Intracellular Temperature Research Articles

Polydopamine Nanoparticles as an Organic and Biodegradable Multitasking Tool for Neuroprotection and Remote Neuronal Stimulation

Oxidative stress represents a common issue in most neurological diseases, causing severe impairments of neuronal cell physiological activity that ultimately lead to neuron loss of function and cellular death. In this work, lipid-coated polydopamine nanoparticles (L-PDNPs) are proposed both as antioxidant and neuroprotective agents, and as a photothermal conversion platform able to stimulate neuronal activity. L-PDNPs showed the ability to counteract reactive oxygen species (ROS) accumulation in differentiated SH-SY5Y, prevented mitochondrial ROS-induced dysfunctions and stimulated neurite outgrowth. Moreover, for the first time in the literature, the photothermal conversion capacity of L-PDNPs was used to increase the intracellular temperature of neuron-like cells through near-infrared (NIR) laser stimulation, and this phenomenon was thoroughly investigated using a fluorescent temperature-sensitive dye and modeled from a mathematical point of view. It was also demonstrated that the increment in temperature caused by the NIR stimulation of L-PDNPs was able to produce a Ca2+ influx in differentiated SH-SY5Y, being, to the best of our knowledge, the first example of organic nanostructures used in such an approach. This work could pave the way to new and exciting applications of polydopamine-based and of other NIR-responsive antioxidant nanomaterials in neuronal research.

A Simple Calibration Method of Anti-Stokes-Stokes Raman Intensity Ratios Using the Water Spectrum for Intracellular Temperature Measurements.

Presented here is a facile and practical method for calibrating anti-Stokes-Stokes intensity ratios in low-frequency Raman spectra that is devised specifically for temperature measurements inside cells. The proposed method uses as an intensity standard the low-frequency Raman spectrum of liquid water, a major molecular component of cells, whose temperature is independently measured with a thermocouple. Rather than calibrating pixel intensities themselves, we obtain a correction factor at each Raman shift in the 20-200 cm-1 region by dividing the anti-Stokes-Stokes intensity ratio calculated theoretically from the Boltzmann factor at the known temperature by that obtained experimentally. The validity of the correction curve so obtained is confirmed by measuring water at other temperatures. The anti-Stokes-Stokes intensity ratios that have been subjected to our calibration are well fitted with the Boltzmann factor within ∼1% errors and yield water temperatures in fairly good agreement with the thermocouple temperature (an average difference ∼1 ℃). The present method requires only 15 min of spectral acquisition time for calibration, which is 50 times shorter than that in a recently reported calibration method using the pure rotational Raman spectrum of N2. We envision that it will be an effective asset in Raman thermometry and its applications to cellular thermogenesis and thermoregulation.

Evaluation of a Novel Missense Mutation in ABCB4 Gene Causing Progressive Familial Intrahepatic Cholestasis Type 3.

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a hepatic disorder occurring predominantly in childhood and is difficult to diagnose. PFIC3, being a rare autosomal recessive disease, is caused by genetic mutations in both alleles of ABCB4, resulting in the disruption of the bile secretory pathway. The identification of pathogenic effects resulting from different mutations in ABCB4 is the key to revealing the internal cause of disease. These mutations cause truncation, instability, misfolding, and impaired trafficking of the MDR3 protein. Here, we reported a girl, with a history of intrahepatic cholestasis and progressive liver cirrhosis, with an elevated gamma-glutamyltransferase level. Genetic screening via whole exome sequencing found a novel homozygous missense mutation ABCB4:c.1195G>C:p.V399L, and the patient was diagnosed with PFIC3. Various computational tools predicted the variant to be deleterious and evolutionary conserved. For functional characterization studies, plasmids, encoding ABCB4 wild-type and selected established mutant constructs, were expressed in human embryonic kidney (HEK-293T) and hepatocellular carcinoma (HepG2) cells. In vitro expression analysis observed a reduced expression of mutant protein compared to wild-type protein. We found that ABCB4 wild type was localized at the apical canalicular membrane, while mutant p.V399L showed intracellular retention. Intracellular mistrafficking proteins usually undergo proteasomal or lysosomal degradation. We found that after treatment with proteasomal inhibitor MG132 and lysosomal inhibitor bafilomycin A1, MDR3 expression of V399L was significantly increased. A decrease in MDR3 expression of mutant V399L protein may be a result of proteasomal or lysosomal degradation. Pharmacological modulator cyclosporin A and intracellular low temperature (30°C) treatment significantly rescued both the folding defect and the active maturation of the mutant protein. Our study identified a novel pathogenic mutation which expanded the mutational spectrum of the ABCB4 gene and may contribute to understanding the molecular basis of PFIC3. Therefore, genetic screening plays a conclusive role in the diagnosis of rare heterogenic disorders like PFIC3.

