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Objectives: Thermal dose in tumor tissue is a key factor for regional hyperthermia (HT) combined with chemotherapy and for drug delivery using thermosensitive liposomes (TSL). It influences therapy outcome, affects the accumulation of liposomes, and triggers the content release from TSL in the target tissue. For the development and clinical application of TSL, noninvasive visualization is of critical importance. For this purpose, TSL loaded with MRI contrast agent (CA) have been developed. With increase in temperature, the CA is released from TSL at the phase transition temperature Tm resulting in a relaxation time change, which allows MRI monitoring. The purpose of this study was to examine the feasibility of an in vivo application and MR characterization of Gd-DTPA-BMA-loaded phosphatidylglyceroglycerol-TSL (Gd-TSL) at mild HT conditions in tumor tissue using a clinically relevant setting., Material and Methods: Gd-TSL were characterized in vitro with varying thermal doses between 37 degrees C and 45 degrees C and distinct solvents by MR at 0.5 T and 1.5 T. In vivo studies were performed in C57BL/6 mice bearing BFS-1 fibrosarcomas at 1.5 T. One tumor-bearing leg was immersed in a temperature-controlled water bath (T). Gd-TSL (Tm = 43.5 +/- 0.2 degrees C) were injected either intratumorally or intravenously at T = 37.3 +/- 0.1 degrees C or T = 42.5 +/- 0.3 degrees C. As a control, nonliposomal Gd-DTPA-BMA was injected intravenously at T = 43.1 +/- 0.3 degrees C. A second tumor on the contralateral limb, which remained unheated, served as a control. CA release was monitored by T1-weighted spin-echo., Results: The in vitro characterization demonstrated at heated and unheated samples a strong increase in T1-relaxivity of Gd-TSL solutions from 0.4 mM-1 s-1 (37.5 degrees C) to 4.2 mM-1 s-1 (43.3 degrees C) at 0.5 T. Thermal dose and solvent affected the rate of relaxation time change significantly. A fast and complete release was observed in samples with serum, whereas Gd-TSL in glucose was only partially released within 1 hour. A dedicated experimental setup was developed for standardized in vivo investigation. Tumor signal intensity changes were detectable in all animals. After intratumoral injection of Gd-TSL, the signal increased heterogeneously (max., +52% +/- 25%) within 3 minutes after temperature increase and decreased strongly thereafter, whereas after i.v. injection, the signal increased homogeneously (+19% +/- 3%) within 2 minutes persisting thereafter. The unheated control tumors on the contralateral legs showed a 10% +/- 3% signal increase within 2 minutes. Injection at 37 degrees C showed a continuous signal increase in "heated" and unheated tumors of up to 8% to 10%. Nonliposomal CA injection demonstrated that tumors were well perfused during HT., Conclusion: HT-induced CA release from Gd-TSL was monitored and characterized by MRI after i.v. injection in tumor-bearing mice. Higher temperatures resulted in higher signal changes. Immediately after i.v. injection, heated tumor tissue was distinguishable from unheated tumor tissue. The Gd-TSL appears to be suitable for MR monitoring of HT tumor treatment in a clinical MRI setting independent of field strength.

Thermosensitive liposomes (TSL) with encapsulated proton (1H) magnetic resonance (MR) contrast agents have been proposed for noninvasive online temperature monitoring during tumor treatment using chemotherapy combined with hyperthermia (HT). The technique exploits the fact that water exchange between the TSL interior and exterior is increased and/or the encapsulated 1H MR contrast agent is released near the gel-to-liquid crystalline phase transition temperature (Tm) of TSL and thus shortens the 1H MR relaxation time of tissue. In this work, newly developed, phosphatidylglyceroglycerol (DPPGOG)-based TSL with encapsulated 1H MR longitudinal relaxation time (T1)-shortening gadodiamide (Gd-DTPA-BMA) were characterized in vitro by measuring the temperature dependence of the T1 of these gadodiamide-containing DPPGOG-TSL samples between 30 and 50 degrees C. The measurements revealed that the T1 nonlinearly slightly decreased with increasing temperature from 30 to 37 degrees C, mainly due to increased water exchange between the gadodiamide-containing DPPGOG-TSL interior and exterior with the exception of negligible gadodiamide release. This implies that gadodiamide-containing DPPGOG-TSL were stable at temperatures < or =37 degrees C, which was also confirmed by an independent stability study. From 37 to 44 degrees C, the T1 nonlinearly markedly decreased with increasing temperature since encapsulated gadodiamide was rapidly released. Above 44 degrees C, gadodiamide was completely released and the T1 was directly proportional to temperature while heated from 44 to 50 degrees C and cooled from 50 to 30 degrees C, respectively. Additionally, gadodiamide release was theoretically quantified and this calculated concentration was consistent with the actually released amount directly obtained from the cooling course of empty DPPGOG-TSL with completely released gadodiamide., ((c) 2008 John Wiley & Sons, Ltd.)

