Heat is of fundamental importance in many cellular processes such as cell metabolism, cell division and gene expression. (1-3) Accurate and noninvasive monitoring of temperature changes in individual cells could thus help clarify intricate cellular processes and develop new applications in biology and medicine. Here we report the use of green fluorescent proteins (GFP) as thermal nanoprobes suited for intracellular temperature mapping. Temperature probing is achieved by monitoring the fluorescence polarization anisotropy of GFP. The method is tested on GFP-transfected HeLa and U-87 MG cancer cell lines where we monitored the heat delivery by photothermal heating of gold nanorods surrounding the cells. A spatial resolution of 300 nm and a temperature accuracy of about 0.4 °C are achieved. Benefiting from its full compatibility with widely used GFP-transfected cells, this approach provides a noninvasive tool for fundamental and applied research in areas ranging from molecular biology to therapeutic and diagnostic studies.
The facile synthesis and photophysical properties of three non-hydrolysable thioglycosylated porphyrinoids are reported. Starting from meso perfluorophenylporphyrin, the non-hydrolysable thioglycosylated porphyrin (PGlc4), chlorin (CGlc4), isobacteriochlorin (IGlc4), and bacteriochlorin (BGlc4) can be made in 2–3 steps. The ability to append a wide range of targeting agents onto the perfluorophenyl moieties, the chemical stability, and the ability to fine-tune the photophysical properties of the chromophores make this a suitable platform for development of biochemical tags, diagnostics, or as photodynamic therapeutic agents. Compared to the porphyrin in phosphate buffered saline, CGlc4 has a markedly greater absorbance of red light near 650 nm and a 6-fold increase in fluorescence quantum yield; whereas IGlc4 has broad Q bands and a 12-fold increase in fluorescence quantum yield. BGlc4 has a similar fluorescence quantum yield to PGlc4, (<10%) but the lowest energy absorption/emission peaks of BGlc4 are considerably red shifted to near 730 nm with a nearly 50-fold greater absorbance, which may allow this conjugate to be an effective PDT agent. The uptake of CGlc4, IGlc4, and BGlc4 derivatives into cells such as human breast cancer cells MDA-MB-231 and K:Molv NIH 3T3 mouse fibroblast cells can be observed at nM concentrations. Photobleaching under these conditions is minimal.
Controlling and monitoring temperature at the single cell level has become pivotal in biology and medicine. Indeed, temperature influences many intracellular processes and is also involved as an activator in novel therapies. Aiming to assist such developments, several approaches have recently been proposed to probe cell temperature in vitro. None of them have so far been extended to a living organism. Here we present the first in vivo intracellular temperature imaging. Our technique relies on measuring the fluorescence polarization anisotropy of green fluorescent protein (GFP) on a set of GFP expressing neurons in Caenorhabditis elegans (C. elegans). We demonstrate fast and noninvasive monitoring of subdegree temperature changes on a single neuron induced by local photoheating of gold nanoparticles. This simple and biocompatible technique is envisioned to benefit several fields including hyperthermia treatment, selective drug delivery, thermal regulation of gene expression and neuron laser ablation.
A water-soluble tetra-S-glycosylated porphyrin (P-Glu4) is absorbed by MDA-MB-231 human breast cancer cells whereupon irradiation with visible light causes necrosis or apoptosis depending on the concentration of the porphyrin and the power of the light. With the same amount of light irradiation power (9.4 W m−2), at 10–20 μM concentrations necrosis is predominantly observed, while at <10 μM concentrations, apoptosis is the principal cause of cell death. Of the various possible pathways for the induction of apoptosis, experiments demonstrate that calcium is released from the endoplasmic reticulum, cytochrome c is liberated from the mitochondria to the cytosol, pro-caspase-3 is activated, poly-(ADP-ribose) polymerase is cleaved, and the chromatin is condensed subsequent to photodynamic treatment of these cells. Confocal microscopy indicates a substantial portion of the P-Glu4 is located in the endoplasmic reticulum at <10 μM. These data indicate that the photodynamic treatment of MDA-MB-231 cells using low concentrations of the P-Glu4 porphyrin and low light induces apoptosis mostly initiated from stress produced to the endoplasmic reticulum.
A characteristic of cancer cells is the generation of lactate from glucose in spite of adequate oxygen for oxidative phosphorylation. This property -known as the "Warburg effect" or aerobic glycolysis -contrasts with anaerobic glycolysis, which is triggered in hypoxic normal cells. The Warburg effect is thought to provide a means for cancer cells to survive under conditions where oxygen is limited and to generate metabolites necessary for cell growth. The shift from oxidative phosphorylation to glycolysis in response to hypoxia is mediated by the production of hypoxiainducible factor (HIF) -a transcription factor family that stimulates the expression of proteins involved in glucose uptake and glycolysis. We reported previously that elevated phospholipase D (PLD) activity in renal and breast cancer cells is required for the expression of the α subunits of HIF1 and HIF2. We report here that the aerobic glycolysis observed in human breast and renal cancer cells is dependent on the elevated PLD activity. Intriguingly, the effect of PLD on the Warburg phenotype was dependent on the mammalian target of rapamycin complex 1 (mTORC1) in the breast cancer cells and on mTORC2 in the renal cancer cells. These data indicate that elevated PLD-mTOR signaling, which is common in human cancer cells, is critical for the metabolic shift to aerobic glycolysis.