Ratiometric fluorescence nanoprobe for monitoring of intracellular temperature and tyrosine based on a dual emissive carbon dots/gold nanohybrid

A novel dual-emission nitrogen doped carbon dots/gold nanohybrid (NCDs-Au) was designed for specific and sensitive ratiometric detection of intracellular temperature and tyrosine. In this probe, a reductive NCDs was successfully prepared with the use of natural biomass Dendrobium officinale as precursor. The new prepared NCDs acted as both reducers and stabilizers to synthesize a novel NCDs-Au nanohybrid by a facile one-step procedure along with a quantum yield of 14.3%. The prepared nanoprobe showed characteristic fluorescence emissions of NCDs and Au NCs with single-wavelength excitation. Notably, the nanoprobe shows an interesting wavelength-dependent dual response to temperature (448 nm) and tyrosine (660 nm), enabling the two targets to be detected proportionally. As an effective temperature sensor, the nanoprobe exhibited good temperature-dependent fluorescence with a sensational linear response from 5 to 75 °C. In addition, the sensor has a linear response toward tyrosine in the range of 0.5–175 μM with a detection limit of 0.19 μM. Moreover, the fluorescent nanoprobe was successfully applied to ratiometricly monitor the variation of temperature or tyrosine level in cells because of the low cytotoxicity, chemical stability and excellent fluorescence properties. These results suggested that the nanoprobe here has provided the possibility for rapidly biosensing with the acceptable selectivity and sensitivity.

TICT-Based Near-Infrared Ratiometric Organic Fluorescent Thermometer for Intracellular Temperature Sensing.

Fluorescent thermometers with near-infrared (NIR) emission play an important role in visualizing the intracellular temperature with high resolution and investigating the cellular functions and biochemical activities. Herein, we designed and synthesized a donor-Π-acceptor luminogen, 2-([1,1'-biphenyl]-4-yl)-3-(4-((E)-4-(diphenylamino)styryl) phenyl) fumaronitrile (TBB) by Suzuki coupling reaction. TBB exhibited twisted intramolecular charge transfer-based NIR emission, aggregation-induced emission, and temperature-sensitive emission features. A ratiometric fluorescent thermometer was constructed by encapsulating thermosensitive NIR fluorophore TBB and Rhodamine 110 dye into an amphiphilic polymer matrix F127 to form TBB&R110@F127 nanoparticles (TRF NPs). TRF NPs showed a good temperature sensitivity of 2.37%·°C-1, wide temperature response ranges from 25 to 65 °C, and excellent temperature-sensitive emission reversibility. Intracellular thermometry experiments indicated that TRF NPs could monitor the cellular temperature change from 25 to 53 °C for Hep-G2 cells under the photothermal therapy agent heating process, indicating the considerable potential applications of TRF NPs in the biological thermometry field.

