Total ice content and lipid saturation determine adipose tissue cryolipolysis by injection of ice-slurry.

OBJECTIVES: Cryolipolysis uses tissue cooling to solidify lipids, preferentially damaging lipid-rich cells. Topical cooling is popular for the reduction of local subcutaneous fat. Injection of biocompatible ice-slurry is a recently introduced alternative. We developed and verified a quantitative model that simulates the heat exchange and phase changes involved, offering insights into ice-slurry injection for treating subcutaneous fat. METHODS: Finite element method was used to model the spatial and temporal progression of heat transfer between adipose tissue and injected ice-slurry, estimating dose-response relationships between properties of the slurry and size of tissue affected by cryolipolysis. Phase changes of both slurry and adipose tissue lipids were considered. An in vivo swine model was used to validate the numerical solutions. Oils with different lipid compositions were exposed to ice-slurry in vitro to evaluate the effects of lipid freezing temperature. Microscopy and nuclear magnetic resonance (NMR) were performed to detect lipid phase changes. RESULTS: A ball of granular ice was deposited at the injection site in subcutaneous fat. Total injected ice content determines both the effective cooling region of tissue, and the duration of tissue cooling. Water's high latent heat of fusion enables tissue cooling long after slurry injection. Slurry temperature affects the rate of tissue cooling. In swine, when 30 ml slurry injection at -3.5°C was compared to 15 ml slurry injection at -4.8°C (both with the same total ice content), the latter led to almost twice faster tissue cooling. NMR showed a large decrease in diffusion upon lipid crystallization; saturated lipids with higher freezing temperatures were more susceptible to solidification after ice-slurry injection. CONCLUSIONS: Total injected ice content determines both the volume of tissue treated by cryolipolysis and the cooling duration after slurry injection, while slurry temperature affects the cooling rate. Lipid saturation, which varies with diet and anatomic location, also has an important influence.

Extracorporeal Removal of Thermosensitive Liposomal Doxorubicin from Systemic Circulation after Tumor Delivery to Reduce Toxicities.

Thermosensitive liposomal doxorubicin (TSL-Dox) combined with localized hyperthermia enables targeted drug delivery. Tumor drug uptake occurs only during hyperthermia. We developed a novel method for removal of systemic TSL-Dox remaining after hyperthermia-triggered delivery to reduce toxicities. The carotid artery and jugular vein of Norway brown rats carrying two subcutaneous BN-175 tumors were catheterized. After allowing the animals to recover, TSL-Dox was infused at 7 mg/kg dose. Drug delivery to one of the tumors was performed by inducing 15 min microwave hyperthermia (43 °C). At the end of hyperthermia, an extracorporeal circuit (ECC) comprising a heating module to release drug from TSL-Dox followed by an activated carbon filter to remove free drug was established for 1 h (n = 3). A computational model simulated TSL-Dox pharmacokinetics, including ECC filtration, and predicted cardiac Dox uptake. In animals receiving ECC, we were able to remove 576 ± 65 mg of Dox (29.7 ± 3.7% of the infused dose) within 1 h, with a 2.9-fold reduction of plasma AUC. Fluorescent monitoring enabled real-time quantification of blood concentration and removed drug. Computational modeling predicted that up to 59% of drug could be removed with an ideal filter, and that cardiac uptake can be reduced up to 7×. We demonstrated removal of drug remaining after tumor delivery, reduced plasma AUC, and reduced cardiac uptake, suggesting reduced toxicity.

Untargeted Large Volume Hyperthermia Reduces Tumor Drug Uptake From Thermosensitive Liposomes.