The photophysical properties of a chlorin, isobacteriochlorin, and bacteriochlorin built on a core tetrapentafluorophenylporphyrin (TPPF20) and the non-hydrolysable para thioglycosylated conjugates of these chromophores are presented. The photophysical characterization of these compounds was done in three different solvents to correlate to different environments in cells and tissues. Compared to TPPF20 these conjugates have greater absorption in the red region of the visible spectrum and greater fluorescence quantum yields. The excited state lifetimes are from 3-11 nsec. The radiative and non-radiative rate constants for deactivation of the excited state were estimated from the fluorescence quantum yield and excited state lifetime. The data indicates that the bacteriochlorin has strong absorption bands near 730 nm and efficiently enters the triplet manifold. The isobacteriochlorin has a 40-70% fluorescence quantum yield depending on solvent, so it may be a good fluorescent tag. The isobacteriochlorins also display enhanced 2-photon absorption, thereby allowing the use of 860 nm light to excite the compound. While the 2-photon cross section of 25 GM units is not large, low light and low chromophore concentrations can induce apoptosis. The glycosylated compounds accumulate in cells and a head and neck squamous carcinoma xenograft tumor model in mice. These compounds are robust to photobleaching.
This version is available at https://strathprints.strath.ac.uk/59696/ Strathprints is designed to allow users to access the research output of the University of Strathclyde. Unless otherwise explicitly stated on the manuscript, Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Please check the manuscript for details of any other licences that may have been applied. You may not engage in further distribution of the material for any profitmaking activities or any commercial gain. You may freely distribute both the url (https://strathprints.strath.ac.uk/) and the content of this paper for research or private study, educational, or not-for-profit purposes without prior permission or charge.Any correspondence concerning this service should be sent to the Strathprints administrator: strathprints@strath.ac.ukThe Strathprints institutional repository (https://strathprints.strath.ac.uk) is a digital archive of University of Strathclyde research outputs. It has been developed to disseminate open access research outputs, expose data about those outputs, and enable the management and persistent access to Strathclyde's intellectual output. conditions. The results shown here pave the way for using the proposed methodology to discern the role of target enzymes in intracellular signaling pathways. 3Enzymes are biological catalysts involved in crucial cellular processes such as signaling pathways, DNA replication, and protein expression. In biotechnology, enzymes are used for manufacturing biosensors, [1] medicines, [2] fuels [3] and foodstuffs. [4] A methodology for inactivating a target enzyme on demand could be useful for discerning the role of the biocatalyst in a particular cell function. [5] A light-triggered inactivation mechanism would be ideal for these applications since it would enable turning off the enzyme activity noninvasively and on demand. Current methods for controlling the activity of enzymes with light require the synthesis of photoresponsive enzyme substrates, i.e. a molecule that changes its conformation upon irradiation at a particular wavelength. [6] While this approach allows a fine control over the activity of a given enzyme, it can only be applied to those enzymes whose substrate can be synthesized in a laboratory. Furthermore, enzymes show exquisite specificity for the conversion of a particular substrate. This means that different lightresponsive molecules must be synthesized for each specific enzyme, which is very timeconsuming. Moreover, supplying synthetic substrates may be cumbersome in intracellular or in vivo studies, in which the photoresponsive molecules may need to permeate cell membranes and compete with naturally occurring substrates in order to inactivate the enzymes. In this context it would be highly desirable to find a generic methodology that allowed inactivating any target enzyme remotely regardless of its substrate specificity and in different biological scenarios, such as in the presence of other enzym...
While cancer is still an implacable disease, many cancers can be cured if they are diagnosed in an early stage. Recently, it was reported that the transformation from normal cells to cancer cells can change their mechanoelastic properties to become softer and more deformable. If some cancer cells are more deformable, then a progressive increase of the volume of softer cancer cells should be induced as an abrupt change in osmolarity is applied. Based on this hypothesis, we developed a sensor that can electronically monitor the volume increase of cancer cells under hyposmotic pressure. By this methodology, K:Molv NIH 3T3 cells, 786-O human kidney carcinoma cells, and MPSC-1 ovarian cancer cells were successfully detected within 30 minutes using on the order of 10 cells. These cancer cells could be detected with the same sensitivity even in the presence of a vast excess of the respective non-cancerous cells (NIH 3T3 cells, Human Embryonic Kidney (HEK) 293 cells, ovarian surface epithelial (OSE) cells). Since the proposed impedimetric sensor could be useful for detecting cancer cells fast and reliably, it could be further implemented in the screening of large populations of tissue samples and the detection of circulating tumor cells for point-of-care applications.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.