Directional Bleb Formation in Spherical Cells under Temperature Gradient

Living cells sense absolute temperature and temporal changes in temperature using biological thermosensors such as ion channels. Here, we reveal, to our knowledge, a novel mechanism of sensing spatial temperature gradients within single cells. Spherical mitotic cells form directional membrane extensions (polar blebs) under sharp temperature gradients (≥∼0.065°C μm⁻¹; 1.3°C temperature difference within a cell), which are created by local heating with a focused 1455-nm laser beam under an optical microscope. On the other hand, multiple nondirectional blebs are formed under gradual temperature gradients or uniform heating. During heating, the distribution of actomyosin complexes becomes inhomogeneous due to a break in the symmetry of its contractile force, highlighting the role of the actomyosin complex as a sensor of local temperature gradients.     

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e., gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.     

Regional hyperthermia by magnetic induction in a beagle dog model: analysis of thermal dosimetry

We have investigated magnetic induction heating techniques for achieving normal tissue hyperthermia in a beagle dog model to clarify the physics and physiology of "regional heating," to develop an animal model of regional heating in humans, and to develop a method of rapid regional heating in dogs for a normal visceral tissue toxicity study. Heating was done with a concentric coil or a coaxial pair of coils applied to the abdominal region, and with or without surface cooling blankets in each case. Thermometers were placed at multiple visceral and subcutaneous sites including an intraarterial thermocouple at the aortic arch level. With either electrode arrangement and no surface cooling, whole-body hyperthermia ( WBH ) at 42 degrees C was produced within 30 to 55 min with 250 W applied power; the 42 degrees C state could be maintained with 40 to 60 W of power. Thermal gradients in these cases reflected nonuniform power deposition superimposed upon arterial temperature elevation. With surface cooling blankets added, systemic heating was significantly reduced, and temperature gradients again reflected the nonuniform power deposition. Regional heating in a dog produces WBH unless sufficient surface cooling is used to provide a heat dissipation rate balancing the heat absorption rate; this latter case best models the use of inductive techniques in humans. The coaxial pair of coils, without surface cooling, produced rapid WBH and the visceral temperature maximum and minimum were within Tesoph + 0.21 degrees C and Tesoph - 0.07 degrees C, respectively (95% confidence index; Tesoph = esophageal temperature). This is an appropriate technique for the proposed toxicity study.     

Modeling of laser coagulation of tissue with MRI temperature monitoring

Light energy from a laser source that is delivered into body tissue via a fiber-optic probe with minimal invasiveness has been used to ablate solid tumors. This thermal coagulation process can be guided and monitored accurately by continuous magnetic resonance imaging (MRI) since the laser energy delivery system does not interfere with MRI. This report deals with mathematical modeling and analysis of laser coagulation of tissue. This model is intended for "real-time" analysis of magnetic resonance images obtained during the coagulation process to guide clinical treatment. A mathematical model is developed to simulate the thermal response of tissue to a laser light heating source. For fast simulation, an approximate solution of the thermal model is used to predict the dynamics of temperature distribution and tissue damage induced by a laser energy line source. The validity of these simulations is tested by comparison with MRI-based temperature data acquired from in vivo experiments in rabbits. The model-simulated temperature distribution and predicted lesion dynamics correspond closely with MRI-based data. These results demonstrate the potential for using this combination of fast modeling and MRI technologies during laser heating of tissue for online prediction of tumor lesion size during laser heating.     

Tissue temperature feedback control of power: the key to successful ablation

Multiple ablation technologies are used to treat atrial fibrillation during cardiac operations. All such ablation technologies use locally induced temperature extremes (>50°C or <-20°C) to kill tissue and create a lesion pattern in the atria which blocks activation pathways that initiate and sustain atrial fibrillation. The technologies used to heat tissue have included radiofrequency (RF), microwave, high-intensity focused ultrasound, and infrared laser. RF accounts for more than 95% of the heating-based ablation technology used by cardiac surgeons. Energy delivery with RF is easier to control than with some other technologies, the heating produced by the energy source is well understood, and manufacturing costs are not excessive. Whichever heating technology is used, control of energy delivery is required to ensure both safe and effective heating of the targeted tissue. All targeted tissue needs to be heated above 50°C to achieve cell death. However, the targeted tissue should not be heated above 100°C, as this can cause perforation due to a steam pop. In addition, adjacent noncardiac tissues must not be damaged during the ablation procedure. The best method to achieve this control uses direct measurement of tissue temperature, because the tissue temperature defines both the safe and effective limits for the ablative process.     