Key events during the cryopreservation of t cells

Background & Aim Slow, controlled cooling is essential for successful cryopreservation of t cells. In practice, samples are typically cooled at a controlled rate of around 1°C min−1 before being transferred at around -80°C to -100°C. However, other than dehydration, the physical and biological events occurring as the temperature is lowered in t cells (and mammalian cells more widely) have been under-studied. Placing samples in the liquid or vapor phase of nitrogen too early could result in rapid cooling rates, leading to intracellular ice formation and cell death. However, there is little basis in the literature for setting the controlled cooling endpoint at -80°C or below. The present work explored the critical physical and biological events during the cooling of t cells (immortalized jurkat t cell line) through a combination of biological and physical measurements (Differential Scanning Calorimetry and Fourier transformed Infra-Red Spectroscopy). Methods, Results & Conclusion During cooling in a VIA Freeze controlled rate freezer, cells were seen to undergo a membrane transition from a liquid crystal to a gel state at -1.0°C ± 0.8°C. Ice nucleation occurred at -7.7°C ± 1.4°C in 1mL cryovials and was characterized by a rapid release of heat and macroscopic solidification of the sample. Cells dehydrate on cooling after ice nucleation to remain in osmotic equilibrium with the extra-cellular channels between ice crystals. A further transition was observed using DSC at -46.9°C ± 1.3°C. This transition was only present in cryovials during cooling when cells were present and is an intracellular glass transition. The intracellular glass transition temperature corresponded to the cells becoming osmotically inactive, reaching their maximally dehydrated state. Biologically samples following controlled cooling and plunged into liquid nitrogen below this temperature showed good post-thaw outcome, whereas samples plunged into liquid nitrogen above this point had substantial reduction in post-thaw viability and function. An extracellular glass transition at -123°C was observed and below this temperature samples could be safely stored and shipped. These results suggest that controlling cooling until -47°C is reached in t cells is critical for a successful cryopreservation, and no biological advantage is conferred by further cooling. Shorter controlled-rate cooling cycles could thus safely be applied, saving time and potentially allowing more cryopreservation cycles to be completed a day.

Graphene Quantum Dots As Intracellular Imaging-Based Temperature Sensors

Non-invasive temperature sensing is necessary for the analysis of biological processes occurring in the human body including cellular enzyme activity, protein expression, and ion regulation. Considering that a variety of such biological processes occur at the microscopic scale, a novel mechanism allowing for the detection of the temperature changes in microscopic environments is desired. One-dimensional graphene quantum dots can serve as agents for such detection: they are promising non-invasive probes that because of their 2-5 nm size and optical sensitivity to temperature change enable sub-cellular resolution imaging. Both biocompatible bottom-up synthesized nitrogen-doped graphene quantum dots and quantum dots produced from reduced graphene oxide via top-down approach exhibit temperature-induced fluorescence variations. This response observed for the first time is utilized for deterministic temperature sensing in bulk suspension as well as inside mammalian cells. Distinctive quenching of quantum dot fluorescence by up to 19.8% is observed, in a temperature range from 25℃ to 49℃, in aqueous solution, while the intensity is restored to the original values as the temperature decreases back to 25℃. A similar trend is observed in vitro in HeLa cells as the cellular temperature is increased from 25℃ to 41℃. Our findings suggest that the temperature-dependent fluorescence quenching of bottom-up and top-down-synthesized graphene quantum dots can serve as a non-invasive, reversible, and deterministic mechanism for temperature sensing in microscopic sub-cellular biological environments.

Label‐Free Imaging of Intracellular Temperature by Using the O−H Stretching Raman Band of Water

AbstractWe propose a label‐free method for measuring intracellular temperature using a Raman image of a cell in the O−H stretching band. Raman spectra of cultured cells and the medium were first measured at various temperatures using a Raman microscope and the intensity ratio of the two regions of the O−H stretching band was calculated. The intensity ratio varies linearly with temperature in both the medium and cells, and the resulting calibration lines allow simultaneous visualization of both intracellular and extracellular temperatures in a label‐free manner. We applied this method to the measurement of temperature changes after the introduction of FCCP (carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone) in living cells. We observed a temperature rise in the cytoplasm and succeeded in obtaining an image of the change in intracellular temperature after the FCCP treatment.

Label-Free Imaging of Intracellular Temperature by Using the O-H Stretching Raman Band of Water.

