Hyperthermia-triggered release of hypoxic cell radiosensitizers from temperature-sensitive liposomes improves radiotherapy efficacy in vitro.

Hypoxia is a characteristic feature of solid tumors and an important cause of resistance to radiotherapy. Hypoxic cell radiosensitizers have been shown to increase radiotherapy efficacy, but dose-limiting side effects prevent their widespread use in the clinic. We propose the encapsulation of hypoxic cell radiosensitizers in temperature-sensitive liposomes (TSL) to target the radiosensitizers specifically to tumors and to avoid unwanted accumulation in healthy tissues. The main objective of the present study is to develop and characterize TSL loaded with the radiosensitizer pimonidazole (PMZ) and to evaluate the in vitro efficacy of free PMZ and PMZ encapsulated in TSL in combination with hyperthermia and radiotherapy. PMZ was actively loaded into TSL at different drug/lipid ratios, and the physicochemical characteristics and the stability of the resulting TSL-PMZ were evaluated. PMZ release was determined at 37 °C and 42 °C in HEPES buffer saline and fetal bovine serum. The concentration-dependent radiosensitizing effect of PMZ was investigated by exposing FaDu cells to different PMZ concentrations under hypoxic conditions followed by exposure to ionizing irradiation. The efficacy of TSL-PMZ in combination with hyperthermia and radiotherapy was determined in vitro, assessing cell survival and DNA damage by means of the clonogenic assay and histone H2AX phosphorylation, respectively. All TSL-PMZ formulations showed high encapsulation efficiencies and were stable for 30 d upon storage at 4 °C and 20 °C. Fast PMZ release was observed at 42 °C, regardless of the drug/lipid ratio. Increasing the PMZ concentration significantly enhanced the effect of ionizing irradiation. Pre-heated TSL-PMZ in combination with radiotherapy caused a 14.3-fold increase in cell death as compared to radiotherapy treatment alone. In conclusion, our results indicate that TSL-PMZ in combination with hyperthermia can assist in improving the efficacy of radiotherapy under hypoxic conditions.

Triggered radiosensitizer delivery using thermosensitive liposomes and hyperthermia improves efficacy of radiotherapy: An in vitro proof of concept study.

INTRODUCTION: To increase the efficacy of chemoradiation and decrease its toxicity in normal tissue, a new concept is proposed, local radiosensitizer delivery, which combines triggered release of a radiosensitizer from thermosensitive liposomes with local hyperthermia and radiotherapy. Here, key aspects of this concept were investigated in vitro I) the effect of hyperthermia on the enhancement of radiotherapy by ThermoDox (thermosensitive liposome containing doxorubicin), II) the concentration dependence of the radiosensitizing effect of doxorubicin and III) the sequence of doxorubicin, hyperthermia and radiotherapy maximizing the radiosensitizing effect. METHODS: Survival of HT1080 (human fibrosarcoma) cells was measured after exposure to ThermoDox or doxorubicin for 60 minutes, at 37 or 43°C, with or without irradiation. Furthermore, cell survival was measured for cells exposed to different doxorubicin concentrations and radiation doses. Finally, cell survival was measured after applying doxorubicin and/or hyperthermia before or after irradiation. Cell survival was measured by clonogenic assay. In addition, DNA damage was assessed by γH2AX staining. RESULTS: Exposure of cells to doxorubicin at 37°C resulted in cell death, but exposure to ThermoDox at 37°C did not. In contrast, ThermoDox and doxorubicin at 43°C resulted in similar cytotoxicity, and in combination with irradiation caused a similar enhancement of cell kill due to radiation. Doxorubicin enhanced the radiation effect in a small, but significant, concentration-dependent manner. Hyperthermia showed the strongest enhancement of radiation effect when applied after irradiation. In contrast, doxorubicin enhanced radiation effect only when applied before irradiation. Concurrent doxorubicin and hyperthermia immediately before or after irradiation showed equal enhancement of radiation effect. CONCLUSION: In vitro, ThermoDox resulted in cytotoxicity and enhancement of irradiation effect only in combination with hyperthermia. Therefore hyperthermia-triggered radiosensitizer release from thermosensitive liposomes may ultimately serve to limit toxicities due to the radiosensitizer in unheated normal tissue and result in enhanced efficacy in the heated tumor.