The use of DNA molecular beacons as nanoscale temperature probes for microchip-based biosensors

Today's biosensors and drug delivery devices are increasingly incorporating lithographically patterned circuitry that is placed within microns of the biological molecules to be detected or released. Elevated temperatures due to Joule heating from the underlying circuitry cannot only reduce device performance, but also alter the biological activity of such molecules (i.e. binding, enzymatic, folding). As a consequence, biochip design and characterization will increasingly require local measurements of the temperature and temperature gradients on the biofunctionalized surface. We have developed a technique to address this challenge based on the use of DNA molecular beacons as a nanoscale temperature probe. The surface of fused-silica chips with lithographically patterned, current-carrying gold rings have been functionalized with a layer of molecular beacons. We utilize the temperature dependence of the molecular beacons to calibrate the temperature at the center of the rings as a function of applied current from 25 to 50 degrees C. The fluorescent images of the rings reveal the extent of heating to the surrounding chip due to the applied current while resolving temperature gradients over length scales of less than 500nm. Finite element analysis and analytic calculations of the distribution of heat in the vicinity of the current-carrying rings agree well with the experimental results. Thus, molecular beacons are shown to be a viable tool for temperature calibration of micron-sized circuitry and the visualization of submicron temperature gradients.     

An alternative method for delivering exogenous material into developing zebrafish embryos

Non-invasive manipulation of multicellular systems is important for medical and biological research. The ability to introduce, remove, or modify molecules in the intracellular environment is pivotal to our understanding of cellular structure and function. Herein, we report on an alternative method for introducing foreign material into developing embryos using the application of femtosecond (fs) laser pulses. When intense fs laser pulses are focused to a sub-micron spot, transient pores are formed, providing a transport pathway for the delivery of exogenous material into embryonic cells. In this study, zebrafish embryos were used as a model system to demonstrate the non-invasiveness of this applied delivery tool. Utilizing optically induced transient pores chorionated and dechorionated zebrafish embryos were successfully loaded with a fluorescent reporter molecule (fluorescein isothiocyanate), Streptavidin-conjugated quantum dots or DNA (Simian-CMV-EGFP). Pore formation was independent of the targeted location, with both blastomere-yolk interface and blastomere pores competent for delivery. Long-term survival of laser manipulated embryos to pec-fin stage was 89% and 100% for dechorionated and chorionated embryos, respectively. To our knowledge, this is the first report of DNA delivery into zebrafish embryos utilizing fs laser pulses.     

Laser surgery of zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) embryos using femtosecond laser pulses: Optimal parameters for exogenous material delivery,and the laser's effect on short- and long-term development

Background  

Femtosecond (fs) laser pulses have recently received wide interest as an alternative tool for manipulating living biological systems. In various model organisms the excision of cellular components and the intracellular delivery of foreign exogenous materials have been reported. However, the effect of the applied fs laser pulses on cell viability and development has yet to be determined. Using the zebrafish (Danio rerio) as our animal model system, we address both the short- and long-term developmental changes following laser surgery on zebrafish embryonic cells.     

Fundamental limits to position determination by concentration gradients

Position determination in biological systems is often achieved through protein concentration gradients. Measuring the local concentration of such a protein with a spatially varying distribution allows the measurement of position within the system. For these systems to work effectively, position determination must be robust to noise. Here, we calculate fundamental limits to the precision of position determination by concentration gradients due to unavoidable biochemical noise perturbing the gradients. We focus on gradient proteins with first-order reaction kinetics. Systems of this type have been experimentally characterised in both developmental and cell biology settings. For a single gradient we show that, through time-averaging, great precision potentially can be achieved even with very low protein copy numbers. As a second example, we investigate the ability of a system with oppositely directed gradients to find its centre. With this mechanism, positional precision close to the centre improves more slowly with increasing averaging time, and so longer averaging times or higher copy numbers are required for high precision. For both single and double gradients, we demonstrate the existence of optimal length scales for the gradients for which precision is maximized, as well as analyze how precision depends on the size of the concentration-measuring apparatus. These results provide fundamental constraints on the positional precision supplied by concentration gradients in various contexts, including both in developmental biology and also within a single cell.     

