Thermosensitive Liposomes

Thermosensitive liposomes (TSLs) are a drug delivery system for targeted delivery that release the encapsulated drug when heated to fever temperatures (∼40–42°C). Combined with localized hyperthermia, TSLs allow precise drug delivery to a targeted region. While mostly investigated as cancer therapy, other applications including treatment of local infections and wound healing have been explored. Over the last ∼40 years, numerous TSL formulations and payloads have been investigated. As with other nanoparticles, the addition of targeting molecules to TSL has been examined to improve targeted delivery. TSL release kinetics and plasma stability are two important factors that affect efficacy, and new formulations often aim to further improve on these properties. The possibility of encapsulating a magnetic resonance (MR) contrast agent that is released together with the encapsulated drug allows for visualization of drug delivery with MR imaging. Various heating modalities have been examined in combination with TSL. Since the goal is to expose a defined tissue region to uniform temperatures within the range where TSLs release (typically ∼40–43°C), the choice of an appropriate heating modality has considerable impact on treatment efficacy. Several ongoing clinical trials with TSL as cancer therapy suggest the potential for clinical impact in the near future

Monitoring Focal Laser Ablation of Prostate Cancer with Interstitial Thermal and Optical Probes

Prostate cancer is diagnosed in almost 200,000 men in the U.S. annually. With approximately 30,000 deaths, the mortality to incidence ratio is relatively low in comparison to other common cancers. Moreover, the primary treatment modalities, radical prostatectomy and radiation therapy, are associated with a substantial reduction in quality of life. Consequently, there is a growing interest in focal therapy which aims to treat the target tumor while minimizing damage to surrounding tissue.Focal laser ablation (FLA) is a form of focal therapy in which a laser fiber is inserted into a target tumor and oncologic control is achieved through thermally induced coagulative necrosis. Many groups have performed FLA using magnetic resonance imaging (MRI) for laser fiber targeting and real-time feedback. While this approach has shown promise, we contend that the resource intensive nature of MRI will forever preclude widespread adoption of FLA.This thesis presents a concerted effort to translate FLA from the MRI suite to the urology clinic. To this end we performed a clinical trial using magnetic resonance – ultrasound fusion guidance and interstitial thermal probes for treatment targeting and monitoring respectively. This approach proved to be safe and potentially effective; however, the utility of thermal probes was found to be inherently limited due to the need for empirically derived thermal damage models.In an effort to provide an improved monitoring modality we developed an interstitial optical monitoring system that directly assesses the state of tissue based on laser-tissue interaction. To correlate the optical signal and the growth of the coagulation zone we created a tissue mimicking phantom which simulates the thermal and optical response of prostatic tissue and facilitates visualization of the coagulation zone on MRI. FLA was performed in the phantom under MRI surveillance with simultaneous interstitial optical monitoring resulting in the development of a real-time feedback algorithm. The algorithm was subsequently tested in ex vivo bovine tissue and was capable of identifying the coagulation border with a mean absolute error of 0.3�0.1mm. Further work is necessary to demonstrate the utility of interstitial optical monitoring in vivo

Development of Targeted Liposomal Formulation Approaches for Enhanced Colorectal Cancer Therapy

Colorectal cancer (CRC) is the 4th most commonly detected cancer in the USA. Despite promising advances, the 5-year survival rate for the metastatic disease remains dismal (40⁰C with ultrasound contrast agents and bacterial attachments can improve the real-time chemo-immunotherapy of CRC. Towards these goals, we investigated the following specific aims in murine models of colon cancer: 1) Develop echogenic-LTSL (E-LTSL) for real-time ultrasound-enhanced reporting of tumor temperature and doxorubicin delivery, 2) Utilize tumor homing Salmonella typhimurium for LTSL delivery and enhanced chemo-immunotherapy with High Intensity Focused Ultrasound (HIFU) tumor heating (~42°C), and 3) Investigate the ability of magnetic bacteria Magnetospirillim magneticum (AMB-1) to aid LTSL tumor drug delivery under magnetic guidance. Our data showed that intratumoral vascular contrast of E-LTSL as a function of temperature and doxorubicin delivery was strongly correlated, enabling robust estimation of temporal variation in colon tumor temperature and drug delivery. LTSL attachment didn’t impact Salmonella viability and improved chemo-immunotherapy outcomes in murine colon cancers by promoting the population of M1 macrophages with HIFU heating. Finally, the use of magnetic guidance for AMB-LTSL significantly reduced the colon cancer viability by enhancing cellular and tumor localizations of doxorubicin. In conclusion, we found that multifunctional LTSL formulations significantly improved the CRC treatment outcomes in murine models by aiding the real-time monitoring and removing the resistive and suppressive tumor microenvironment features

How sonoporation disrupts cellular structural integrity: morphological and cytoskeletal observations