GOAL: The impact of hyperthermia (HT) method on tumor drug uptake with thermosensitive liposomes (TSL) is not well understood. METHODS: We created realistic three-dimensional (3-D) computer models that simulate TSL-encapsulated doxorubicin (TSL-DOX) delivery in mouse tumors with three HT methods (thermistor probe (T), laser (L) and water bath (WB), at 15 min and 60 min HT duration), with corroborating in vivo studies. RESULTS: Average computer model-predicted tumor drug concentrations (µg/g) were 8.8(T, 15 min), 21.0(T, 60 min), 14.1(L, 15 min), 25.2(L, 60 min), 9.4(WB, 15 min), and 8.7(WB, 60 min). Tumor fluorescence was increased by 2.6 × (T) and 1.6 × (L) when HT duration was extended from 15 to 60 min (p < 0.05), with no increase for WB HT. Pharmacokinetic analysis confirmed that water bath HT causes rapid depletion of encapsulated TSL-DOX in systemic circulation due to the large heated tissue volume. CONCLUSIONS: Untargeted large volume HT causes poor tumor drug uptake from TSL.

Computer simulations of an irrigated radiofrequency cardiac ablation catheter and experimental validation by infrared imaging.

PURPOSE: To develop and validate a three-dimensional (3-D) computer model based on accurate geometry of an irrigated cardiac radiofrequency (RF) ablation catheter with microwave radiometry capability, and to test catheter performance. METHODS: A computer model was developed based on CAD geometry of a RF cardiac ablation catheter prototype to simulate electromagnetic heating, heat transfer, and computational fluid dynamics (blood flow, open irrigation, and natural convection). Parametric studies were performed; blood flow velocity (0-25 cm/s) and irrigation flow (0-40 ml/min) varied, both with perpendicular (PE) and parallel (PA) catheter orientations relative to tissue. Tissue Agar phantom studies were performed under similar conditions, and temperature maps were recorded via infrared camera. Computer model simulations were performed with constant voltage and with voltage adjusted to achieve maximum tissue temperatures of 95-105 °C. RESULTS: Model predicted thermal lesion width at 5 W power was 5.8-6.4 mm (PE)/6.5-6.6 mm (PA), and lesion depth was 4.0-4.3 mm (PE)/4.0-4.1 mm (PA). Compared to phantom studies, the mean errors of the computer model were as follows: 6.2 °C(PE)/4.3 °C (PA) for maximum gel temperature, 0.7 mm (10.9%) (PE)/0.1 mm (0.8%) (PA) for lesion width, and 0.3 mm (7.7%)(PE)/0.7 mm (19.1%) (PA) for lesion depth. For temperature-controlled ablation, model predicted thermal lesion width was 7-9.2 mm (PE)/8.6-9.2 mm (PA), and lesion depth was 4.3-5.5 mm (PE)/3.4-5.4 mm (PA). CONCLUSIONS: Computer models were able to reproduce device performance and to enable device evaluation under varying conditions. Temperature controlled ablation of irrigated catheters enables optimal tissue temperatures independent of patient-specific conditions such as blood flow.

Closed-loop trans-skull ultrasound hyperthermia leads to improved drug delivery from thermosensitive drugs and promotes changes in vascular transport dynamics in brain tumors.