We propose a label-free method for measuring intracellular temperature using a Raman image of a cell in the O-H stretching band. Raman spectra of cultured cells and the medium were first measured at various temperatures using a Raman microscope and the intensity ratio of the two regions of the O-H stretching band was calculated. The intensity ratio varies linearly with temperature in both the medium and cells, and the resulting calibration lines allow simultaneous visualization of both intracellular and extracellular temperatures in a label-free manner. We applied this method to the measurement of temperature changes after the introduction of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) in living cells. We observed a temperature rise in the cytoplasm and succeeded in obtaining an image of the change in intracellular temperature after the FCCP treatment.

Heterodimerization of TALK Subunits

The potassium channels form a large and diverse family of ions channels. The 15 members that constitute the Two Pore domain Potassium channels (K2P) subfamily contribute to resting membrane potential and are involved in physiological functions such as depression, migraine, pain or neuronal protection. These K2P channels have been classified into six subfamilies. Most of these channels respond to a diverse regulatory inputs that include extracellular and intracellular pH, temperature, membrane stretch and noxious chemicals. Their membrane topology is distinguishable from the others K+ channels by the presence of two pore and 4 transmembrane domains per subunit. As a consequence of this singular topology, two subunits of K2P channels assemble to constitute a functional channel. Recently, we have shown that different subunits can organize as dimers among the same subfamily members. These heterodimers present different properties and regulations compared to the ones of each homomers. This heteromultimerization represent an alternative to the regulation of relative expression levels. Here we report the heterodimerization between members of TALK subfamily (TWIK-Related AlkalinepH-ActivatedK+channel): TALK-1, TALK-2 and TASK-2. Although, these channels belong to the same subfamily, they have different sensitivity to extracellular pH variations and differential subcellular distribution. TALK-2 is localized at the plasma membrane, whereas TALK-1 channel is mostly in the ER membrane and has been recently implicated in the control of intracellular Ca2+ homeostasis. TASK-2, for its part, is equally distributed to the membrane and in the ER. Using PLA in HEK293 and dominant negative assays in oocytes, we show that TALK-1, TALK-2 and TASK-2 subunits assemble and form active heterodimerics channels with dinstinctive electrophysiological and trafficking properties. Such results emphasize the complexity of this K2P channel family and the need to take into account the co-distribution of each subfamily members within the tissue of interest.

Dual-emission fluorescent silicon nanoparticle-based nanothermometer for ratiometric detection of intracellular temperature in living cells.

In this article, we present a kind of dual-emission fluorescent nanothermometer, which is made of europium (Eu3+)-doped silicon nanoparticles (Eu@SiNPs), allowing the detection of intracellular temperature in living cells with high accuracy. In particular, the presented SiNP-based thermometer features dual-emission fluorescence (blue (455 nm) and red (620 nm) emission), negligible toxicity (cell viability of treated cells remains above 90% during 24 h of treatment) and robust photostability in living cells (i.e., preserving >90% of fluorescence intensity after 45 min of continuous UV irradiation). More significantly, the fluorescence intensity of the Eu@SiNPs exhibits a linear ratiometric temperature response in a broad range from 25 to 70 °C. Taking advantage of these attractive merits, the Eu@SiNP-based nanothermometer is able to accurately (∼4.5% change per °C) determine dynamic changes in intracellular temperature in a quantitative and long-term (i.e., 30 min) manner.

AIE-active non-conjugated poly(N-vinylcaprolactam) as a fluorescent thermometer for intracellular temperature imaging.

Since temperature is one of the most significant physiological parameters that dictate the cellular status of living organisms, accurate intracellular temperature measurement is crucial and a valuable biomarker for the diagnosis and treatment of diseases. Herein, we introduce the foremost example of a non-conjugated polymer as a next generation fluorescent thermometer which is capable of addressing the key shortcomings including toxicity and thermal-induced fluorescence quenching associated with π-π conjugated system-based thermometers developed so far. We revealed, for the first time, the unique photophysical and aggregation-induced emission (AIE) characteristics of well-known thermoresponsive poly(N-vinylcaprolactam) (PNVCL) devoid of any classical fluorophore entity. PNVCL underwent a coil to globular conformational transition in an aqueous medium and appeared to be fluorescent above its lower critical solution temperature (LCST) near body temperature (38 °C). Eventually, this intriguing aspect enabled higher cellular uptake of PNVCL at the LCST boundary. By virtue of the AIE effect, the thermo-induced aggregation phenomenon has been ingeniously utilized to apply PNVCL as a novel fluorescent thermometer for intracellular temperature determination.

