Cellular Internalization Pathways Research Articles

The characteristics, biodistribution, magnetic resonance imaging and biodegradability of superparamagnetic core–shell nanoparticles

An efficient contrast agent for magnetic resonance imaging (MRI) is essential to enhance the detection and characterization of lesions within the body. In this study, we described the development of biodegradable nanoparticles with a core–shell structure to formulate superparamagnetic iron oxide (CSNP–SPIO) for MRI. The developed nanoparticles were composed of a hydrophobic PLGA core and a positively-charged glycol chitosan shell. The results obtained by transmission electron microscopy, energy dispersive X-ray analysis, electron energy loss spectroscopy, and X-ray diffraction measurement confirmed that the prepared nanoparticles had a core–shell structure with SPIO in their core area. No aggregation of nanoparticles was observed during storage in water, as a result of the electrostatic repulsion between the positively-charged nanoparticles. The magnetic properties of nanoparticles were examined by a vibrating sample magnetometer and a superconducting quantum interference device; the results showed that the superparamagnetism of SPIO was preserved after the CSNP–SPIO formulation. In tracking their cellular internalization pathway, we found that CSNP–SPIO accumulated in lysosomes. In the biodistribution study, a high level of radioactivity was observed in the liver shortly after administration of the 99mTc-labeled CSNP–SPIO intravenously. Once taken up by the liver cells, the liver turned dark on T 2* images. Following cellular internalization, CSNP–SPIO were broken down gradually; therefore, with time increasing, a significant decrease in the darkness of the liver on T 2* images was found. The aforementioned results indicate that the developed CSNP–SPIO can serve as an efficient MRI contrast agent and could be degraded after serving their imaging function.

Localization and dynamics of magnetic transfection complexes

Abstract summary Magnetic targeting is a technique that rapidly accumulates complexes containing drugs or gene vectors at target sites by means of a magnetic field. The effect of magnetic targeting on the cellular internalization pathway has not yet been elucidated. Using widefield fluorescence microscopy, we studied the uptake dynamics of magnetic lipoplexes in vitro. By single particle tracking, three modes of motion were found: directed motion on the cell membrane, confined diffusion and directed motion along microtubules.

The effect of particle design on cellular internalization pathways

The interaction of particles with cells is known to be strongly influenced by particle size, but little is known about the interdependent role that size, shape, and surface chemistry have on cellular internalization and intracellular trafficking. We report on the internalization of specially designed, monodisperse hydrogel particles into HeLa cells as a function of size, shape, and surface charge. We employ a top-down particle fabrication technique called PRINT that is able to generate uniform populations of organic micro- and nanoparticles with complete control of size, shape, and surface chemistry. Evidence of particle internalization was obtained by using conventional biological techniques and transmission electron microscopy. These findings suggest that HeLa cells readily internalize nonspherical particles with dimensions as large as 3 mum by using several different mechanisms of endocytosis. Moreover, it was found that rod-like particles enjoy an appreciable advantage when it comes to internalization rates, reminiscent of the advantage that many rod-like bacteria have for internalization in nonphagocytic cells.

Microfabricated Particles for Engineered Drug Therapies: Elucidation into the Mechanisms of Cellular Internalization of PRINT Particles

To investigate the cellular internalization pathways of shape- and size-specific particles as a function of zeta potential in different cell types. A top-down particle fabrication technique called PRINT was utilized to fabricate monodisperse 1 microm cylindrical particles. Cellular internalization of these PRINT particles was monitored using confocal microscopy, flow cytometry, and transmission electron microscopy. The endocytic pathway used by 1 microm cationic PRINT particles was evaluated using different inhibitory strategies. Cytotoxicity assays were used to determine the toxicity of both cationic and anionic PRINT particles in multiple cell types. Particle internalization was confirmed using confocal microscopy, flow cytometry and transmission electron microscopy. The mechanism of internalization of positively charged PRINT particles was found to be predominantly clathrin-mediated endocytosis and macropinocytosis with very few particles utilizing a caveolae-mediated endocytic pathway. The exposed charge on the surface of the particles had a significant effect on the rate of endocytosis in all cell types tested, except for the macrophage cells. No significant cytotoxicity was observed for all PRINT particles used in the present study. Cylindrical 1 microm PRINT particles were readily internalized into HeLa, NIH 3T3, OVCAR-3, MCF-7, and RAW 264.7 cells. Particles with a positive zeta potential exhibited an enhanced rate of endocytosis compared to negatively charged particles with identical sizes and shapes. It was found that PRINT particles with a positive zeta potential were endocytosed into HeLa cells using predominantely clathrin-mediated and macropinocytotic pathways.

The effect of particle design on cellular internalization pathways

The effect of particle design on cellular internalization pathways

Cellular internalization of cytolethal distending toxin: a new end to a known pathway

The cytolethal distending toxins (CDTs) are unique in their ability to induce DNA damage, activate checkpoint responses and cause cell cycle arrest or apoptosis in intoxicated cells. However, little is known about their cellular internalization pathway. We demonstrate that binding of the Haemophilus ducreyi CDT (HdCDT) on the plasma membrane of sensitive cells was abolished by cholesterol extraction with methyl-beta-cyclodextrin. The toxin was internalized via the Golgi complex, and retrogradely transported to the endoplasmic reticulum (ER), as assessed by N-linked glycosylation. Further translocation from the ER did not require the ER-associated degradation (ERAD) pathway, and was Derlin-1 independent. The genotoxic activity of HdCDT was dependent on its internalization and its DNase activity, as induction of DNA double-stranded breaks was prevented in Brefeldin A-treated cells and in cells exposed to a catalytically inactive toxin. Our data contribute to a better understanding of the CDT mode of action and highlight two important aspects of the biology of this bacterial toxin family: (i) HdCDT translocation from the ER to the nucleus does not involve the classical pathways followed by other retrogradely transported toxins and (ii) toxin internalization is crucial for execution of its genotoxic activity.

Cytolethal distending toxins.

The cytolethal distending toxins (CDTs) constitute the most recently discovered family of bacterial protein toxins. CDTs are unique among bacterial toxins as they have the ability to induce DNA double strand breaks (DSBs) in both proliferating and nonproliferating cells, thereby causing irreversible cell cycle arrest or death of the target cells. CDTs are encoded by three linked genes ( cdtA, cdtB and cdtC) which have been identified among a variety of Gram-negative pathogenic bacteria. All three of these gene products are required to constitute the fully active holotoxin, and this is in agreement with the recently determined crystal structure of CDT. The CdtB component has functional homology with mammalian deoxyribonuclease I (DNase I). Mutation of the conserved sites necessary for this catalytic activity prevents the induction of DSBs as well as all subsequent intoxication responses of target cells. CDT is endocytosed via clathrin-coated pits and requires an intact Golgi complex to exert the cytotoxic activity. Several issues remain to be elucidated regarding CDT biology, such as the detailed function(s) of the CdtA and CdtC subunits, the identity of the cell surface receptor(s) for CDT, the final steps in the cellular internalization pathway, and a molecular understanding of how CDT interacts with DNA. Moreover, the role of CDTs in the pathogenesis of diseases still remains unclear.