Effective drug delivery in brain tumors remains a major challenge in oncology. Although local hyperthermia and stimuli-responsive delivery systems, such as thermosensitive liposomes, represent promising strategies to locally enhance drug delivery in solid tumors and improve outcomes, their application in intracranial malignancies remains unexplored. We hypothesized that the combined abilities of closed-loop trans-skull Magnetic Resonance Imaging guided Focused Ultrasound (MRgFUS) hyperthermia with those of thermosensitive drugs can alleviate challenges in drug delivery and improve survival in gliomas. Methods: To conduct our investigations, we first designed a closed loop MR-guided Focused Ultrasound (MRgFUS) system for localized trans-skull hyperthermia (ΔT < 0.5 °C) in rodents and established safety thresholds in healthy mice. To assess the abilities of the developed system and proposed therapeutic strategy for FUS-triggered chemotherapy release we employed thermosensitive liposomal Dox (TSL-Dox) and tested it in two different glioma tumor models (F98 in rats and GL261 in mice). To quantify Dox delivery and changes in the transvascular transport dynamics in the tumor microenvironment we combined fluorescent microscopy, dynamic contrast enhanced MRI (DCE-MRI), and physiologically based pharmacokinetic (PBPK) modeling. Lastly, to assess the therapeutic efficacy of the system and of the proposed therapeutic strategy we performed a survival study in the GL261 glioma bearing mice. Results: The developed closed-loop trans-skull MRgFUS-hyperthermia system that operated at 1.7 MHz, a frequency that maximized the brain (FUS-focus) to skull temperature ratio in mice, was able to attain and maintain the desired focal temperature within a narrow range. Histological evidence (H&E and Nissl) suggests that focal temperature at 41.5 ± 0.5 °C for 10 min is below the threshold for tissue damage. Quantitative analysis of doxorubicin delivery from TSLs with MRgFUS-hyperthermia demonstrated 3.5-fold improvement in cellular uptake in GL261 glioma mouse tumors (p < 0.001) and 5-fold increase in delivery in F98 glioma rat tumors (p < 0.05), as compared to controls (TSL-Dox-only). Moreover, PBPK modeling of drug transport that was calibrated using the experimental data indicated that thermal stress could lead to significant improvement in the transvascular transport (2.3-fold increase in the vessel diffusion coefficient; P < 0.001), in addition to promoting targeted Dox release. Prospective experimental investigations with DCE-MRI during FUS-hyperthermia, supported these findings and provided evidence that moderate thermal stress (≈41 °C for up to 10 min) can promote acute changes in the vascular transport dynamics in the brain tumor microenvironment (Ktrans value for control vs. FUS was 0.0097 and 0.0148 min-1, respectively; p = 0.026). Crucially, survival analysis demonstrated significant improvement in the survival in the TSL-Dox-FUS group as compared to TSL-Dox-only group (p < 0.05), providing supporting evidence on the therapeutic potential of the proposed strategy. Conclusions: Our investigations demonstrated that spatially controlled thermal stress can be attained and sustained in the mouse brain, using a trans-skull closed-loop MRgFUS system, and used to promote the effective delivery of chemotherapy in gliomas from thermosensitive drugs. This system also allowed us to conduct mechanistic investigations that resulted in the refinement of our understanding on the role of thermal stress in augmenting mass and drug transport in brain tumors. Overall, our study established a new paradigm for effective drug delivery in brain tumors based on closed-loop ultrasound-mediated thermal stress and thermosensitive drugs.

Computer Modeling of Drug Delivery with Thermosensitive Liposomes in a Realistic Three-Dimensional Geometry.

Thermosensitive liposomes (TSL) are nanoparticles that can encapsulate therapeutic drugs, and release those drugs when exposed to hyperthermic temperatures (>40 °C). Combined with localized hyperthermia, TSL enable focused drug delivery. In this study, we created a three-dimensional (3D) computer model for simulating delivery with TSL-encapsulated doxorubicin (TSL-Dox) to mouse tumors. A mouse hind limb was scanned by a 3D scanner and the resulting geometry was imported into finite element modeling software, with a virtual tumor added. Then, heating by a surface probe was simulated. Further, a drug delivery model was coupled to the heat transfer model to simulate drug delivery kinetics. For comparison, experimental studies in gel phantoms and in vivo fluorescence imaging studies in mice carrying lung tumor xenografts were performed. We report the tissue temperature profile, drug concentration profile and compare the experimental studies with the computer model. The thermistor produced very localized heating that resulted in highest drug delivery to regions near the probe. The average tumor temperature was 38.2˚C (range 34.4-43.4˚C), and produced an average tumor drug concentration of 11.8 µg/g (0.3-28.1 µg/g) after 15 min heating, and 25.6 µg/g (0.3-52 µg/g), after 60 min heating. The computer model reproduced the temperature profile compared to phantom experiments (mean error 0.71 °C, range 0.59-1.25 °C), as well as drug delivery profile as compared to in vivo studies. Our results suggest feasibility of using this approach to model drug delivery in preclinical studies with accurate model geometry.

Real-time fluorescence imaging for visualization and drug uptake prediction during drug delivery by thermosensitive liposomes.