Influence of cholesterol inclusion on the doxorubicin release characteristics of lysolipid-based thermosensitive liposomes.

Fast hyperthermia (i.e. 39-42 °C) triggered doxorubicin release from lysolipid-containing thermosensitive liposomes (LTSL) in the tumor vasculature has been demonstrated to result in considerable enhancement of bioavailable drug levels in heated tumor tissue in preclinical tumor models. However, there is also significant leakage of doxorubicin already at 37 °C in the bloodstream, making these LTSL less efficient and increasing the risk for systemic toxicity. In conventional liposomes, cholesterol is incorporated in the bilayer to increase the stability of the liposomes. Here, we investigate the effect of cholesterol inclusion on the doxorubicin release characteristics of LTSL at 37 °C and hyperthermic temperatures. For this purpose, three LTSL formulations with 0, 5 and 10 mol% cholesterol were prepared. Inclusion of cholesterol reduced the undesired doxorubicin leakage at 37 °C in Hepes-buffered saline (HBS) as well as in fetal bovine serum (FBS). The incorporation of cholesterol in the LTSL bilayers did not influence the hyperthermia-triggered release property of the LTSL. These results were supported by DSC measurements. Therefore, in conclusion, our data indicate that cholesterol inclusion in LTSL offers a simple solution to the problem of significant leakage of doxorubicin from LTSL already at 37 °C in the bloodstream.

A stimuli responsive liposome loaded hydrogel provides flexible on-demand release of therapeutic agents.

Lysolipid-based thermosensitive liposomes (LTSL) embedded in a chitosan-based thermoresponsive hydrogel matrix (denoted Lipogel) represents a novel approach for the spatiotemporal release of therapeutic agents. The entrapment of drug-loaded liposomes in an injectable hydrogel permits local liposome retention, thus providing a prolonged release in target tissues. Moreover, release can be controlled through the use of a minimally invasive external hyperthermic stimulus. Temporal control of release is particularly important for complex multi-step physiological processes, such as angiogenesis, in which different signals are required at different times in order to produce a robust vasculature. In the present work, we demonstrate the ability of Lipogel to provide a flexible, easily modifiable release platform. It is possible to tune the release kinetics of different drugs providing a passive release of one therapeutic agent loaded within the gel and activating the release of a second LTSL encapsulated agent via a hyperthermic stimulus. In addition, it was possible to modify the drug dosage within Lipogel by varying the duration of hyperthermia. This can allow for adaption of drug dosing in real time. As an in vitro proof of concept with this system, we investigated Lipogels ability to recruit stem cells and then elevate their production of vascular endothelial growth factor (VEGF) by controlling the release of a pro-angiogenic drug, desferroxamine (DFO) with an external hyperthermic stimulus. Initial cell recruitment was accomplished by the passive release of hepatocyte growth factor (HGF) from the hydrogel, inducing a migratory response in cells, followed by the delayed release of DFO from thermosensitive liposomes, resulting in a significant increase in VEGF expression. This delayed release could be controlled up to 14days. Moreover, by changing the duration of the hyperthermic pulse, a fine control over the amount of DFO released was achieved. The ability to trigger the release of therapeutic agents at a specific timepoint and control dosing level through changes in duration of hyperthermia enables sequential multi-dose profiles. STATEMENT OF SIGNIFICANCE: This paper details the development of a heat responsive liposome loaded hydrogel for the controlled release of pro-angiogenic therapeutics. Lysolipid-based thermosensitive liposomes (LTSLs) embedded in a chitosan-based thermoresponsive hydrogel matrix represents a novel approach for the spatiotemporal release of therapeutic agents. This hydrogel platform demonstrates remarkable flexibility in terms of drug scheduling and sequencing, enabling the release of multiple agents and the ability to control drug dosing in a minimally invasive fashion. The possibility to tune the release kinetics of different drugs independently represents an innovative platform to utilise for a variety of treatments. This approach allows a significant degree of flexibility in achieving a desired release profile via a minimally invasive stimulus, enabling treatments to be tuned in response to changing symptoms and complications.

T1 and T2 temperature dependence of female human breast adipose tissue at 1.5 T: groundwork for monitoring thermal therapies in the breast.

