Enhanced cellular internalization of near-infrared fluorescent single-walled carbon nanotubes facilitated by a transfection reagent.

Functionalized single-walled carbon nanotubes (SWCNTs) hold immense potential for diverse biomedical applications due to their biocompatibility and optical properties, including near-infrared fluorescence. Specifically, SWCNTs have been utilized to target cells as a vehicle for drug delivery and gene therapy, and as sensors for various intracellular biomarkers. While the main internalization route of SWCNTs into cells is endocytosis, methods for enhancing the cellular uptake of SWCNTs are of great importance. In this research, we demonstrate the use of a transfecting reagent for promoting cell internalization of functionalized SWCNTs. We explore different types of SWCNT functionalization, namely single-stranded DNA (ssDNA) or polyethylene glycol (PEG)-lipids, and two different cell types, embryonic kidney cells and adenocarcinoma cells. We show that internalizing PEGylated functionalized SWCNTs is enhanced in the presence of the transfecting reagent, where the effect is more pronounced for negatively charged PEG-lipid. However, ssDNA-SWCNTs tend to form aggregates in the presence of the transfecting reagent, rendering it unsuitable for promoting internalization. For all cases, cellular uptake is visualized by near-infrared fluorescence microscopy, showing that the SWCNTs are typically localized within the lysosome. Generally, cellular internalization was higher in the adenocarcinoma cells, thereby paving new avenues for drug delivery and sensing in malignant cells.

Sperm-cell DNA fragmentation prediction using label-free quantitative phase imaging and deep learning.

In intracytoplasmic sperm injection (ICSI), a single sperm cell is selected and injected into an egg. The quality of the chosen sperm and specifically its DNA fragmentation have a significant effect on the fertilization success rate. However, there is no method today to measure the DNA fragmentation of live and unstained cells during ICSI. We present a new method to predict the DNA fragmentation of sperm cells using multi-layer stain-free imaging data, including quantitative phase imaging, and lightweight deep learning architectures. The DNA fragmentation ground truth is achieved by staining the cells with acridine orange and imaging them via fluorescence microscopy. Our prediction model is based on the MobileNet convolutional neural network architecture combined with confidence measurement determined by distances between vectors in the latent space. Our results show that the mean absolute error for cells with high prediction confidence is 0.05 and the 90th percentile mean absolute error is 0.1, where the range of DNA fragmentation score is [0,1]. In the future, this model may be applied to improve cell selection by embryologists during ICSI.

Monitoring the Activity and Inhibition of Cholinesterase Enzymes using Single-Walled Carbon Nanotube Fluorescent Sensors.

Cholinesterase enzymes are involved in a wide range of bodily functions, and their disruption is linked to pathologies such as neurodegenerative diseases and cancer. While cholinesterase inhibitors are used as drug treatments for diseases such as Alzheimer and dementia at therapeutic doses, acute exposure to high doses, found in pesticides and nerve agents, can be lethal. Therefore, measuring cholinesterase activity is important for numerous applications ranging from the search for novel treatments for neurodegenerative disorders to the on-site detection of potential health hazards. Here, we present the development of a near-infrared (near-IR) fluorescent single-walled carbon nanotube (SWCNT) optical sensor for cholinesterase activity and demonstrate the detection of both acetylcholinesterase and butyrylcholinesterase, as well as their inhibition. We show sub U L-1 sensitivity, demonstrate the optical response at the level of individual nanosensors, and showcase an optical signal output in the 900-1400 nm range, which overlaps with the biological transparency window. To the best of our knowledge, this is the longest wavelength cholinesterase activity sensor reported to date. Our near-IR fluorescence-based approach opens new avenues for spatiotemporal-resolved detection of cholinesterase activity, with numerous applications such as advancing the research of the cholinergic system, detecting on-site potential health hazards, and measuring biomarkers in real-time.

Changing gears from chemical adhesion of cells to flat substrata toward engulfment of micro-protrusions by active mechanisms.

