Fluorescently Labeled PLGA Nanoparticles for Visualization In Vitro and In Vivo: The Importance of Dye Properties

Abstract

Fluorescently labeled nanoparticles are widely used for evaluating their distribution in the biological environment. However, dye leakage can lead to misinterpretations of the nanoparticles’ biodistribution. To better understand the interactions of dyes and nanoparticles and their biological environment, we explored PLGA nanoparticles labeled with four widely used dyes encapsulated (coumarin 6, rhodamine 123, DiI) or bound covalently to the polymer (Cy5.5.). The DiI label was stable in both aqueous and lipophilic environments, whereas the quick release of coumarin 6 was observed in model media containing albumin (42%) or liposomes (62%), which could be explained by the different affinity of these dyes to the polymer and lipophilic structures and which we also confirmed by computational modeling (log PDPPC/PLGA: DiI—2.3, Cou6—0.7). The importance of these factors was demonstrated by in vivo neuroimaging (ICON) of the rat retina using double-labeled Cy5.5/Cou6-nanoparticles: encapsulated Cou6 quickly leaked into the tissue, whereas the stably bound Cy.5.5 label remained associated with the vessels. This observation is a good example of the possible misinterpretation of imaging results because the coumarin 6 distribution creates the impression that nanoparticles effectively crossed the blood–retina barrier, whereas in fact no signal from the core material was found beyond the blood vessels.

Robust Chemical Strategy for Stably Labeling Polyester-Based Nanoparticles with BODIPY Fluorophores

“…Because nanocarrier labeling by the physical entrapment of hydrophobic small-molecule dyes remains a popular approach due to the simplicity and lack of requirement for chemically modifying the particle-forming components, we also included in these studies NPs made of plain PLA with the incorporation of a non-functionalized, strongly hydrophobic BODIPY 505/515 dye (log P O/W = 4.3) . BODIPY 505/515 is analogous in its structure and fluorescence characteristics to BODIPY FL, whereas its hydrophobicity is comparable to that of another popular probe used for non-covalent NP labeling, Coumarin 6 (log P O/W = 5.0) . NPs with different sizes (280 and 140 nm, Figure A) and narrow, non-overlapping size distributions were prepared by the emulsification–solvent evaporation method using either a water-immiscible or a partially water-miscible volatile organic phase composition (chloroform or 1:1 chloroform/tetrahydrofuran, respectively) to allow particle size modulation without changing the amounts and ratios of the particle-forming components .…”

“…These results show that a reliable NP labeling strategy based on stable association of the probe with the carrier requires its incorporation in a molecular form that essentially has no diffusivity through the particle matrix, thus preventing its redistribution on a time scale comparable to that of the particle disintegration (or, for any practical purposes, greater than the duration of experiments involving the labeled formulation). Although this condition can theoretically be reasonably satisfied with small-molecule probes possessing extremely high hydrophobicities, the suitability of this labeling approach has to be reexamined for each specific nanocarrier formulation and experimental settings . In a recently reported example, a small-molecule fluorophore with a log P O/W greater than 10 was shown to rapidly partition from polyester-based NPs coming in contact with lipid membranes .…”

Pitfalls in methods to study colocalization of nanoparticles in mouse macrophage lysosomes

“…The dye encapsulation might change the physicochemical properties of NP and the fluorescence properties of a dye, for example, by inducing fluorescence quenching or enhancing emission. 26,69,70 An experimental design was implemented to compare the colocalization of NP with similar sizes but different fluorophores. J774.A1 cells were initially exposed to 59 nm SiO 2 -BDP FL NP and 59 nm SiO 2 -Rhodamine B (RhoB) NP for a period of 13 h. The medium containing NP was subsequently removed and cells were washed.…”

Fluorescent Nanosystems for Drug Tracking and Theranostics: Recent Applications in the Ocular Field

“…Assessing the affinity of the dye to the polymer and the lipophilic structures could be useful in scaling up these issues. Although C6 has not proved to be an ideal label, it aided in explaining the phenomenon whereby drugs are delivered to tissues through encapsulation in nanocarriers without involving any nanoparticle penetration [ 120 ]. Similar results were obtained by Zhang et al tracking in vivo the distribution of PLGA-NPs in the retinal blood circulation after intravenous injection.…”

“…Moreover, rapid clearance remains a challenge for nanosystems as they need to release their payload before being eliminated from the eye. Many studies focus on assessing the distribution in various tissues once the formulation has been instilled into the eye [ 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 134 , 137 , 138 , 139 ]. Unfortunately, few studies focus on assessing how mechanisms including blinking, tear drainage and ocular metabolism may interact with nanosystems [ 66 , 115 ].…”