Objective: Thermosensitive liposomal doxorubicin (TSL-Dox) is a promising stimuli-responsive nanoparticle drug delivery system that rapidly releases the contained drug in response to hyperthermia (HT) (>40 °C). Combined with localized heating, TSL-Dox allows highly localized delivery. The goals of this study were to demonstrate that real-time fluorescence imaging can visualize drug uptake during delivery, and can predict tumor drug uptake. Methods: Nude mice carrying subcutaneous tumors (Lewis lung carcinoma) were anesthetized and injected with TSL-Dox (5 mg/kg dose). Localized HT was induced by heating tumors for 15, 30 or 60 min via a custom-designed HT probe placed superficially at the tumor location. In vivo fluorescence imaging (excitation 523 nm, emission 610 nm) was performed before, during, and for 5 min following HT. After imaging, tumors were extracted, drug uptake was quantified by high-performance liquid chromatography, and correlated with in vivo fluorescence. Plasma samples were obtained before and after HT to measure TSL-Dox pharmacokinetics. Results: Local drug uptake could be visualized in real-time during HT. Compared to unheated control tumors, fluorescence of heated tumors increased by 4.6-fold (15 min HT), 9.3-fold (30 min HT), and 13.2-fold (60 min HT). HT duration predicted tumor drug uptake (p = .02), with tumor drug concentrations of 4.2 ± 1.3 µg/g (no HT), 7.1 ± 5.9 µg/g (15 min HT), 14.1 ± 6.7 µg/g (30 min HT) and 21.4 ± 12.6 µg/g (60 min HT). There was good correlation (R2 = 0.67) between fluorescence of the tumor region and tumor drug uptake. Conclusions: Real-time in vivo fluorescence imaging can visualize drug uptake during delivery, and can predict tumor drug uptake.

In vitro Measurement of Release Kinetics of Temperature Sensitive Liposomes with a Fluorescence Imaging System.

Temperature sensitive liposomes (TSL) are a promising type of nanoparticles for localized drug delivery. TSL typically release the contained drug at mild hyperthermic temperatures (40-42 °C). Combined with localized hyperthermia, this allows for local drug delivery. In vitro characterization of TSL involves measurements of drug release at varying temperatures, but current methods are inadequate due to low temporal resolution of ~8 - 10 seconds. We present a novel method for measuring the drug release with sub-second temporal resolution. In the proposed system, the TSL entrapping the fluorescent drug (Doxorubicin) are pumped through a capillary tube. The tube is rapidly heated to a desired temperature via Peltier element. Since fluorescence increases as drug is released from TSL, drug release kinetics can be measured via fluorescent imaging. By fitting exponential models, we calculated the time constants of drug release at temperatures of 39.5, 40.5 and 41.5.C were about 6.09, 2.06 and 1.03 seconds, respectively. Our initial tests show that the developed system can measure TSL release at subsecond resolution, and thus allow adequate in vitro evaluation of TSL formulations.

Thermosensitive Liposomes for Image-Guided Drug Delivery.

Liposomes have been employed as cancer therapy clinically since the 1990s, with the primary benefit of reduced toxicity but no appreciable efficacy improvement. Thermosensitive liposomes (TSLs) are specifically formulated such that they release the encapsulated drug when exposed to hyperthermic temperatures in the fever range (~40-42°C) and have been investigated as cancer therapy for several decades, with first clinical trials initiated in the last decade. Combined with localized hyperthermia, TSLs allow precise drug delivery to a targeted region. Typically, the targeted tissue is exposed to localized hyperthermia facilitated by an image-guided hyperthermia device. Thus, TSLs enable image-guided drug delivery where drug is delivered to a tissue region identified by medical imaging. Recent TSL formulations are based on the more recent paradigm of intravascular triggered release, where drug is released rapidly (within seconds) while TSLs pass through the vasculature of the heated tissue region. The drug released within the blood then extravasates and is taken up by cancer cells. These TSLs enable up to 20-30 times higher tumor drug uptake compared to infusion of unencapsulated drug, and the dose locally delivered to the heated region can be modulated based on heating duration. This chapter reviews various TSL formulations, the different anticancer agents that have been encapsulated, as well as targeted cancer types. Further, the various hyperthermia devices that have been used for image-guided hyperthermia are reviewed, focusing on those that have been employed in human patients.