Rapid Temperature Jump by Infrared Diode Laser Irradiation for Patch-Clamp Studies

Several thermal TRP ion channels have recently been identified. These channels are directly gated by temperature, but the mechanisms have remained elusive. Studies of their temperature gating have been impeded by lack of methods for rapid alteration of temperature in live cells. As a result, only measurements of steady-state properties have been possible. To solve the problem, we have developed an optical approach that uses recently available infrared diode lasers as heat sources. By restricting laser irradiation around a single cell, our approach can produce constant temperature jumps over 50°C in submilliseconds. Experiments with several heat-gated ion channels (TRPV1-3) show its applicability for rapid temperature perturbation in both single cells and membrane patches. Compared with other laser heating approaches such as those by Raman-shifting of the Nd:YAG fundamentals, our approach has the advantage of being cost effective and applicable to live cells while providing an adequate resolution for time-resolved detection of channel activation.     

Evidence for localized cell heating induced by infrared optical tweezers.

The confinement of liposomes and Chinese hamster ovary (CHO) cells by infrared (IR) optical tweezers is shown to result in sample heating and temperature increases by several degrees centigrade, as measured by a noninvasive, spatially resolved fluorescence detection technique. For micron-sized spherical liposome vesicles having bilayer membranes composed of the phospholipid 1,2-diacyl-pentadecanoyl-glycero-phosphocholine (15-OPC), a temperature rise of approximately 1.45 +/- 0.15 degrees C/100 mW is observed when the vesicles are held stationary with a 1.064 microns optical tweezers having a power density of approximately 10(7) W/cm2 and a focused spot size of approximately 0.8 micron. The increase in sample temperature is found to scale linearly with applied optical power in the 40 to 250 mW range. Under the same trapping conditions, CHO cells exhibit an average temperature rise of nearly 1.15 +/- 0.25 degrees C/100 mW. The extent of cell heating induced by infrared tweezers confinement can be described by a heat conduction model that accounts for the absorption of infrared (IR) laser radiation in the aqueous cell core and membrane regions, respectively. The observed results are relevant to the assessment of the noninvasive nature of infrared trapping beams in micromanipulation applications and cell physiological studies.     

A continuous wave laser T-jump apparatus and its application to chemical reactions in hemoglobin single crystals

A laser temperature jump apparatus is constructed where the T-jump is achieved by means of the direct absorption of continuous laser radiation of low intensity by a solid sample. The final temperature in the irradiated volume element is reached when the absorbed radiation power equals the dissipation of heat by heat conduction. The time range from the beginning of irradiation to the stationary state depends on the geometry of the irradiated volume element and is less than 10 ms. The heating laser beam is simultaneously used to detect the relaxation to the new chemical equilibrium in the sample. Relaxation processes with relaxation rates between 10(2) s-1 and less than 10(-3) s-1 on samples with volumes less than 10(-3) mm3 may be investigated using this T-jump method. One application of this method is the determination of reaction rates of ligand reactions in hemoglobin single crystals. Rate constants obtained for the reaction of thiocyanate with crystallized horse methemoglobin are presented.     

Operation and pressure distribution of immobilized cell hollow fiber bioreactors

It has been cited in the literature on hollow fiber systems that pressure gradients persist, and the transmembrane flux of the hollow fiber system is dependent on the pattern of the pressure gradients. The pattern can be used to its advantage in immobilized enzyme systems. However, with immobilized living cell systems, the pressure gradients lead to a nonuniform environment within the hollow fiber cartridge and not necessarily favorable results. This article provides pertinent pressure-drop data on hollow fiber cartridges which are in flow configurations typical of immobilized cell culture work. The results illuminate operational problems that may arise in the culture of either anchorage dependent or independent cells. Possible solutions with crossflow systems are suggested.     