A quantum thermometric sensing and analysis system using fluorescent nanodiamonds for the evaluation of living stem cell functions according to intracellular temperature.

Intracellular thermometry techniques play an important role in elucidating the relationship between the intracellular temperature and stem cell functions. However, there have been few reports on thermometry techniques that can detect the intracellular temperature of cells during several days of incubation. In this study, we developed a novel quantum thermometric sensing and analysis system (QTAS) using fluorescent nanodiamonds (FNDs). FNDs could label adipose tissue-derived stem cells (ASCs) at high efficiency with 24 h of incubation, and no cytotoxicity was observed in ASCs labeled with less than 500 μg mL−1 of FNDs. The peak of FNDs was confirmed at approximately 2.87 GHz with the characteristic fluorescence spectra of NV centers that could be optically detected (optically detected magnetic resonance [ODMR]). The ODMR peak clearly shifted to the high-frequency side as the temperature decreased and gave a mean temperature dependence of −(77.6 ± 11.0) kHz °C−1, thus the intracellular temperature of living ASCs during several days of culturing could be precisely measured using the QTAS. Moreover, the intracellular temperature was found to influence the production of growth factors and the degree of differentiation into adipocytes and osteocytes. These data suggest that the QTAS can be used to investigate the relationship between intracellular temperature and cellular functions.

A simple yet effective AIE-based fluorescent nano-thermometer for temperature mapping in living cells using fluorescence lifetime imaging microscopy.

We designed and synthesized a novel nano-thermometer using aggregation-induced-emission (AIE) dye as the reporter and household butter as the matrix. This temperature nanosensor showed decreased fluorescence intensities (∼2%/°C) and shorter fluorescence lifetimes (∼0.11 ns/°C) upon increasing the environmental temperature in the physiological temperature range. Such fluorescence responses were reversible and independent of the environmental pH and ionic strength. The application of these nano-thermometers in temperature sensing in living cells using fluorescence lifetime imaging microscopy (FLIM) was also demonstrated. To the best of our knowledge, this is the first example of AIE-based nano-thermometer for temperature sensing in living cells. This work also provides us with a simple and low-cost method for rapid fabrication of an effective nanosensor based on AIE mechanism.

Cytoplasmic convection currents and intracellular temperature gradients.

Intracellular thermometry has recently demonstrated temperatures in the nucleus, mitochondria, and centrosome to be significantly higher than those of the cytoplasm and cell membrane. This local thermogenesis and the resulting temperature gradient could facilitate the development of persistent, self-organizing convection currents in the cytoplasm of large eukaryotes. Using 3-dimensional computational simulations of intracellular fluid motion, we quantify the convective velocities that could result from the temperature differences observed experimentally. Based on these velocities, we identify the conditions necessary for this temperature-driven bulk flow to dominate over random thermal diffusive motion at the scale of a single eukaryotic cell. With temperature gradients of the order 1°C and diffusion coefficients comparable to those described in the literature, Péclet numbers ≥ 1 are feasible and permit comparable or greater effects of convection than diffusion in determining intracellular mass flux. In addition to the temperature gradient, the resulting flow patterns would also depend on the spatial localization of the heat source, the shape of the cell membrane, and the complex intracellular structure including the cytoskeleton. While this intracellular convection would be highly context-dependent, in certain settings, convective motion could provide a previously unrecognized mechanism for directed, bulk transport within eukaryotic cells.

Cell Membrane-Coated Magnetic Nanocubes with a Homotypic Targeting Ability Increase Intracellular Temperature due to ROS Scavenging and Act as a Versatile Theranostic System for Glioblastoma Multiforme.

