Fluorescently Labeled PLGA Nanoparticles for Visualization In Vitro and In Vivo: The Importance of Dye Properties (original) (raw)
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Korean journal of radiology
Poly(lactide-co-glycolide) (PLGA) nanoparticles are promising materials for the development of new drug-releasing systems. The purpose of this study was to evaluate the retention time of materials loaded in nanoparticles as compared with that of the material alone by imaging in nude mice. Mice (n = 20) were injected with 0.1 mL fluorescent material 1,1'-dioctadecyl-3,3,3',3' tetramethylindotricarbocyanine iodide (DiR)-loaded PLGA nanoparticles (200 nm) into the right paraspinal muscle, and the same volume of pure DiR solution was injected into the left paraspinal muscle. Fluorescence images were obtained using an optical imaging system. Fluorescent images were taken 1 day after the injection, and seven more images were taken at 1-week intervals. Image analysis was done with ImageJ program, and one region of interest was chosen manually, which corresponded to the highest signal-intensity area of fluorescence signal intensity. After 7 weeks, 12 mice showed a right-sided do...
Dual-Labelled Nanoparticles Inform on the Stability of Fluorescent Labels In Vivo
Pharmaceutics
Fluorescent labelling is commonly used to monitor the biodistribution of nanomedicines. However, meaningful interpretation of the results requires that the fluorescent label remains attached to the nanomedicine. In this work, we explore the stability of three fluorophores (BODIPY650, Cyanine 5 and AZ647) attached to polymeric hydrophobic biodegradable anchors. Using dual-labelled poly(ethylene glycol)-b-poly(lactic acid) (PEG-PLA) nanoparticles that are both radioactive and fluorescent, we investigated how the properties of the fluorophores impact the stability of the labelling in vitro and in vivo. Results suggest that the more hydrophilic dye (AZ647) is released faster from nanoparticles, and that this instability results in misinterpretation of in vivo data. While hydrophobic dyes are likely more suitable to track nanoparticles in biological environments, quenching of the fluorescence inside the nanoparticles can also introduce artefacts. Altogether, this work raises awareness ab...
Labeling nanoparticles: Dye leakage and altered cellular uptake
Cytometry Part A, 2016
In vitro and in vivo behavior of nanoparticles (NPs) is often studied by tracing the NPs with fluorescent dyes. This requires stable incorporation of dyes within the NPs, as dye leakage may give a wrong interpretation of NP biodistribution, cellular uptake and intracellular distribution. Furthermore, NP labeling with trace amounts of dye should not alter NP properties such as interactions with cells or tissues.
European Journal of Pharmaceutics and Biopharmaceutics, 2020
During the last few decades, extensive efforts has been made to design nanocarriers to transport drugs into the central nervous system (CNS). However, its efficacy is limited due to the presence of the Blood-Brain Barrier (BBB) which greatly reduces drug penetration making Drug Delivery Systems (DDS) necessary. Polymeric nanoparticles (NPs) have been reported to be appropriate for this purpose and in particular, poly(lactic-co-glycolic acid) (PLGA) has been used for its ability to entrap small molecule drugs with great efficiency and the ease with which it functionalizes NPs. Despite the fact that their synthetic identity has been studied in depth, the biological identity of such manufactured polymers still remains unknown as does their biodistribution and in vivo fate. This biological identity is a result of their interaction with blood proteins, the so-called "protein corona" which tends to alter the behavior of polymeric nanoparticles in the body. The aim of the present research is to identify the proteins bounded to polymeric nanoparticles designed to selectively interact with the BBB. For this purpose, four different PLGA NPs were prepared and analyzed: (i) "PLGA@Drug," in which a model drug was encapsulated in its core; (ii) "8D3-PLGA" NPs where the PLGA surface was functionalized with a monoclonal anti-transferrin receptor antibody (8D3 mAb) in order to specifically target the BBB; (iii) "8D3-PLGA@Drug" in which the PLGA@Drug surface was functionalized using the same antibody described above and (iv) bare PLGA NPs which were used as a control. Once the anticipated protein corona NPs were obtained, proteins decorating both bare and functionalized PLGA NPs were isolated and analyzed. Apart from the indistinct interaction with PLGA NPs with the most abundant serum proteins, specific proteins could also be identified in the case of functionalized PLGA NPs. These findings may provide valuable insight into designing novel vehicles based on PLGA NPs for crossing the BBB.