The T1 and T2 temperature dependence of female breast adipose tissue was investigated at 1.5 T in order to evaluate the applicability of relaxation-based MR thermometry in fat for the monitoring of thermal therapies in the breast. Relaxation times T1 , T2 and T2TSE (the apparent T2 measured using a turbo spin echo readout sequence) were measured in seven fresh adipose breast samples for temperatures from 25 to 65 °C. Spectral water suppression was used to reduce the influence of the residual water signal. The temperature dependence of the relaxation times was characterized. The expected maximum temperature measurement errors based on average calibration lines were calculated. In addition, the heating-cooling reversibility was investigated for two samples. The T1 and T2TSE temperature (T) dependence could be fitted well with an exponential function of 1/T. A linear relationship between T2 and temperature was found. The temperature coefficients (mean ± inter-sample standard deviation) of T1 and T2TSE increased from 25 °C (dT1/dT = 5.35 ± 0.08 ms/°C, dT2TSE/dT = 3.82 ± 0.06 ms/°C) to 65 °C (dT1 /dT = 9.50 ± 0.16 ms/°C, dT2TSE/dT = 7.99 ± 0.38 ms/°C). The temperature coefficient of T2 was 0.90 ± 0.03 ms/°C. The temperature-induced changes in the relaxation times were found to be reversible after heating to 65 °C. Given the small inter-sample variation of the temperature coefficients, relaxation-based MR thermometry appears to be feasible in breast adipose tissue, and may be used as an adjunct to proton resonance frequency shift (PRFS) thermometry in aqueous tissue (glandular + tumor).

Absolute MR thermometry using nanocarriers.

Accurate time-resolved temperature mapping is crucial for the safe use of hyperthermia-mediated drug delivery. We here propose a magnetic resonance imaging temperature mapping method in which drug delivery systems serve not only to improve tumor targeting, but also as an accurate and absolute nano-thermometer. This method is based on the temperature-dependent chemical shift difference between water protons and the protons in different groups of drug delivery systems. We show that the chemical shift of the protons in the ethylene oxide group in polyethylene glycol (PEG) is temperature-independent, whereas the proton resonance of water decreases with increasing temperature. The frequency difference between both resonances is linear and does not depend on pH and physiological salt conditions. In addition, we show that the proton resonance of the methyl group in N-(2-hydroxypropyl)-methacrylamide (HPMA) is temperature-independent. Therefore, PEGylated liposomes, polymeric mPEG-b-pHPMAm-Lac2 micelles and HPMA copolymers can provide a temperature-independent reference frequency for absolute magnetic resonance (MR) thermometry. Subsequently, we show that multigradient echo MR imaging with PEGylated liposomes in situ allows accurate, time-resolved temperature mapping. In conclusion, nanocarrier materials may serve as highly versatile tools for tumor-targeted drug delivery, acting not only as hyperthermia-responsive drug delivery systems, but also as accurate and precise nano-thermometers.

Arrhenius analysis of the relationship between hyperthermia and Hsp70 promoter activation: a comparison between ex vivo and in vivo data.

PURPOSE: Tight regulation of gene expression in the region where therapy is necessary and for the duration required to achieve a therapeutic effect and to minimise systemic toxicity is very important for clinical applications of gene therapy. Hyperthermia in combination with a temperature sensitive heat shock protein (Hsp70) promoter presents a unique approach allowing non-invasive spatio-temporal control of transgene expression. In this study we investigated the in vivo and ex vivo relationship between temperature and duration of thermal stress with respect to the resulting gene expression using an Arrhenius analysis. MATERIALS AND METHODS: A transgenic mouse expressing the luciferase reporter gene under the transcriptional control of a thermosensitive promoter was used to assure identical genotype for in vivo (mouse leg) and ex vivo (bone marrow mononuclear and embryonic fibroblast cells) studies. The mouse leg and cells were heated at different temperatures and different exposure times. Bioluminescence imaging and in vitro enzymatic assay were used to measure the resulting transgene expression. RESULTS: We showed that temperature-induced Hsp70 promoter activation was modulated by both temperature as well as duration of hyperthermia. The relationship between temperature and duration of hyperthermia and the resulting reporter gene expression can be modelled by an Arrhenius analysis for both in vivo as well as ex vivo. CONCLUSIONS: However, the increase in reporter gene expression after elevating the temperature of the thermal stress with 1°C is not comparable for in vivo and ex vivo situations. This information may be valuable for optimising clinical gene therapy protocols.