Liposomes and local hyperthermia: selective delivery of methotrexate to heated tumors.

Liposomes with phase transitions a few degrees above physiological temperature delivered more than four times as much methotrexate to murine tumors heated to 42 degrees C as to unheated control tumors. Most of the accumulated drug appeared to be intracellular and bound to dihydrofolate reductase, the enzyme blocked by methotrexate in its role as an antineoplastic agent.

Neurite orientation dispersion and density imaging can detect presymptomatic axonal degeneration in the spinal cord of ALS mice.

Neurite orientation dispersion and density imaging (NODDI), a MRI multi-shell diffusion technique, has offered new insights for the study of microstructural changes in neurodegenerative diseases. Mainly, the present study aimed to determine the connection between NODDI-derived parameters and changes in white matter (WM) abnormalities at early stages of amyotrophic lateral sclerosis (ALS). Spinal cords from ALS mice (G93A-SOD1 mice) were scanned in a Bruker Avance III HD 17.6T magnet. Fluorescent axonal-tagged mice (YFP, G93A-SOD1 mice) were used for quantitative histological analysis. NODDI showed a decrease in intra-cellular volume fraction (-24%) and increases in orientation dispersion index (+35%) and isotropic volume fraction (+33%). In addition, histoathological results demonstrated a reductions in axonal area (-11%) and myelin content (-29%). A histological decrease in WM intra-axonal space (-71%) and an increase in the extra-axonal compartment (+22%) were also detected. Our studies demonstrate that NODDI may be a suitable technique for detecting presymptomatic spinal cord WM microstructural degeneration in ALS.

Effects of local tumor hyperthermia on the growth of solid mouse tumors.

The sensitivity to local tumor hyperthermia (43 degrees, 60 min) of a spectrum of eight different solid mouse tumors (Lewis lung carcinoma, M5076 ovarian carcinoma, colon carcinoma 38, colon carcinoma 26, mammary adenocarcinoma C3HBA, mammary adenocarcinoma 16C, glioma 26, and B16 melanoma) was investigated. A microwave (2.45-GHz) apparatus produced localized heating of the tumors without generation of whole-body hyperthermia. The temperature at the center of the heated tumors was regulated to within +/- 0.1 degrees while the temperature uniformity within the tumor was +/- 0.5 degrees. The local hyperthermia treatments reduced the size and retarded the growth of the treated tumors compared with control values for each of the tumors tested. The faster-growing Lewis lung carcinoma and B16 melanoma were the least responsive to treatment, while the slower-growing colon 38 and M5076 ovarian carcinomas were the most responsive. Multiple treatments resulted in longer grwoth delays and greater tumor growth inhibition than did single treatments. No consistent difference in life span between the control and treated groups was measured, and only five of 188 treated animals were cured.

Enhancement of bleomycin activity against Lewis lung tumors in mice by local hyperthermia.

The cytotoxicity of the drug bleomycin in vitro has previously been shown to be enhanced by hyperthermia. This report demonstrates in vivo a more than additive interaction between local tumor hyperthermia (43 degrees, 60 min) and bleomycin (15 mg/kg s.c.) against s.c.-implanted Lewis lung carcinomas in mice. Local hyperthermia was produced by the application of 2450-MHz microwaves to the region of the tumor without induction of significant whole-body hyperthermia. The combined drug and heat treatments were applied to tumors on Days 4, 7, and 10 following implantation. The response of the tumors to simultaneous treatment was a 17-day growth delay compared with controls, whereas the local hyperthermia and bleomycin individually resulted in only 3- and 4-day growth delays, respectively. If the two treatments were given either 4 or 24 hr apart only an additive effect on growth delay was observed.

Rapid measurement of drug release from temperature-sensitive liposomes by electron paramagnetic resonance and radioisotope techniques.

We have developed two new methods for quantifying drug release from temperature-sensitive liposomes. Large unilamellar vesicles were made by the reverse phase evaporation process. They contained a water-soluble electron paramagnetic resonance probe, trimethyl-4-amino-2,2,6,6-tetramethyl piperidine N-oxyl and the radioisotope cytosine-[3H]1-beta-D-arabinofuranoside in their aqueous compartment. Release of the electron paramagnetic resonance probe was measured by placing the liposomes in a solution of a spin label quenching agent, potassium ferricyanide, and monitoring the reduction in signal strength. The measurement of radioisotope released involved rapid ultracentrifugation of the liposomes after which the supernatant was tested for the presence of radioactivity. Both methods were found to be rapid and convenient ways of measuring drug release from temperature-sensitive liposomes and both methods gave comparable results. The radioisotope assay provides a direct measurement of drug leakage, whereas the electron spin resonance assay provides a continuous marker for liposome stability as a function of temperature.