In an MRI hyperthermia hybrid system, T1 changes are investigated for monitoring thermal therapy at 0.2 T. The water bolus, which is needed for power transmission and cooling of the skin, limits MR image quality by signal compression and artifacts. Superparamagnetic ferrofluid in different concentration was investigated with MR relaxometry and MRI methods. We found that using ferrofluid in a low concentration of 70-90 ppm magnetite the water signal can be suppressed without susceptibility artifacts. With our method of signal suppression, a significant improvement of spatial and temporal resolution is possible. The ferrofluid is stable and allows RF heating at 100 MHz. This method of signal extinction may also be useful for other experimental setups where suppression of water is necessary.

MR-thermometry methods have been developed for the guidance and control of thermal therapies such as thermal ablation or regional hyperthermia. However, they are limited to the measurement of temperature changes and, thus, cannot be used to assess absolute temperature values. Paramagnetic thermosensitive liposomes are innovative contrast agents offering the potential to overcome these limitations. They are composed of a gadolinium- or manganese-based compound enclosed by a phospholipid membrane with a distinct gel-to-liquid crystalline phase transition temperature (Tm). At this temperature, the phospholipid membrane changes from a gel-phase to a liquid-crystalline phase which is associated with an increased transmembrane permeability towards solutes and water. Under these conditions, both types of paramagnetic thermosensitive liposomes demonstrate a significant increase in longitudinal (T1) relaxivity, attributed to the release of paramagnetic material from the liposome and/or to the increased water exchange rate between the liposome interior and exterior. Paramagnetic thermosensitive liposomes have already been successfully studied in animal models and have demonstrated a clear correlation between tissue temperature changes and signal intensity changes in MRI. Nevertheless, before entering clinical trials they have to be studied in more detail with regard to dose, pharmacokinetics and toxicity.

The purpose of this study was to characterize T(1), particularly in the hyperthermia temperature range (ca. 37-44 degrees C), in order to control regional hyperthermia with MR monitoring using 0.2 Tesla, and to improve T(1) mapping. A single-slice and a new multislice "T One by Multiple Read-Out Pulses" (TOMROP) pulse sequence were used for fast T(1) mapping in a clinical MRI hyperthermia hybrid system. Temporal stability, temperature sensitivity, and reversibility of T(1) were investigated in a polyamidacryl gel phantom and in samples of muscle and adipose tissues from turkey and pig, and verified in patients. In the gel phantom a high linear correlation between T(1) and temperature (R(2) = 0.97) was observed. In muscle and adipose tissue, T(1) and temperature had a linear relationship below a breakpoint of 43 degrees C. Above this breakpoint muscle tissue showed irreversible tissue changes; these effects were not visible in adipose tissue. The ex vivo results were confirmed in vivo under clinical conditions. T(1) mapping allows the characterization of hyperthermia-related tissue response in healthy tissue. T(1), in combination with fast mapping, is suitable for controlling regional hyperthermia at 0.2 T within the hybrid system., (Copyright 2002 Wiley-Liss, Inc.)

