 ± 5 | 358 ± 5 | 0.13 ± 0.01 | −2.5 ± 0.4 | - | - | 73 | - | |
1.5% PLGA-Cy5 NP | 330 ± 7 | 345 ± 5 | 0.09 ± 0.05 | −2.6 ± 0.3 | - | - | 73 | - | |
2% PLGA-Cy5 NP | 304 ± 14 | 372 ± 9 | 0.12 ± 0.02 | −2.5 ± 0.3 | - | - | 85 | - | |
4% PLGA-Cy5 NP | 309 ± 3 | 249 ± 6 | 0.08 ± 0.01 | −3.2 ± 0.5 | - | - | 66 | - | |
7% PLGA-Cy5 NP | 361 ± 10 | 600 ± 6 | 0.29 ± 0.01 | −2.5 ± 1.6 | - | - | 66 | - | |
9% PLGA-Cy5 NP | 170 ± 21 | 631 ± 5 | 0.21 ± 0.01 | −2.1 ± 0.3 | - | - | 68 | - | |
1% Pep-Cy3 NP | 260 ± 12 | 253 ± 2 | 0.08 ± 0.02 | −2.7 ± 0.3 | 30 | 0.30 | 56 | - | |
FRET NP 0.8:1 (1% Pep-Cy3: 9% PLGA-Cy5) | 227 ± 26 | 236 ± 3 | 0.05 ± 0.01 | −2.1 ± 0.3 | 70 | 0.70 | 0.56:1 | 38 | 60 |
FRET NP 2:1 (1% Pep-Cy3: 4% PLGA-Cy5) | 276 ± 5 | 240 ± 4 | 0.10 ± 0.03 | −2.5 ± 0.5 | 24 | 0.24 | 0.43:1 | 48 | 26 |
FRET NP 4:1 (1% Pep-Cy3: 2% PLGA-Cy5) | 295 ± 8 | 259 ± 2 | 0.09 ± 0.02 | −2.6 ± 0.4 | 30 | 0.30 | 1.11:1 | 60 | 16 |
FRET NP 5:1 (1% Pep-Cy3: 1.5% PLGA-Cy5) | 265 ± 6 | 217 ± 6 | 0.07 ± 0.04 | −3.7 ± 1.0 | 16 | 0.16 | 0.77:1 | 58 | 14 |
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Liu, M.; Lau, C.Y.J.; Cabello, I.T.; Garssen, J.; Willemsen, L.E.M.; Hennink, W.E.; van Nostrum, C.F. 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. Pharmaceuticals 2023, 16, 818. https://doi.org/10.3390/ph16060818
Liu M, Lau CYJ, Cabello IT, Garssen J, Willemsen LEM, Hennink WE, van Nostrum CF. 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. Pharmaceuticals. 2023; 16(6):818. https://doi.org/10.3390/ph16060818
Chicago/Turabian StyleLiu, Mengshan, Chun Yin Jerry Lau, Irene Trillo Cabello, Johan Garssen, Linette E. M. Willemsen, Wim E. Hennink, and Cornelus F. van Nostrum. 2023. "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" Pharmaceuticals 16, no. 6: 818. https://doi.org/10.3390/ph16060818
APA StyleLiu, M., Lau, C. Y. J., Cabello, I. T., Garssen, J., Willemsen, L. E. M., Hennink, W. E., & van Nostrum, C. F. (2023). 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. Pharmaceuticals, 16(6), 818. https://doi.org/10.3390/ph16060818