RNase A effects on sedimentation and DNA binding properties of dexamethasone-receptor complexes from HeLa cell cytosol

Dexamethasone-receptor complexes from HeLa cell cytosol sediment at 7.4S in low salt sucrose gradients, and at 3.8S in high salt gradients. If cytosol is heated at 25 degrees C, receptor complexes sediment at 6.9S in low salt, and at 3.6S in high salt gradients. RNase A treatment at 25 degrees C, instead, results in receptor complexes which sediment in low salt gradients as two major forms at 6.5 and 4.8S. Receptor complexes from RNase A-treated cytosols sediment as their counterparts from untreated cytosols in high salt gradients. Although the shift in sedimentation properties of receptor complexes at 2 degrees C is induced by RNase A, and not by other low molecular weight basic proteins or RNase T1, the effect can be also obtained by inactive RNase A. The catalytically active enzyme, however, is required to observe 6.5 and 4.8S complexes after cytosol incubations at 25 degrees C. Placental ribonuclease inhibitor prevents the appearance of RNase A-induced receptor forms at 25 degrees C, but not at 2 degrees C. Moreover, this inhibitor can prevent the 7.4 to 6.9S shift in sedimentation coefficient of receptor complexes caused by cytosol heating. Dexamethasone-receptor complexes from HeLa cell cytosol show low levels of binding to DNA-cellulose, and heating at 25 degrees C is required to observe a six-fold increase in DNA binding levels. RNase A treatment of cytosols at 2 degrees C does not result in significant enhancement in receptor complex binding to DNA. If RNase A treatment is carried out at 25 degrees C, however, DNA binding levels of receptor complexes increased by 25% over the values observed with control heated cytosol. This effect cannot be observed if RNase T1 substitutes for RNase A. Placental ribonuclease inhibitor can prevent the temperature-dependent increase in DNA binding properties of dexamethasone-receptor complexes either in the presence or absence of exogenous RNase A. These findings indicate that exogenous RNases can perturb the structure of dexamethasone-receptor complexes without being involved in the transformation process.     

Apparatus for exposing cell membranes to rapid temperature transients

We seek to determine whether cell membranes contain sensors that trigger a downstream response to temperature excursions. To do this, we have developed a novel apparatus for exposing a cell membrane to an extremely rapid temperature excursion in the nanosecond range. Cells are plated on a gold surface that is back-heated by a pulsed laser and cooled by conduction of heat into the glass substrate and the liquid medium. Analysis using the heat diffusion equation shows that the greatest temperature rise is localized within a region tens of nanometres thick, suitable for specifically heating a cell membrane without heating the remainder of a cell. We refer to this device as a nanosecond hotplate.This paper was submitted as a record of the 2002 Australian Biophysical Society     

Mechanisms of laser nanoparticle-based techniques for gene transfection—a calculation study

Cell plasma membranes can be transiently permeabilized to uptake exogenous molecules with high efficiency using a laser nanoparticle-based gene transfection technique. In combination with experimental results, a theoretical model is set up to calculate the temperature distribution and variance around the nanoparticles. This paper also provides a thorough discussion of the underlying mechanisms of cell permeabilization. We find that, rapid heating of the particles and the accompanying extreme temperature rise can lead to microbubble formation around laser-heated particles, which is the origin of photoacoustic effects and other nonlinear optical responses. This transient heat is also capable of causing protein denaturation through thermal inactivation and photochemistry. Furthermore, the dynamic mode that involves the overlapping of bubbles is presented. This mode can significantly increase the plasma membrane permeability of the cells without affecting their viability.     

Laser-induced heating of dextran-coated mesocapsules containing indocyanine green

Indocyanine green (ICG) is a photosensitive reagent with clinically relevant diagnostic and therapeutic applications. Recently, ICG has been investigated for its utility as an exogenous chromophore during laser-induced heating. However, ICG's effectiveness remains hindered by its molecular instability, rapid circulation kinetics, and nonspecific systemic distribution. To overcome these limitations, we have encapsulated ICG within dextran-coated mesocapsules (MCs). Our objective in this study was to explore the ability of MCs to induce thermal damage in response to laser irradiation. To simulate tumorous tissue targeted with MCs, cylindrical phantoms were prepared consisting of gelatin, intralipid emulsion, and various concentrations of MCs. The phantoms were embedded within fresh chicken breast tissue representing surrounding normal tissue. The tissue models were irradiated at lambda = 808 nm for 10 min at constant power (P = 4.2 W). Five hypodermic thermocouples were used to record the temperature at various depths below the tissue surface and transverse distances from the laser beam central axis during irradiation. Temperature profiles were processed to remove the baseline temperature and influence of light absorption by the thermocouple and subsequently used to calculate a damage index based on the Arrhenius damage integral. Tissue models containing MCs experienced a maximum temperature change of 18.5 degrees C. Damage index calculations showed that the heat generation from MCs at these parameters is sufficient to induce thermal damage, while no damage was predicted in the absence of MCs. ICG maintains its heat-generating capabilities in response to NIR laser irradiation when encapsulated within MCs. Such encapsulation provides a potentially useful methodology for laser-induced therapeutic strategies.     