A novel in vitro method for studying the permeation kinetics of superficially applied liposomes or vesicles through layers of human skin or keratinocytes on a solid support is presented, employing attenuated total reflection infrared spectroscopy. The method is applied to investigate transport kinetics of unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) through cultured human keratinocyte layers and through human skin. We find a strong resemblance of the qualitative features of the permeation kinetics of small unilamellar DMPC vesicles for skin and keratinocytes. Detailed studies of the vesicles transport through keratinocyte layers show that DMPC vesicles with an average diameter of 55 nm can readily permeate through the layer at 37 degrees C with a diffusion constant of D = (4.0 +/- 0.8) x 10(-15) m2/second, whereas larger vesicles of twice that diameter do not permeate at all. In contrast, liposomes containing a chemical permeation enhancer permeate through the layer significantly faster [D = (7.0 +/- 0.5) x 10(-15) m2/second] than the small DMPC vesicles despite their five-times-larger diameter. Moreover, the transport of the DMPC vesicles depends drastically on their phase state. No permeation was observed for small DMPC vesicles at a temperature of 10 degrees C when the lipid is in the crystalline phase state. Our results indicate that keratinocyte culture layers can pose a significant permeation barrier for vesicles. The permeation mechanism can be explained by diffusion of the vesicles through small pores and gaps in the layer, presumably driven by transdermal osmotic gradients.

The transfer of lipids between small unilamellar vesicles of either dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), or dioctadecyl diammonium bromide (DODAB) and a single bilayer on a solid support of chain perdeuterated DMPC-d54 has been studied by time-resolved ATR infrared spectroscopy, deuterium NMR, and DSC. The IR method was used for measuring the transfer kinetics and the amount of lipid transferred to the supported bilayer, while NMR was employed for the assessment of molecular order and for the occurrence of lipid asymmetries due to the transfer. We find that the composition of a supported planar dimyristoylphosphatidylcholine (DMPC-d54) bilayer can be modified by incubation with high concentrations of sonicated vesicles consisting of the donor lipid. Three cases were studied. First, the incubation was done with DMPC as donor lipid. The kinetics of this process is double exponential and comparatively slow, with a half-time in the range of several hours. The activation energy was estimated as 50 +/- 2 kJ/mol. In a second set of measurements, cationic DODAB or anionic DMPG was used as donor lipid. The kinetics of this transfer is 1 order of magnitude faster than for DMPC and can be described by a single exponential. For DMPG transfer, we obtained an activation energy of 35 +/- 2 kJ/mol. Independent of the headgroup charge of the donor lipid, 25-35% of the (acceptor) DMPC in the supported bilayer is not accessible for exchange with the donor lipid. The transfer of either DMPG or DODAB causes drastic changes of the phase transition behavior of the supported bilayer without significantly altering the lipid packing density of the lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction of myelin basic protein (MBP) with single bilayers on a solid support (planar and spherical support) is studied by deuterium nuclear magnetic resonance (2H-NMR), differential scanning calorimetry (DSC) and polarized attenuated total reflection infrared spectroscopy (ATR-IR). The single bilayer consisted of either DMPC or of a binary mixture of DMPC with 10-20 mol% of an acidic phospholipid (DMPG, DMPS or DMPA). All methods applied indicate that MBP strongly interacts with the binary lipid systems but not with the pure DMPC bilayers. The interaction is predominantly electrostatic in nature and does not depend on the choice of a particular acidic lipid (for the binary systems). In particular, the results give no indication for a hydrophobic interaction of MBP with the membrane. Our data provide evidence that, in contrast to previous findings, no demixing and/or domain formation in the binary systems is induced due to the MBP coupling. The infrared order parameter was determined for both lipid components of the binary systems and shows a remarkable change for both lipids due to the interaction with MBP while the NMR order parameter remained essentially unchanged. This is discussed in terms of the different timescales characteristic for both methods. The single supported bilayer responds to the MBP coupling as a whole although only 50% of the bilayer surface is accessible to the protein, indicating a strong coupling between the two bilayer leaflets via the hydrophobic chain region. Moreover, the asymmetric coupling of MBP to the single supported bilayer does not result in a significant redistribution of lipids between the two bilayer leaflets. NMR relaxation time measurements in the headgroup and chain region of DMPG and DMPC suggest that the lateral diffusion coefficient of the acidic lipid decreases significantly due to the coupling with MBP while the zwitterionic DMPC is not affected.