Posters: no. 1Control ID: 1672429OBJECTIVES: In considering sonoporation for drug delivery applications, it is essential to understand how living cells respond to this puncturing force. Here we seek to investigate the effects of sonoporation on cellular structural integrity. We hypothesize that the membrane morphology and cytoskeletal behavior of sonoporated cells under recovery would inherently differ from that of normal viable cells. METHODS: A customized and calibrated exposure platform was developed for this work, and the ZR-75-30 breast carcinoma cells were used as the cell model. The cells were exposed to either single or multiple pulses of 1 MHz ultrasound (pulse length: 30 or 100 cycles; PRF: 1kHz; duration: up to 60s) with 0.45 MPa spatial-averaged peak negative pressure and in the presence of lipid-shelled microbubbles. Confocal microscopy was used to examine insitu the structural integrity of sonoporated cells (identified as ones with exogenous fluorescent marker internalization). For investigations on membrane morphology, FM 4-64 was used as the membrane dye (red), and calcein was used as the sonoporation marker (green); for studies on cytoskeletal behavior, CellLight (green) and propidium iodide (red) were used to respectively label actin filaments and sonoporated cells. Observation started from before exposure to up to 2 h after exposure, and confocal images were acquired at real-time frame rates. Cellular structural features and their temporal kinetics were quantitatively analyzed to assess the consistency of trends amongst a group of cells. RESULTS: Sonoporated cells exhibited membrane shrinkage (decreased by 61% in a cell’s cross-sectional area) and intracellular lipid accumulation (381% increase compared to control) over a 2 h period. The morphological repression of sonoporated cells was also found to correspond with post-sonoporation cytoskeletal processes: actin depolymerization was observed as soon as pores were induced on the membrane. These results show that cellular structural integrity is indeed disrupted over the course of sonoporation. CONCLUSIONS: Our investigation shows that the biophysical impact of sonoporation is by no means limited to the induction of membrane pores: e.g. structural integrity is concomitantly affected in the process. This prompts the need for further fundamental studies to unravel the complex sequence of biological events involved in sonoporation.postprin

A study on the change in plasma membrane potential during sonoporation

Posters: no. 4Control ID: 1680329OBJECTIVES: There has been validated that the correlation of sonoporation with calcium transients is generated by ultrasound-mediated microbubbles activity. Besides calcium, other ionic flows are likely involved in sonoporation. Our hypothesis is the cell electrophysiological properties are related to the intracellular delivery by ultrasound and microbubbles. In this study, a real-time live cell imaging platform is used to determine whether plasma membrane potential change is related to the sonoporation process at the cellular level. METHODS: Hela cells were cultured in DMEM supplemented with 10% FBS in Opticell Chamber at 37 °C and 5% CO2, and reached 80% confluency before experiments. The Calcein Blue-AM, DiBAC4(3) loaded cells in the Opticell chamber filled with PI solution and Sonovue microbubbles were immerged in a water tank on a inverted fluorescence microscope. Pulsed ultrasound (1MHz freq., 20 cycles, 20Hz PRF, 0.2-0.5MPa PNP) was irradiated at the angle of 45° to the region of interest for 1s.The real-time fluorescence imaging for different probes was acquired by a cooled CCD camera every 20s for 10min. The time-lapse fluorescence images were quantitatively analyzed to evaluate the correlation of cell viability, intracellular delivery with plasma membrane potential change. RESULTS: Our preliminary data showed that the PI fluorescence, which indicated intracellular delivery, was immediately accumulated in cells adjacent to microbubbles after exposure, suggesting that their membranes were damaged by ultrasound-activated microbubbles. However, the fluorescence reached its highest level within 4 to 6 minutes and was unchanged thereafter, indicating the membrane was gradually repaired within this period. Furthermore, using DIBAC4(3), which detected the change in the cell membrane potential, we found that the loss of membrane potential might be associated with intracellular delivery, because the PI fluorescence accumulation was usually accompanied with the change in DIBAC4 (3) fluorescence. CONCLUSIONS: Our study suggests that there may be a linkage between the cell membrane potential change and intracellular delivery mediated by ultrasound and microbubbles. We also suggest that other ionic flows or ion channels may be involved in the cell membrane potential change in sonoporation. Further efforts to explore the cellular mechanism of this phenomenon will improve our understanding of sonoporation.postprin

Developmental delays and subcellular stress as downstream effects of sonoporation