In this study, hybrid nanocubes composed of magnetite (Fe3 O4 ) and manganese dioxide (MnO2 ), coated with U-251 MG cell-derived membranes (CM-NCubes) are synthesized. The CM-NCubes demonstrate a concentration-dependent oxygen generation (up to 15%), and, for the first time in the literature, an intracellular increase of temperature (6 °C) due to the exothermic scavenging reaction of hydrogen peroxide (H2 O2 ) is showed. Internalization studies demonstrate that the CM-NCubes are internalized much faster and at a higher extent by the homotypic U-251 MG cell line compared to other cerebral cell lines. The ability of the CM-NCubes to cross an in vitro model of the blood-brain barrier is also assessed. The CM-NCubes show the ability to respond to a static magnet and to accumulate in cells even under flowing conditions. Moreover, it is demonstrated that 500 µg mL-1 of sorafenib-loaded or unloaded CM-NCubes are able to induce cell death by apoptosis in U-251 MG spheroids that are used as a tumor model, after their exposure to an alternating magnetic field (AMF). Finally, it is shown that the combination of sorafenib and AMF induces a higher enzymatic activity of caspase 3 and caspase 9, probably due to an increment in reactive oxygen species by means of hyperthermia.

Abstract 450: Adipose Tissue Browning Induced by Natriuretic Peptide Exerts Thermogenic Actions and Improves Insulin Resistance in an in vivo Model of Diet-Induced Obese Mice

Although natriuretic peptides (NPs) classically act on renal and cardiovascular systems, increasing evidence suggest that NPs also largely coordinate inter-organ metabolic cross-talk with adipose tissues. We recently reported that A-type NP (ANP) raises intracellular temperature in cultured adipocytes in a low-temperature sensitive manner. We herein investigated whether ANP exerts a significant impact on adipose tissues in vivo, using diet-induced obese mice. C57BL/6 mice were exposed to high-fat diet (HFD) or normal-fat diet (NFD) for 13 weeks, and treated with or without ANP infusion for 3 weeks (0.5 ug/kg/min via osmotic-pump). The ANP infusion significantly increased its serum concentration both in NFD and HFD group. Histological analyses revealed that HFD induced increased brown adipose tissue (BAT) cell size with accumulation of lipid droplets (whitening). ANP treatment significantly suppressed this whitening response (re-browning). Similarly, HFD induced enlarged inguinal white adipose tissue (WAT) lipid droplets, which was significantly reduced by ANP. This was associated with a substantial increase in uncoupling protein-1(UCP1) expression in WAT confirmed by immunohistochemical analysis (browning, n=3 each) and UCP1mRNA analysis (NFD+NP 2.7±0.4 fold vs NFD, P<0.01; HFD+NP 6.4±2.2 fold vs HFD, P<0.05, n=5 each). To determine the functional significance of browning effects of ANP, intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were performed and showed that ANP treatment substantially improved insulin resistance in HFD (p<0.05, n=9 each). Moreover, cold tolerance test (at 4°C for 4 hours) demonstrated that the ANP-treated mice were tolerant to cold exposure, which was more salient in HFD with ANP (Decrease in rectal temperature from baseline [°C]: NFD+ANP –2.4±0.6, HFD –2.5±0.4, HFD+ANP –2.1±0.4, versus NFD –9.2±2.2, P<0.01 each, n=9 each). In conclusion, ANP induces WAT browning and preserves BAT function in obese models, leading to in vivo thermogenesis as well as improvement in insulin resistance. These findings reveal the compensatory roles of natriuretic peptides against the core body temperature fall and insulin resistance in a state of heart failure.

Quadruply-labeled serum albumin as a biodegradable nanosensor for simultaneous fluorescence imaging of intracellular pH values, oxygen and temperature.