The effects of A23187 on the phospholipid phase transition of large unilamellar vesicles (LUVs) as detected by ultrasound spectroscopy.

The effect of the hydrophobic Ca2+ ionophore, A23187, on the phospholipid dynamics of large unilamellar vesicle (LUVs: 4: 1 (w/w) mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG] membranes, as a function of A23187 content, was investigated using techniques sensitive to the phospholipid phase transition. The ultrasonic absorption per wavelength, alpha lambda, was determined with a double crystal acoustic interferometer, as a function of temperature and frequency for LUVs in the vicinity of their phospholipid phase transition. Differential scanning calorimetry (DSC) and electron spin resonance (ESR) were also employed to probe the thermodynamics and molecular environment of the hydrocarbon side chains. With increasing A23187 content, the phase transition temperature (Tm) of the LUV suspensions remained near 42.0 degrees C, while the amplitude of alpha lambda at the phase transition increased dramatically. At Tm the relaxation frequency, where alpha lambda max occurs, decreased with A23187 content, suggesting that the relaxation rate of the event responsible for the absorption of ultrasound decreased. The ESR studies showed no change in the fluidity of the bilayer with the inclusion of 2 and 5 mol% A23187 in the C-12 region of the bilayer. Therefore, A23187 in LUV membranes slows the structural relaxation of the hydrocarbon side chains of the phospholipid bilayer at the phase transition.

Effects of divalent cations on the ultrasonic absorption coefficient of negatively charged liposomes (LUV) near their phase transition temperature.

The ultrasonic absorption coefficient per wavelength (alpha lambda), as a function of temperature and frequency, was determined for large unilamellar vesicles (LUV) in the vicinity of their phospholipid phase transition temperature, using a double crystal acoustic interferometer. (The vesicles were composed of a 4:1 (w/w) mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). It has been found that alpha lambda reaches a maximum (alpha lambda)max at the phase transition temperature (tm) of the phospholipids in the bilayer, at an ultrasonic relaxation frequency of 2.1 MHz. Divalent cations (Ca2+ and Mg2+), added to LUV suspensions, shifted (alpha lambda)max to higher temperatures, dependent upon the concentration of divalent cation. It was also found that the shape of the alpha lambda versus t curve was significantly changed, representing changes in the Van't Hoff enthalpy of the transition, and therefore, the cooperative unit of the transition. This suggests that divalent cations interact individually with the negatively charged phospholipid headgroups of DPPG and with DPPC headgroups, thus decreasing the cooperative unit of the transition. The observed upward shift in tm suggests an interaction that increases the activation energy and, therefore, the temperature of the phase transition. However, alpha lambda as a function of frequency did not change with the addition of divalent cations and, thus, the relaxation time of the event responsible for the absorption of ultrasound is not changed by the addition of divalent cations.

The effects of 2H2O on the phase transition of large unilamellar vesicle (LUV) suspensions as detected by ultrasound spectroscopy and electron spin resonance.

The importance of water in the molecular dynamics of large unilamellar vesicle (LUV) suspensions, in which increasing portions of the water were replaced by 2H2O, was investigated. Determinations of the ultrasonic absorption coefficient per wavelength, alpha lambda, were performed as a function of temperature and frequency for LUVs (LUVs: 4:1 (w/w) mixture of dipalmitoylphosphatidylcholine, DPPC, and dipalmitoylphosphatidylglycerol, DPPG) in the vicinity of their phospholipid phase transition, using a double crystal acoustic interferometer. Electron spin resonance (ESR) and differential scanning calorimetry (DSC) were also employed to probe this system. When increasing portions of the aqueous content of the LUV suspensions were replaced by 2H2O the phase transition temperature increased from 42.0 degrees C to 42.9 degrees C (indicating an increase in the activation energy of the transition), and the amplitude of alpha lambda at the phase transition increased. However, alpha lambda max as a function of frequency at the phase transition did not change with the addition of 2H2O, indicating that the relaxation time of the event responsible for the absorption of ultrasound was unaffected. The increase in the activation energy of the transition with the addition of 2H2O suggested that the mobility of phospholipids near the membrane/aqueous interface was changed. Electron spin resonance (ESR) experiments on LUVs with nitroxide spin probes positioned at the membrane/aqueous interface (5-doxyl stearate and CAT16) showed that LUVs in 2H2O have a broader splitting, Amax, at the membrane/aqueous interface than do LUVs in H2O. These results suggest that 2H2O changes the mobility and/or structure of the phospholipids in the region of the membrane/aqueous interface. This difference in Amax was not seen for the probe PC-12-doxyl stearate, which resides at the C-12 position of the bilayer.

