Quantification by Luminescence Tracking of Red Emissive Gold Nanoparticles in Cells

Abstract

Optical microscopy techniques are ideal for live cell imaging for real-time nanoparticle tracking of nanoparticle localization. However, the quantification of nanoparticle uptake is usually evaluated by analytical methods that require cell isolation. Luminescent labeling of gold nanoparticles with transition metal probes yields particles with attractive photophysical properties, enabling cellular tracking using confocal and time-resolved microscopies. In the current study, gold nanoparticles coated with a red-luminescent ruthenium transition metal complex are used to quantify and track particle uptake and localization. Analysis of the red-luminescence signal from particles is used as a metric of cellular uptake, which correlates to total cellular gold and ruthenium content, independently measured and correlated by inductively coupled plasma mass spectrometry. Tracking of the luminescence signal provides evidence of direct diffusion of the nanoparticles across the cytoplasmic membrane with particles observed in the cytoplasm and mitochondria as nonclustered “free” nanoparticles. Electron microscopy and inhibition studies identified macropinocytosis of clusters of particles into endosomes as the major mechanism of uptake. Nanoparticles were tracked inside GFP-tagged cells by following the red-luminescence signal of the ruthenium complex. Tracking of the particles demonstrates their initial location in early endosomes and, later, in lysosomes and autophagosomes. Colocalization was quantified by calculating the Pearson’s correlation coefficient between red and green luminescence signals and confirmed by electron microscopy. Accumulation of particles in autophagosomes correlated with biochemical evidence of active autophagy, but there was no evidence of detachment of the luminescent label or breakup of the gold core. Instead, accumulation of particles in autophagosomes caused organelle swelling, breakdown of the surrounding membranes, and endosomal release of the nanoparticles into the cytoplasm. The phenomenon of endosomal release has important consequences for the toxicity, cellular targeting, and therapeutic future applications of gold nanoparticles.

Designing Functional Bionanoconstructs for Effective In Vivo Targeting

“…This is particularly true in cases where the bionanoconstruct has been designed to act as a carrier of molecules of interest, such as drugs, nucleic acids, or contrast agents. The cellular uptake and intracellular distribution of nanomaterials and their bioconjugates are commonly assessed by techniques such as flow cytometry (Figure c), confocal laser scanning microscopy, transmission electron microscopy, Raman spectroscopy, and inductively coupled mass spectrometry. − When performing any receptor interaction, cellular uptake, or intracellular distribution study, consideration must be given as to whether the microenvironment of the target is adequately represented, to ensure correct interpretation of the bionanoconstruct’s activity. The conditions of the microenvironment surrounding the target will influence the physicochemical properties of the bionanoconstruct, which in turn determine whether the construct is recognized by its target and internalized by the cell, and by which intracellular route the construct is trafficked …”

Metal peptide conjugates in cell and tissue imaging and biosensing

“…Other approaches to improving permeability have focused on tuning the lipophilicity, charge and solubility of the complex which in turn can influence cellular uptake and accumulation, as mentioned previously. The use of nanocarriers [95][96][97][98], liposomes [99], dendrimers [100], sugars/carbohydrates [101,102], polyethyleneglycol (PEG) chains [81,82], vitamins [103][104][105], antibodies [106], lipophilic moieties such as triphenylphosphonium (TPP) [107], amino acids [108] and cell-penetrating peptides (CPPs) [81,[109][110][111] has also been shown to increase solubility and improve membrane permeability, facilitating reliable uptake of complexes within cells for a range of applications. Recent reviews describe the preparation and application of ruthenium bioconjugates [112] and vectorisation strategies of metal complex luminophores [3].…”

Synthetic DNA for Cell Surface Engineering: Experimental Comparison between Click Conjugation and Lipid Insertion in Terms of Cell Viability, Engineering Efficiency, and Displaying Stability

“…Cell surface engineering is promising for various applications such as cancer immunotherapy, tissue engineering, cell delivery, and biomolecular sensing. − It can be achieved by transferring exogenous genetic materials into host cells for protein synthesis and transport to the cell membrane . Protein display on the cell surface can last for a long period as the cells can continuously express exogenous genes.…”