Microfluidic delivery of small molecules into mammalian cells based on hydrodynamic focusing

Microfluidics-based cell assays offer high levels of automation and integration, and allow multiple assays to be run in parallel, based on reduced sample volumes. These characteristics make them attractive for studies associated with drug discovery. Controlled delivery of drug molecules or other exogenous materials into cells is a critical issue that needs to be addressed before microfluidics can serve as a viable platform for drug screening and studies. In this study, we report the application of hydrodynamic focusing for controlled delivery of small molecules into cells immobilized on the substrate of a microfluidic device. We delivered calcein AM which was permeant to the cell membrane into cells, and monitored its enzymatic conversion into fluorescent calcein during and after the delivery. Different ratios of the sample flow to the side flow were tested to determine how the conditions of hydrodynamic focusing affected the delivery. A 3D numerical model was developed to help understand the fluid flow, molecular diffusion due to hydrodynamic focusing in the microfluidic channel. The results from the simulation indicated that the calcein AM concentration on the outer surface of a cell was determined by the conditions of hydrodynamic focusing. By comparing the results from the simulation with those from the experiment, we found that the calcein AM concentration on the cell outer surface correlated very well with the amount of the molecules delivered into the cell. This suggests that hydrodynamic focusing provides an effective way for potentially quantitative delivery of exogenous molecules into cells at the single cell or subcellular level. We expect that our technique will pave the way to high-throughput drug screening and delivery on a microfluidic platform.     

Laser hyperthermia of tumors using gold nanoparticles monitored by optical coherence tomography and acoustic thermometry

Local laser hyperthermia of grafted RShM-5 tumors in mice with the use of plasmon resonant gold nanoparticles has been carried out. Accumulation of particles in the tumor was monitored in vivo noninvasively by optical coherence tomography. Thereby it was determined that the maximal content of nanoparticles in the tumor was reached within 5 h after intravenous administration, and laser hyperthermia was performed at this time. Monitoring the tumor temperature during the treatment by IR thermography and acoustic thermometry showed that the nanoparticles provided efficient temperature elevation inside the tumor as well as more selective heating. Local laser hyperthermia with gold nanoparticles, but not the laser exposure alone, substantially inhibited tumor growth in several days after a single session.     

Potassium and sodium movements in the Ehrlich mouse ascites tumor cell

Studies have been conducted on the movements of sodium and potassium into and out of the Ehrlich ascites tumor cell. Under steady state conditions, at 22 degrees C., in the absence of an exogenous source of glucose, the cell flux for both potassium and sodium averaged 0.8 microM10(7) cells/hr, or 3.0 pM/cm.(2)/sec. The cell can accumulate potassium and extrude sodium against electrochemical gradients for both ions. It is possible under the experimental conditions reported to separate the transport systems for these two ions. Thus, it has been shown that under conditions of low temperature with a diminished metabolism, net fluxes for the two ions are different. Also, following periods of 24 hours at 2 degrees C., an exogenous source of glucose enhances the accumulation of potassium sevenfold while sodium extrusion is uninfluenced by the presence of glucose. Similarly potassium exchange rates are temperature-dependent, with Q(10) values as high as 5, while exchange rates for sodium are temperature-insensitive, with Q(10) values of 1.2 to 1.6. Glycolysis has been eliminated as an energy source for the transport processes since these processes go on in the absence of an exogenous source of glucose. It is estimated that a maximum of 0.3 per cent of the energy derived from the total oxidative metabolism of glucose would be required to support independent transport of potassium and sodium.