Ultrasonic evidence for structural relaxation in large unilamellar liposomes.

The ultrasonic absorption of large unilamellar vesicles (average diameter 0.2 micron) was determined in the frequency range 0.5-5 MHz. The liposomes were composed of a 4:1 mixture by weight of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylglycerol. They were studied with and without cholesterol or gramicidin incorporated into the bilayer. A large increase in absorption occurs at the solid to liquid-crystalline phase transition temperature (42 degrees C) of the pure lipid vesicles. This increase in absorption is interpreted as a structural relaxation of the 'melting' fatty acid chains occurring with an average relaxation time of 76 ns. The liposomes were also found to be extremely permeable near the transition temperature. Essentially complete release of cytosine arabinoside, a small water-soluble molecule, occurred at 42 degrees C. Addition of cholesterol or gramicidin to the bilayer of the liposomes broadened the ultrasonic absorption and reduced the efflux of cytosine arabinoside at the phase transition. No increase in absorption was observed at the transition temperature in the presence of 50 mol% of cholesterol. Gramicidin, in addition to broadening the transition, slows the isomerization of bonds in the hydrocarbon chains of the lipids. A concentration of 5 mol% gramicidin increased the average relaxation time to 211 ns.

A potential method for local drug and dye delivery in the ocular vasculature.

We propose a new drug and dye delivery system that would allow repeated release of substances in the ocular vasculature by an externally controlled mechanism. The substances are encapsulated in heat-sensitive liposomes, which are lysed by locally applying a heat pulse produced by an argon laser. The system was tested by investigating the release of carboxyfluorescein encapsulated in the liposomes. The liposome suspension was incubated at 37 degrees or 38.5 degrees C and irradiated at different powers and pulse durations. The amount of dye released was monitored by fluorophotometry and compared with the concentration obtained when the liposomes were lysed at their transition temperature of 41 degrees C. The results showed that 85% of the encapsulated substance can be released. Moreover, a dramatic contrast was observed between the fluorescence before and after the lysis. Presently the energy density is higher than but close to the maximal permissible exposure for humans. The release mechanism with the short laser pulse appeared to be similar to that present when liposomes were heated slowly.

Feasibility of blood flow measurement by externally controlled dye delivery.

We are developing a new method of delivering substances locally and repeatedly in the retinal vasculature under external control. This delivery system is based on encapsulating the substance in heat-sensitive lipsomes, which are injected intravenously and lysed by a heat pulse delivered by a laser. The feasibility of using this system with dyes and creating a sharp dye front was tested in vitro and in vivo. The results indicate that the background fluorescence of intact liposomes is minimal but in contrast a dramatic increase in fluorescence is achieved where the dye is released. In vivo tests indicated that only the selected vascular branch fluoresced. Moreover, a sharp dye front could be obtained repeatedly and preserved over significant distances. The presence of a sharp dye front allowed measurements, in vitro, of blood velocity which correlated well (r = 0.985, P less than 0.001) with the average blood velocity values calculated from the known flow rate.

Liposome encapsulated MnCl2 as a liver specific contrast agent for magnetic resonance imaging.