The construction of multiple fluorescent nanosensors for intracellular studies is a challenging task because spectral overlap of indicator probes can lead to cross-talk and mutual interference. This work describes biodegradable nanosensors that can simultaneously measure three intracellular parameters (temperature, pH and oxygen concentration). Bovine serum albumin (BSA) is selected as the scaffold to construct the triple nanosensor by covalent immobilization four fluorophores on BSA. The following luminophores were used: (a) fluorescein as a probe for pH values, (b) a platinum(II) porphyrin complex for oxygen; (c) a europium(III) clathrate complex for temperature, and (d) a rhodamine B as a reference dye. The nanoparticles have a size of 20nm and show excellent biocompatibility and good brightness. The nanosensors were used for ratiometric imaging of intracellular pH values, oxygen and temperature in HeLa cells. The triple nanosensor responds reversibly and this can be used for real-time tracing of these key parameters. Owing to their biodegradable feature, the use of this kind of triple nanosensor reduce the stress on cellular activities because less nanosensors can be used to gather the total information. Graphical abstractA triple nanosensor for simultaneously ratiometrically sensing intracellular pH, oxygen and temperature values was constructed by covalently labelling four fluorophores on a single serum albumin protein. The nanosensor shows good sensitivity, biocompatibility, is biodegradable and suitable for continuously measuring these important parameters.

Transient heat release during induced mitochondrial proton uncoupling

Non-shivering thermogenesis through mitochondrial proton uncoupling is one of the dominant thermoregulatory mechanisms crucial for normal cellular functions. The metabolic pathway for intracellular temperature rise has widely been considered as steady-state substrate oxidation. Here, we show that a transient proton motive force (pmf) dissipation is more dominant than steady-state substrate oxidation in stimulated thermogenesis. Using transient intracellular thermometry during stimulated proton uncoupling in neurons of Aplysia californica, we observe temperature spikes of ~7.5 K that decay over two time scales: a rapid decay of ~4.8 K over ~1 s followed by a slower decay over ~17 s. The rapid decay correlates well in time with transient electrical heating from proton transport across the mitochondrial inner membrane. Beyond ~33 s, we do not observe any heating from intracellular sources, including substrate oxidation and pmf dissipation. Our measurements demonstrate the utility of transient thermometry in better understanding the thermochemistry of mitochondrial metabolism.

Flower-like Mn-Doped Magnetic Nanoparticles Functionalized with αvβ3-Integrin-Ligand to Efficiently Induce Intracellular Heat after Alternating Magnetic Field Exposition, Triggering Glioma Cell Death.

Despite the potential of magnetic nanoparticles (NPs) to mediate intracellular hyperthermia when exposed to an alternating magnetic field (AMF), several studies indicate that the intracellular heating capacity of magnetic NPs depends on factors such as cytoplasm viscosity, nanoparticle aggregation within subcellular compartments, and dipolar interactions. In this work, we report the design and synthesis of monodispersed flowerlike superparamagnetic manganese iron oxide NPs with maximized SAR (specific absorption rate) and evaluate their efficacy as intracellular heaters in the human tumor-derived glioblastoma cell line U87MG. Three main strategies to tune the particle anisotropy of the core and the surface to reach the maximum heating efficiency were adopted: (1) varying the crystalline anisotropy by inserting a low amount of Mn2+ in the inverse spinel structure, (2) varying the NP shape to add an additional anisotropy source while keeping the superparamagnetic behavior, and (3) maximizing NP-cell affinity through conjugation with a biological targeting molecule to reach the NP concentration required to increase the temperature within the cell. We investigate possible effects produced by these improved NPs under the AMF (f = 96 kHz, H = 47 kA/m) exposure in the glioblastoma cell line U87MG by monitoring the expression of hsp70 gene and reactive oxygen species (ROS) production, as both effects have been described to be induced by increasing the intracellular temperature. The induced cell responses include cellular membrane permeabilization and rupture with concomitant high ROS appearance and hsp70 expression, followed by cell death. The responses were largely limited to cells that contained the NPs exposed to the AMF. Our results indicate that the developed strategies to optimize particle anisotropy in this work are a promising guidance to improve the heating efficiency of magnetic NPs in the human glioma cell line.