Intercellular Transportation of Quantum Dots Mediated by Membrane Nanotubes (original) (raw)
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Imaging the lateral diffusion of membrane molecules with quantum dots
2007
This protocol describes a sensitive approach to tracking the motion of membrane molecules such as lipids and proteins with molecular resolution in live cells. This technique makes use of fluorescent semiconductor nanocrystals, quantum dots (QDs), as a probe to detect membrane molecules of interest. The photostability and brightness of QDs allow them to be tracked at a single particle level for longer periods than previous fluorophores, such as fluorescent proteins and organic dyes. QDs are bound to the extracellular part of the object to be followed, and their movements can be recorded with a fluorescence microscope equipped with a spectral lamp and a sensitive cooled charge-coupled device camera. The experimental procedure described for neurons below takes about 45 min. This technique is applicable to various cultured cells.
Dynamics and mechanisms of quantum dot nanoparticle cellular uptake
Journal of Nanobiotechnology, 2010
Background: The rapid growth of the nanotechnology industry and the wide application of various nanomaterials have raised concerns over their impact on the environment and human health. Yet little is known about the mechanism of cellular uptake and cytotoxicity of nanoparticles. An array of nanomaterials has recently been introduced into cancer research promising for remarkable improvements in diagnosis and treatment of the disease. Among them, quantum dots (QDs) distinguish themselves in offering many intrinsic photophysical properties that are desirable for targeted imaging and drug delivery.
Delivery and visualization of proteins conjugated to quantum dots in cardiac myocytes
Journal of Molecular and Cellular Cardiology, 2008
The design of a novel transduction complex has permitted the introduction of protein-quantum dot conjugates into the cytoplasm of living cells. Appropriate subcellular localization of quantum dot conjugated cardiac troponin C to the myofibrils and a nuclear peptide to the nucleus were attained in living cardiac myocytes using this approach. This new methodology opens the possibility for live tracking of exogenous proteins and study of protein dynamics.
Scientific Reports, 2013
Protein transport is an important phenomenon in biological systems. Proteins are transported via several mechanisms to reach their destined compartment of cell for its complete function. One such mechanism is the microtubule mediated protein transport. Up to now, there are no reports on synthetic systems mimicking the biological protein transport mechanism. Here we report a highly efficient method of mimicking the microtubule mediated protein transport using newly designed biotinylated peptides encompassing a microtubule-associated sequence (MTAS) and a nuclear localization signaling (NLS) sequence, and their final conjugation with streptavidin-coated CdSe/ZnS quantum dots (QDs). Our results demonstrate that these novel bio-conjugated QDs enhance the endosomal escape and promote targeted delivery into the nucleus of human mesenchymal stem cells via microtubules. Mimicking the cellular transport mechanism in stem cells is highly desirable for diagnostics, targeting and therapeutic applications, opening up new avenues in the area of drug delivery. I n the mammalian cell, the cell membrane acts as a barrier to cargoes (ions, small molecules or macromolecules). In higher order biological systems, a nuclear localization signal (NLS) is essential for targeting macromolecular cargoes to the nucleus. Studies on stem cells hold much promise for human regenerative medicine. Recent advances in the field of nanoparticles (NPs) generated novel applications in biomedicine including regenerative medicine. Applications such as imaging, diagnostics and drug delivery (the so-called theranostics) require precise targeting approach for their successes 1-4 . NPs have been engineered to induce membrane receptor internalization, followed by downstream signaling and subsequent cellular responses. NP-mediated cellular response is size-dependent 5 . Gold NPs have been shown to be a powerful vehicle for drug delivery 6 . The presence of Herceptin-gold NP complexes within endosomes showed that receptor-mediated uptake is the most probable mechanism. There are several biological barriers at the cellular level that the engineered NPs must overcome, starting from cell membrane to sub-cellular compartment (cytosol, mitochondria or nucleus). Several endocytotic mechanisms can be engaged to facilitate the internalization of a carrier. In clathrin-mediated endocytosis, endosomal escape must occur before fusion with a lysosome to prevent degradation of the cargo by the harsh lysosomal conditions. More importantly, the endosomal escape is usually necessary to allow access of the carrier to the desired sub-cellular compartment 7 . For example, the uptake of TAT-functionalized Au NPs was enhanced in HeLa cells and the particles initially found in the cytosol, nucleus, mitochondria and later within densely filled vesicles were released, negotiating intracellular membrane barriers quite freely, including the possibility of direct membrane transfer 8 .