Journal of Controlled Release, 2013
Numerous studies on nanocarriers use fluorescent dye labeling to investigate their biodistribution or cellular trafficking. However, when the fluorescence dye is not grafted to the nanocarrier, the question of the stability of the labeling arises. How can it be validated that the fluorescence observed during an experiment corresponds to the nanocarriers, and not to the free dye released from the nanocarriers? Studying the integrity of the labeling is challenging. Therefore, an innovative approach to confirm the labeling stability was developed, based on the transfer of a fluorescent dye from its hosting nanocarrier to a lipophilic compartment. Lipid nanocapsules (LNC) and triglyceride oil were used as models. The protocol involved mixing of LNC suspension and oil, and then separation by centrifugation. The quality of the separation was controlled by light scattering, using the derived count rate tool. Dye transfer from loaded LNCs to the lipophilic compartment or from a lipophilic compartment containing dye to non-loaded LNC was investigated by varying the nature of the dye and the oil, the oil volume and the LNC dilution. Tensiometry was used to define the dye location in the nanocarrier. Results showed that when dyes such as Nile Red and Coumarin-6 are located in oily core, the transfer occurred in a partition-dependent manner. In contrast, when the dye was entrapped in the surfactant shell of LNCs such as lipophilic indocarbocyanines (i.e. DiO, DiI and DiD), no transfer was observed. Dye diffusion was also observed in cell culture, with Nile Red inside lipid bodies of HEI-OC1 cells, without uptake of LNCs. In contrast, DiO-loaded LNCs had to be internalized to observe fluorescence inside the cells, providing a further confirmation of the absence of transfer in this case, and the stability of fluorescence labeling of the LNCs.
The International Journal of Artificial Organs, 2011
Fluorescent-labeled aliphatic polyesters are essential materials for in vitro and in vivo studies of the behavior of these biodegradable polymers in interaction with cells or in a body. In particular, the direct cellular localization of drug delivery systems based on these materials allows better understanding of the internalization mechanism and determination of the pharmacokinetics. Polylactide-co-glycolide (PLGA) is a rapidly degradable copolymer widely used in pharmaceutics and nanomedecine. It was prepared by ring-opening polymerization of lactide and glycolide in order to obtain a well-defined material to investigate conditions allowing the covalent linkage of a fluorescent dye (fluorescein) while preserving the macromolecular characteristics of the polymer. The success of the functionalization was ascertained by proton nuclear magnetic resonance (1 H NMR), size-exclusion chromatography (SEC) and fluorescence spectroscopy.
Pharmaceutical Research, 2004
Purpose. To delineate the characteristics and mechanisms of up- take of biodegradable poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles in primary cultured rabbit conjunctival epithelial cells (RCECs). Methods. Poly(d,l-lactide-co-glycolide) nanoparticles (PLGA 50:50, 100 nm in diameter) containing 6-coumarin (as a fluorescent marker) were used. The effect of size was studied using various particle sizes (100 nm, 800 nm, and 10 μm). The effect of cytochalasin D, nocodazole, and metabolic inhibitors on nanoparticle uptake was investigated. The capability of nanoparticles to enhance the uptake of an encapsulated protein, BSA bound to Texas red (TR-BSA), was evaluated. Results. Maximal uptake of nanoparticles at 37°C occurred at 2 h, and 100-nm particles had the highest uptake in RCECs in comparison with 800-nm and 10-μm particles. Nanoparticle uptake was saturable over the 0.1-4 mg/ml concentration range. Nanoparticle uptake was confirmed by confocal microscopy and was inhibited significantly by coumarin-free nanoparticles (of similar size), by lower incubation temperature, and by the presence of metabolic inhibitors and cytochalasin D. The uptake of encapsulated TR-BSA in RCECs at 4 h was 28% higher than free BSA application. Conclusion. Our findings suggest that PLGA nanoparticle uptake in primary cultured rabbit conjunctival epithelial cells occurs most likely by adsorptive-type endocytosis.