Localized delivery of therapeutic doxorubicin dose across the canine blood-brain barrier with hyperthermia and temperature sensitive liposomes.

Most drugs cannot penetrate the blood-brain barrier (BBB), greatly limiting the use of anti-cancer agents for brain cancer therapy. Temperature sensitive liposomes (TSL) are nanoparticles that rapidly release the contained drug in response to hyperthermia (>40 °C). Since hyperthermia also transiently opens the BBB, we hypothesized that localized hyperthermia can achieve drug delivery across the BBB when combined with TSL. TSL-encapsulated doxorubicin (TSL-Dox) was infused intravenously over 30 min at a dose of 0.94 mg/kg in anesthetized beagles (age ∼17 months). Following, a hyperthermia probe was placed 5-10 mm deep through one of four 3-mm skull burr holes. Hyperthermia was performed randomized for 15 or 30 min, at either 45 or 50 °C. Blood was drawn every 30 min to measure TSL-Dox pharmacokinetics. Nonsurvival studies were performed in four dogs, where brain tissue at the hyperthermia location was extracted following treatment to quantify doxorubicin uptake via high-performance liquid chromatography (HPLC) and to visualize cellular uptake via fluorescence microscopy. Survival studies for 6 weeks were performed in five dogs treated by a single hyperthermia application. Local doxorubicin delivery correlated with hyperthermia duration and ranged from 0.11 to 0.74 µg/g of brain tissue at the hyperthermia locations, with undetectable drug uptake in unheated tissue. Fluorescence microscopy demonstrated doxorubicin delivery across the BBB. Histopathology in Haematoxylin & Eosin (H&E) stained samples demonstrated localized damage near the probe. No animals in the survival group demonstrated significant neurological deficits. This study demonstrates that localized doxorubicin delivery to the brain can be facilitated by TSL-Dox with localized hyperthermia with no significant neurological deficits.

Thermal Therapy Approaches for Treatment of Brain Tumors in Animals and Humans.

Primary brain tumors are often aggressive, with short survival from time of diagnosis even with standard of care therapies such as surgery, chemotherapy, and radiation therapy. Thermal therapies have been extensively investigated as both primary and adjuvant therapy. Although thermal therapies are not yet widely used clinically, there have been several promising approaches demonstrated in both animals and humans. This review presents thermal therapy approaches in animal and human studies, including both hyperthermia (temperatures ~42°C-45°C) and thermal ablation (temperatures > 50°C). Hyperthermia is primarily used as adjuvant to chemotherapy and radiotherapy, and is the most widely studied radiation sensitizer where enhanced efficacy has been shown in human patients with brain cancer. Hyperthermia has additional beneficial effects such as immunogenic effects, and opening of the bloodbrain barrier to potentially enhance drug delivery, for example in combination with nanoparticle drug delivery systems. Thermal ablation uses high temperatures for direct local tumor destruction, and it found its way into clinical use as laser interstitial thermal therapy (LITT). This review presents various hyperthermia and ablation approaches, including a review of different devices and methods that have been used for thermal therapies, such as radiofrequency/microwaves, laser, high-intensity focused ultrasound, and magnetic nanoparticles. Current research efforts include the combination of advanced thermal therapy devices, such as focused ultrasound with radiation, as well as the use of thermal therapies to enhance chemotherapy delivery across the blood-brain barrier.

Development of a radiofrequency ablation platform in a clinically relevant murine model of hepatocellular cancer.