Microelectrode arrays increasingly serve to extracellularly record in parallel electrical activity from many excitable cells without inflicting damage to the cells by insertion of microelectrodes. Nevertheless, apart from rare cases they suffer from a low signal to noise ratio. The limiting factor for effective electrical coupling is the low seal resistance formed between the plasma membrane and the electronic device. Using transmission electron microscope analysis we recently reported that cultured Aplysia neurons engulf protruding micron size gold spines forming tight apposition which significantly improves the electrical coupling in comparison with flat electrodes (Hai et al 2009 Spine-shaped gold protrusions improve the adherence and electrical coupling of neurons with the surface of micro-electronic devices J. R. Soc. Interface 6 1153-65). However, the use of a transmission electron microscope to measure the extracellular cleft formed between the plasma membrane and the gold-spine surface may be inaccurate as chemical fixation may generate structural artifacts. Using live confocal microscope imaging we report here that cultured Aplysia neurons engulf protruding spine-shaped gold structures functionalized by an RGD-based peptide and to a significantly lesser extent by poly-l-lysine. The cytoskeletal elements actin and associated protein cortactin are shown to organize around the stalks of the engulfed gold spines in the form of rings. Neurons grown on the gold-spine matrix display varying growth patterns but maintain normal electrophysiological properties and form functioning synapses. It is concluded that the matrices of functionalized gold spines provide an improved substrate for the assembly of neuro-electronic hybrids.

Local calcium-dependent mechanisms determine whether a cut axonal end assembles a retarded endbulb or competent growth cone.

The transformation of a cut axonal end into a growth cone (GC), after axotomy, is a critical event in the cascade leading to regeneration. In an earlier series of studies we analyzed the cellular cascades that transform a cut axonal end into a competent GC. We found that axotomy of cultured Aplysia neurons leads to a transient elevation of the free intracellular Ca2+ concentration ([Ca2+]i), calpain activation and localized proteolysis of submembranal spectrin. These events are associated with the formation of distinct microtubule (MT) based vesicle traps that accumulate anterogradely transported vesicles that fuse with the spectrin free plasma membrane in support of the growth process (Erez, H., Malkinson, G., Prager-Khoutorsky, M., De Zeeuw, C.I., Hoogenraad, C.C., and Spira, M.E. 2007. Formation of microtubule-based traps controls the sorting and concentration of vesicles to restricted sites of regenerating neurons after axotomy. J. Cell Biol. 176: 497-507.; Erez, H., and Spira, M.E. 2008. Local self-assembly mechanisms underlie the differential transformation of the proximal and distal cut axonal ends into functional and aberrant growth cones. J. Comp. Neurol. 507: spc1.). Here we report that under conditions that limit calcium influx into the cut axonal end, axotomy leads to the formation of endbulbs (EBs) rather than to competent GCs. Under these conditions typical MT based vesicle traps are not formed, and Golgi derived vesicles concentrate at the very tip of the cut axon. Since under these conditions the spectrin barrier is not cleaved, vesicle fusion with the plasma membrane and actin polymerization are retarded and growth processes are impaired. We conclude that the immediate assembly of competent GC or an EB after axotomy is the outcome of autonomous local events that are shaped by the magnitudes of the [Ca2+]i gradients at the site of injury.

Calcium-induced exocytosis from actomyosin-driven, motile varicosities formed by dynamic clusters of organelles.

Varicosities are ubiquitous neuronal structures that appear as local swellings along neurites of invertebrate and vertebrate neurons. Surprisingly little is known about their cell biology. We use here cultured Aplysia neurons and demonstrate that varicosities are motile compartments that contain large clusters of organelles. The content of varicosities propagate along neurites within the plasma membrane "sleeve", split and merge, or wobble in place. Confocal imaging, retrospective immunolabeling, electron microscopy and pharmacological perturbations reveal that the motility of the varicosities' organelle content occurs in concert with an actin scaffold and is generated by actomyosin motors. Despite the motility of these organelle clusters within the cytoplasm along the neurites, elevation of the free intracellular calcium concentration within varicosities by trains of action potentials induces exocytosis followed by membrane retrieval. Our observations demonstrate that varicosities formed in the absence of postsynaptic cells behave as "ready to go" prefabricated presynaptic terminals. We suggest that the varicosities' motility serves to increase the probability of encountering a postsynaptic cell and to rapidly form a functional synapse.