We examined the usefulness of MnCl2 entrapped in liposomes as a liver specific contrast agent for magnetic resonance imaging. Toxicity experiments showed that the effective dose for imaging experiments was 7 to 11 times lower than the LD50 of free MnCl2. In rats with implanted liver tumors, liposome encapsulated MnCl2 caused a two- to three-fold increase in the relaxation rate of the liver while having little effect on the relaxation rate of tumor tissue. T1 weighted, magnetic resonance images obtained at 0.5 T of an R3230 adenocarcinoma implanted in the liver showed an increase in the signal intensity of both normal liver and tumor tissue after the injection of free MnCl2 (25 mumole/kg). However, after the injection of liposome encapsulated MnCl2 (40 mumole/kg) the liver exhibited a marked increase in signal intensity with little change in the signal intensity of the tumor tissue. These results suggest that liposome encapsulated MnCl2 has excellent potential as a liver specific contrast agent for the improved detection of liver metastases.

Detection and quantitation in rat tissues of the superparamagnetic magnetic resonance contrast agent dextran magnetite as demonstrated by electron spin resonance spectroscopy.

RATIONALE AND OBJECTIVES: The compound studies in this article is a superparamagnetic macromolecular complex of magnetite cores coated with hydrophilic dextran, which is under active investigation as a contrast agent for magnetic resonance imaging (MRI) in liver and spleen. The biodistribution of paramagnetic compounds is problematic and is usually studied by histochemical reactions or by radiolabeling the compound under study. The purpose of this article is to show how electron spin resonance (ESR) spectroscopy detects dextran magnetite (DM) particles in tissues. METHODS: DM injected intravenously in the experimental animal was detected in some reticulo-endothelial organs by ESR. The spectroscopic study was validated using electron microscopy and electron-probe microanalysis. RESULTS: DM exhibits an ESR spectrum; ESR delineated the distribution of DM distribution in liver, spleen, bone marrow, and blood as a function of time. The blood clearance was biphasic, dependent on the size of particles. CONCLUSIONS: ESR spectroscopy is a highly sensitive and reproducible method of studying DM distribution.

Microwave-stimulated drug release from liposomes.

Microwaves (2450 MHz) are shown to stimulate the release of an aqueous chemotherapeutic drug from phospholipid vesicles. This effect occurs at temperatures below the membrane phase transition temperature of 41 degrees C where these liposomes are normally not leaky. In buffered saline, microwave exposure (60 mW/g) triggers the onset of drug release at 33 degrees C, whereas in plasma a near maximal release is observed as low as 27 degrees C. Significantly, this drug release is enhanced by oxygen and is attenuated by antioxidants. These results demonstrate that phospholipids in artificial membranes devoid of protein are influenced by nonionizing electromagnetic radiation, and that this interaction can be modulated by two physiologically important factors, plasma and oxygen. Such a permeability effect may provide a means for investigating microwave interactions with ordered membrane bilayers.

Magnetic field-induced drug permeability in liposome vesicles.

Liposome vesicles maintained in a uniform static magnetic field release a chemotherapeutic drug (ARA-C, MW = 243) at temperatures approaching the phase-transition region where these liposomes are not normally leaky. Drug release is rapid, and a maximum difference between treated and unexposed liposomes of 30% of the total maximal release of ARA-C was observed within 1 min in a magnetic field. Dose-effect studies conducted between 0.01 and 7.5 T (1 T = 10(4) G) reveal that this permeability effect has a sigmoidal dependence on magnetic flux density. The ED50 is 15 mT, with a 95% confidence interval of 6.50-34.9 mT. Magnetic field exposures were conducted using a superconducting magnet with the liposomes maintained at +/- 0.08 degrees C. For comparison, samarium-cobalt permanent magnets induced a comparable drug release at 0.4 T. These results indicate that a static magnetic field of 10 mT or greater can increase passive transport in phospholipid membrane bilayers maintained at or near their membrane phase-transition temperature. Lipid clustering which occurs at prephase-transition temperatures may predispose phospholipid domains to diamagnetic orientation in a magnetic field and thereby facilitate drug release.

Temperature-dependent permeability of large unilamellar liposomes.

The temperature-dependent drug leakage from liposomes composed of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (4:1, by weight) was studied. Experiments were performed in Hepes buffer and 50% fetal calf serum. Large unilamellar liposomes were formed by the reverse phase evaporation process and extruded through a series of polycarbonate membranes with pore sizes of 0.4, 0.2, 0.1 and 0.08 micron. The release of the water soluble radioisotopes cytosine 1-beta-D-[3H]arabinofuranoside and [3H]inulin from the aqueous compartment of these liposomes was measured as a function of time and temperature. Both radioisotopes were released at temperatures near 42 degrees C, the solid-to-liquid-crystalline phase transition temperature of these lipids. The percent drug release decreased as the size of the liposomes was reduced. This effect was more pronounced in Hepes buffer than serum. The release of both radioisotopes was greatest at 40 degrees C in Hepes buffer and at 43 degrees C in 50% fetal calf serum. In addition, the rate of drug release was much faster in serum than in buffer. These results suggest that different drug release processes are occurring in buffer and in serum.