RFA is used in treatment of patients with hepatocellular cancer (HCC); however, tumor location and size often limit therapeutic efficacy. The absence of a realistic animal model and a radiofrequency ablation (RFA) suitable for small animals presents significant obstacles in developing new strategies. To establish a realistic RFA platform that allows the development of effective RFA-integrated treatment in an orthotopic murine model of HCC, a human cardiac radiofrequency generator was modified for murine use. Parameters were optimized and RFA was then performed in normal murine livers and HCCs. The effects of RFA were monitored by measuring the ablation zone and transaminases. The survival of tumor-bearing mice with and without RFA was monitored, ablated normal liver and HCCs were evaluated macroscopically and histologically. We demonstrated that tissue-mimicking media was able to optimize RFA parameters. Utilizing this information we performed RFA in normal and HCC-bearing mice. RFA was applied to hepatic parenchyma and completely destroyed small tumors and part of large tumors. Localized healing of the ablation and normalization of transaminases occurred within 7 days post RFA. RFA treatment extended the survival of small tumor-bearing mice. They survived at least 5 months longer than the controls; however, mice with larger tumors only had a slight therapeutic effect after RFA. Collectively, we performed RFA in murine HCCs and observed a significant therapeutic effect in small tumor-bearing mice. The quick recovery of tumor-bearing mice receiving RFA mimics observations in human subjects. This platform provides us a unique opportunity to study RFA in HCC treatment.

Hematopoietic stem cell-derived cancer-associated fibroblasts are novel contributors to the pro-tumorigenic microenvironment.

Targeting the tumor microenvironment is critical toward improving the effectiveness of cancer therapeutics. Cancer-associated fibroblasts (CAFs) are one of the most abundant cell types of the tumor microenvironment, playing an important role in tumor progression. Multiple origins for CAFs have been proposed including resident fibroblasts, adipocytes, and bone marrow. Our laboratory previously identified a novel hematopoietic stem cell (HSC) origin for CAFs; however, the functional roles of HSC-derived CAFs (HSC-CAFs) in tumor progression have not yet been examined. To test the hypothesis that HSC-CAFs promote tumor progression through contribution to extracellular matrix (ECM) and paracrine production of pro-angiogenic factors, we developed a method to isolate HSC-CAFs. HSC-CAFs were profiled on the basis of their expression of hematopoietic and fibroblastic markers in two murine tumor models. Profiling revealed production of factors associated with ECM deposition and remodeling. Functional in vivo studies showed that co-injection of HSC-CAFs with tumor cells resulted in increased tumor growth rate and significantly larger tumors than tumor cells alone. Immunohistochemical studies revealed increased blood vessel density with co-injection, demonstrating a role for HSC-CAFs in tumor vascularization. Mechanistic in vitro studies indicated that HSC-CAFs play a role in producing vascular endothelial growth factor A and transforming growth factor-ß1 in endothelial tube formation and patterning. In vitro and in vivo findings suggest that HSC-CAFs are a critical component of the tumor microenvironment and suggest that targeting the novel HSC-CAF may be a promising therapeutic strategy.

Mitochondrial electron transport chain complex dysfunction in the colonic mucosa in ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is characterized by an energy deficiency state of the colonic epithelium. This study evaluated mitochondrial electron transport chain (ETC) complex activity in normal and disease mucosa in patients with UC. Alterations in ETC complexes were also investigated in experimental colitis in mice. METHODS: Biopsies were obtained from macroscopically normal and diseased colonic mucosa of 43 patients with UC and 35 controls undergoing screening colonoscopy and ETC complex activity was assayed biochemically. ETC complex activities were also assayed in colonic epithelial cells isolated from Swiss albino mice with dextran sodium sulfate (DSS)-induced colitis at various stages of induction of colitis. Mucosal nitrite levels and protein carbonyl content were determined. RESULTS: The activity of Complex II was significantly decreased in colonic biopsies from UC patients compared with controls, while activities of other mitochondrial complex were normal. Complex II activity was equally decreased in diseased and normal mucosa in UC; the degree of reduction did not correlate with clinical, endoscopic, or histological grading of disease activity. In DSS-fed mice, a reduction in activity of Complex IV and Complex II was observed. Activity of other complex was not affected. Administration of aminoguanidine, an inducible nitric oxide synthase (iNOS) inhibitor, attenuated all parameters of colitis as well as the reductions in Complex IV and Complex II activity. CONCLUSIONS: Reduction in Complex II activity appears to be a specific change in UC, present in quiescent and active disease. Mitochondrial complex dysfunction occurs in DSS colitis in mice and appears to be mediated by nitric oxide.