Project description:Cancer immunotherapies can be guided by cellular imaging techniques, which can identify the presence or absence of immune cell accumulation in the tumor tissue in vivo and in real time. This review summarizes various new and evolving imaging techniques employed for tracking and monitoring of adoptive natural killer cell immunotherapies.
Project description:The present work demonstrates that Cy5.5 conjugated Fe(3)O(4)/SiO(2) core/shell nanoparticles could allow us to control movement of human natural killer cells (NK-92MI) by an external magnetic field. Required concentration of the nanoparticles for the cell manipulation is as low as ~20 ?g Fe/mL. However, the relative ratio of the nanoparticles loaded NK-92MI cells infiltrated into the target tumor site is enhanced by 17-fold by applying magnetic field and their killing activity is still maintained as same as the NK-92MI cells without the nanoparticles. This approach allows us to open alternative clinical treatment with reduced toxicity of the nanoparticles and enhanced infiltration of immunology to the target site.
Project description:Multimodal Gold-speckled silica nanoparticles as contrast agents for noninvasive imaging with magnetic resonance imaging and photoacoustic tomography have been prepared in a simple one-pot synthesis using nonionic microemulsions. Magnetic resonance contrast is provided through gadolinium incorporated in the silica matrix, whereas the photoacoustic signal originates from nonuniform, discontinuous gold nanodomains speckled across the silica surface.
Project description: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.
Project description:The generation of autologous T cells expressing a chimeric antigen receptor (CAR) have revolutionized the field of adoptive cellular therapy. CAR-T cells directed against CD19 have resulted in remarkable clinical responses in patients affected by B-lymphoid malignancies. However, the production of allogeneic CAR-T cells products remains expensive and clinically challenging. Moreover, the toxicity profile of CAR T-cells means that currently these life-saving treatments are only delivered in specialized centers. Therefore, efforts are underway to develop reliable off-the-shelf cellular products with acceptable safety profiles for the treatment of patients with cancer. Natural killer (NK) cells are innate effector lymphocytes with potent antitumor activity. The availability of NK cells from multiple sources and their proven safety profile in the allogeneic setting positions them as attractive contenders for cancer immunotherapy. In this review, we discuss advantages and potential drawbacks of using NK cells as a novel cellular therapy against hematologic malignancies, as well as strategies to further enhance their effector function.
Project description:Gold nanoparticles continue to generate interest for use in several biomedical applications. Recently, researchers have been focusing on exploiting their dual diagnostic/therapeutic theranostic capabilities. Before clinical translation can occur, regulatory agencies will require a greater understanding of their biodistribution and safety profiles post administration. Previously, the real-time identification and tracking of gold nanoparticles in free-flowing vasculature had not been possible without extrinsic labels such as fluorophores. Here, we present a label-free imaging approach to examine gold nanoparticle (AuNP) activity within the vasculature by utilizing multiphoton intravital microscopy. This method employs a commercially available multiphoton microscopy system to visualize the intrinsic luminescent signal produced by a multiphoton absorption-induced luminescence effect observed in single gold nanoparticles at frame rates necessary for capturing real-time blood flow. This is the first demonstration of visualizing unlabeled gold nanoparticles in an unperturbed vascular environment with frame rates fast enough to achieve particle tracking. Nanoparticle blood concentration curves were also evaluated by the tracking of gold nanoparticle flow in vasculature and verified against known pre-injection concentrations. Half-lives of these gold nanoparticle injections ranged between 67 and 140 s. This label-free imaging approach could provide important structural and functional information in real time to aid in the development and effective analysis of new metallic nanoparticles for various clinical applications in an unperturbed environment, while providing further insight into their complex uptake and clearance pathways.
Project description:Synthesis of gold nanorods (Au NRs) using surfactant-mediated seeded growth involves the interplay of parameters such as pH, reducing agent, and surfactant among others. The use of binary surfactant mixtures of cetyltrimethylammonium bromide (CTAB) and oleic acid (OA) has been reported by our group previously to obtain other anisotropic shapes. However, there are no reports investigating the growth kinetics and mechanisms of such shapes. Here, we report for the first time a ternary representation for compact visualization of shape transitions of gold nanoparticles (Au NPs) as a function of reaction parameters. Further, using UV-Vis spectrophotometry, the growth kinetics of these shapes was tracked using an in-house developed technique. The interplay between the experimental parameters and the properties of Au NPs was investigated using statistical analysis which showed that the reducing agent and pH were significant in influencing shape and growth kinetics. We further propose a growth mechanism in which the supersaturation of growth units controls the final shapes obtained.
Project description:Natural killer (NK) cells are key innate immunity effectors that combat viral infections and control several cancer types. For their immune function, human NK cells rely largely on five different cytotoxic proteases, called granzymes (A/B/H/K/M). Granzyme B (GrB) initiates at least three distinct cell death pathways, but key aspects of its function remain unexplored because selective probes that detect its activity are currently lacking. In this study, we used a set of unnatural amino acids to fully map the substrate preferences of GrB, demonstrating previously unknown GrB substrate preferences. We then used these preferences to design substrate-based inhibitors and a GrB-activatable activity-based fluorogenic probe. We show that our GrB probes do not significantly react with caspases, making them ideal for in-depth analyses of GrB localization and function in cells. Using our quenched fluorescence substrate, we observed GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with GrB attacked MDA-MB-231 target cells, and active GrB influenced its target cell-killing efficiency. In summary, we have developed a set of molecular tools for investigating GrB function in NK cells and demonstrate noninvasive visual detection of GrB with an enzyme-activated fluorescent substrate.
Project description:We report here a new technique for the identification and visualization of functional domains in stratified metal-organic frameworks (MOFs). The technique, namely, gold diffusion enabled domain identification, utilizes the diffusion of Au nanoparticles within MOF cavities to track and selectively stain the more Au-philic domain in an MOF particle thereby allowing direct observation of domains, determination of domain sequences, and, in certain cases, domain boundaries under transmission electron microscopy. This method is an excellent tool for studying MOF materials with complex domain hierarchy.
Project description:Analysis of hematopoietic stem cell function in nonhuman primates provides insights that are relevant for human biology and therapeutic strategies. In this study, we applied quantitative genetic barcoding to track the clonal output of transplanted autologous rhesus macaque hematopoietic stem and progenitor cells over a time period of up to 9.5 months. We found that unilineage short-term progenitors reconstituted myeloid and lymphoid lineages at 1 month but were supplanted over time by multilineage clones, initially myeloid restricted, then myeloid-B clones, and then stable myeloid-B-T multilineage, long-term repopulating clones. Surprisingly, reconstitution of the natural killer (NK) cell lineage, and particularly the major CD16(+)/CD56(-) peripheral blood NK compartment, showed limited clonal overlap with T, B, or myeloid lineages, and therefore appears to be ontologically distinct. Thus, in addition to providing insights into clonal behavior over time, our analysis suggests an unexpected paradigm for the relationship between NK cells and other hematopoietic lineages in primates.