Ultrasonic absorption and permeability for liposomes near phase transition.

The specific ultrasonic absorption coefficient per wavelength as a function of temperature in the vicinity of the phase transition of liposomes, composed of a 4:1 mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG), of different sizes was determined using an acoustic interferometer. Small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) yielded results similar to those in the literature, viz., an absorption maximum at the transition temperature. Seven intermediate sizes including several size distributions of large unilamellar vesicles (LUV) were studied, yielding information on size dependencies of the temperatures at which the peaks occur, the widths at half peak amplitude, and the peak amplitudes. All liposome sizes except the SUV exhibited approximately the same transition temperature as did the largest MLV. The widths of the peaks were inversely related to liposome size, with a strong dependence for the smallest vesicles and an approach to independence for the largest vesicles. The amplitudes of the peaks exhibited a general increase with size with two exceptions, viz., the SUV and the vesicles with average diameters of 90-100 nm. It was also found that the membrane permeability increased near the transition temperature. The temperature dependencies of ultrasonic absorption and membrane permeability are compared.

Ultrasound interaction with large unilamellar vesicles at the phospholipid phase transition: perturbation by phospholipid side chain substitution with deuterium.

The ultrasonic absorption, alpha lambda, as a function of temperature and frequency was determined in large unilamellar vesicles (LUVs) in which specific phospholipid side chains were deuterated. Deuteration significantly altered the temperature and frequency dependence of alpha lambda. The frequency change was especially marked, with decreased frequency and broadening of the ultrasound relaxation, even with only minor changes in the phase transition temperature. Deuteration decreased the Tm and enthalpy of the lipid phase transition, as shown by differential scanning calorimetry, whereas electron spin resonance showed that at and above the lipid phase transition, no differences in the mobility as a function of temperature were observed. These results show that the observed increase in ultrasonic absorption in LUVs at the phospholipid phase transition arises from the interaction of ultrasound with the hydrophobic side chains, probably coupling with structural reorganization of small domains of molecules, a process which is maximized at the phase transition temperature.

Feedback control of the nuclear magnetization state: modeling and control design.

Magnetic resonance (MR) images are obtained by observing the fluctuations in nuclear magnetization produced by sequences of RF pulses and applied magnetic field gradients. The design of a pulse sequence is based on the expected response of the nuclear magnetization. Currently, all pulse sequence parameters are loaded into the pulse programmer before the start of the sequence and remain unchanged until the completion of the sequence. A fundamentally different approach is considered, whereby the sequence parameters are adjusted between successive RF excitation pulses. In this manner, the nuclear magnetization is regulated to a desired state by using measurements of the magnetization to adjust the amplitude and duration of the RF pulses. Feedback control of the nuclear magnetization state may be advantageous in certain types of MR experiments, for example those requiring a repeated spin-echo or gated MR image acquisition. As a first step in developing this approach a simple scheme for regulating the angle between the bulk magnetization and the axis applied static magnetic field is presented. The behavior of the closed-loop system is explored using computer simulations.

Feedback control of the nuclear magnetization state: experimental results.

In a previous paper (Proc. Soc. Magn. Reson. Med., San Francisco, CA, USA, 1991), the authors investigated feedback control of the nuclear magnetization state. This strategy facilitates the optimization of the pulse parameters during the imaging sequence. In this work, results from a series of experiments used to test the theoretical predictions are presented. Two groups of experiments were performed using a commercial imager and a small sample of distilled water. In the first group of experiments the control objective is to regulate the magnetization orientation to a constant reference angle. The results show that a closed-loop system produces the desired orientation. In contrast, an open-loop input results in an appreciable error between desired and measured orientation. A second group of experiments shows that a closed-loop system can force the magnetization orientation to track a desired trajectory. In both groups, the closed-loop experimental and simulation results are in excellent agreement. The application of feedback control to MRI and spectroscopy is discussed.