2016
The cytoplasm is a highly complex and heterogeneous medium that is structured by the cytoskeleton. Cytoskeletal organization and dynamics are known to modulate cytoplasmic transport processes, but how local transport dynamics depends on the highly heterogeneous intracellular organization of F-actin and microtubules is poorly understood. Here we use a novel delivery and functionalization strategy to utilize quantum dots (QDs) as probes for transport dynamics in different sub-cellular environments. Rapid imaging of non-functionalized QDs revealed two populations with a 100-fold difference in diffusion constant. Depolymerization of actin increased the fast diffusing fraction, suggesting that slow QDs are trapped inside the actin network. When nanobody-functionalized QDs were targeted to different kinesin motor proteins and moved over microtubules, they did not experience strong actin-induced transverse displacements, as suggested previously. Only kinesin-1 bound QDs displayed subtle di...
Nanoparticle transport in cellular blood flow
Computers & Fluids, 2018
The biotransport of the intravascular nanoparticle (NP) is influenced by both the complex cellular flow environment and the NP characteristics. Being able to computationally simulate such intricate transport phenomenon with high efficiency is of far-reaching significance to the development of nanotherapeutics, yet challenging due to large length-scale discrepancies between NP and red blood cell (RBC) as well as the complexity mechanism compared to the RBC-enhanced diffusion. For ~500 particles, the Brownian diffusion and RBC-enhanced diffusion are comparable drivers for the particle radial diffusion process.
Intracellular nanoparticle dynamics affected by cytoskeletal integrity
Soft Matter, 2017
The cell interior is a crowded chemical space, which limits the diffusion of molecules and organelles within the cytoplasm, affecting the rates of chemical reactions. We provide insight into the relationship between non-specific intracellular diffusion and cytoskeletal integrity. Quantum dots entered the cell through microinjection and their spatial coordinates were captured by tracking their fluorescence signature as they diffused within the cell cytoplasm. Particle tracking revealed significant enhancement in the mobility of biocompatible quantum dots within fibrosarcoma cells versus their healthy counterparts, fibroblasts, as well as in actin destabilized fibroblasts versus untreated fibroblasts. Analyzing the displacement distributions provided insight into how the heterogeneity of the cell cytoskeleton influences intracellular particle diffusion. We demonstrate that intracellular diffusion of non-specific nanoparticles is enhanced by disrupting the actin network, which has implications for drug delivery efficacy and trafficking.
Mechanisms of Quantum Dot Nanoparticle Cellular Uptake
Toxicological Sciences, 2009
Due to the superior photoemission and photostability characteristics, quantum dots (QD) are novel tools in biological and medical applications. However, the toxicity and mechanism of QD uptake are poorly understood. QD nanoparticles with an emission wavelength of 655 nm are ellipsoid in shape and consist of a cadmium/selenide core with a zinc sulfide shell. We have shown that QD with a carboxylic acid surface coating were recognized by lipid rafts but not by clathrin or caveolae in human epidermal keratinocytes (HEKs). QD were internalized into early endosomes and then transferred to late endosomes or lysosomes. In addition, 24 endocytic interfering agents were used to investigate the mechanism by which QD enter cells. Our results showed that QD endocytic pathways are primarily regulated by the G-proteincoupled receptor associated pathway and low density lipoprotein receptor/scavenger receptor, whereas other endocytic interfering agents may play a role but with less of an inhibitory effect. Lastly, low toxicity of QD was shown with the 20nM dose in HEK at 48 h but not at 24 h by the live/dead cell assay. QD induced more actin filaments formation in the cytoplasm, which is different from the actin depolymerization by cadmium. These findings provide insight into the specific mechanism of QD nanoparticle uptake in cells. The surface coating, size, and charge of QD nanoparticles are important parameters in determining how nanoparticle uptake occurs in mammalian cells for cancer diagnosis and treatment, and drug delivery.