Posters: no. 2Control ID: 1672434OBJECTIVES: The biological impact of sonoporation has often been overlooked. Here we seek to obtain insight into the cytotoxic impact of sonoporation by gaining new perspectives on anti-proliferative characteristics that may emerge within sonoporated cells. We particularly focused on investigating the cell-cycle progression kinetics of sonoporated cells and identifying organelles that may be stressed in the recovery process. METHODS: In line with recommendations on exposure hardware design, an immersion-based ultrasound platform has been developed. It delivers 1 MHz ultrasound pulses (100 cycles; 1 kHz PRF; 60 s total duration) with 0.45 MPa peak negative pressure to a cell chamber that housed HL-60 leukemia cells and lipid-shelled microbubbles at a 10:1 cell-tobubble ratio (for 1e6/ml cell density). Calcein was used to facilitate tracking of sonoporated cells with enhanced uptake of exogenous molecules. The developmental trend of sonoporated cells was quantitatively analyzed using BrdU/DNA flow cytometry that monitors the cell population’s DNA synthesis kinetics. This allowed us to measure the temporal progression of DNA synthesis of sonoporated cells. To investigate whether sonoporation would upset subcellular homeostasis, post-exposure cell samples were also assayed for various proteins using Western blot analysis. Analysis focus was placed on the endoplasmic reticulum (ER): an important organelle with multi-faceted role in cellular functioning. The post-exposure observation time spanned between 0-24 h. RESULTS: Despite maintaining viability, sonoporated cells were found to exhibit delays in cell-cycle progression. Specifically, their DNA synthesis time was lengthened substantially (for HL-60 cells: 8.7 h for control vs 13.4 h for the sonoporated group). This indicates that sonoporated cells were under stress: a phenomenon that is supported by our Western blot assays showing upregulation of ER-resident enzymes (PDI, Ero1), ER stress sensors (PERK, IRE1), and ER-triggered pro-apoptotic signals (CHOP, JNK). CONCLUSIONS: Sonoporation, whilst being able to facilitate internalization of exogenous molecules, may inadvertently elicit a cellular stress response. These findings seem to echo recent calls for reconsideration of efficiency issues in sonoporation-mediated drug delivery. Further efforts would be necessary to improve the efficiency of sonoporation-based biomedical applications where cell death is not desirable.postprin

Real-time imaging of cellular dynamics during low-intensity pulsed ultrasound exposure

Control ID: 1671584Oral Session 5 - Bioeffects of therapeutic ultrasoundOBJECTIVE: Although the therapeutic potential of low-intensity pulsed ultrasound is unquestionable, the wave-matter interactions involved in the process remain to be vaguely characterized. Here we seek to undertake a series of in-situ cellular imaging studies that aim to analyze the mechanical impact of low-intensity pulsed ultrasound on attached fibroblasts from three different aspects: membrane, cytoskeleton, and nucleus. METHODS: Our experimental platform comprised an in-house ultrasound exposure hardware that was coupled to a confocal microscopy system. The waveguided ultrasound beam was geometrically aligned to the microscope’s fieldof-view that corresponds to the center of a polystyrene dish containing fibroblasts. Short ultrasound pulses (5 cycles; 2 kHz PRF) with 0.8 MPa peak acoustic pressure (0.21 W/cm2 SPTA intensity) were delivered over a 10 min period. Live imaging was performed on both membrane (CellMask) and cytoskeleton (actin-GFP, tubulin-RFP) over the entire observation period (up to 30 min after end of exposure). Also, pre- and post-exposure fixed-cell imaging was conducted on the nucleus (Hoechst 33342) and two cytoskeleton components related to stress fibers: F-actin (phalloidin-FITC) and vincullin (Alexa Fluor 647 conjugated). To study whether mechanotransduction was responsible in mediating ultrasound-cell interactions, some experiments were conducted with the addition of gadolinium that blocks stretch-sensitive ion channels. RESULTS: Cell shrinkage was evident over the course of low-intensity pulsed ultrasound exposure. This was accompanied with contraction of actin and tubulin. Also, an increase in central stress fibers was observed at the end of exposure, while the nucleus was found to have decreased in size. Interestingly, after the exposure, a significant rebound in cell volume was observed over a 30 min. period. These effects were not observed in cases with gadolinium blockage of mechanosensitive ion channels. CONCLUSIONS: Our results suggest that low-intensity pulsed ultrasound would transiently induce remodeling of a cell’s membrane and cytoskeleton, and it will lead to repression of nucleus. This indicates that ultrasound after all represents a mechanical stress on cellular membrane. The post-exposure outgrowth phenomenon is also of practical relevance as it may be linked to the stimulatory effects that have been already observed in low-intensity pulsed ultrasound treatments.postprin

Thermal ablation of biological tissues in disease treatment: A review of computational models and future directions

Percutaneous thermal ablation has proved to be an effective modality for treating both benign and malignant tumors in various tissues. Among these modalities, radiofrequency ablation (RFA) is the most promising and widely adopted approach that has been extensively studied in the past decades. Microwave ablation (MWA) is a newly emerging modality that is gaining rapid momentum due to its capability of inducing rapid heating and attaining larger ablation volumes, and its lesser susceptibility to the heat sink effects as compared to RFA. Although the goal of both these therapies is to attain cell death in the target tissue by virtue of heating above 50 oC, their underlying mechanism of action and principles greatly differs. Computational modelling is a powerful tool for studying the effect of electromagnetic interactions within the biological tissues and predicting the treatment outcomes during thermal ablative therapies. Such a priori estimation can assist the clinical practitioners during treatment planning with the goal of attaining successful tumor destruction and preservation of the surrounding healthy tissue and critical structures. This review provides current state-of- the-art developments and associated challenges in the computational modelling of thermal ablative techniques, viz., RFA and MWA, as well as touch upon several promising avenues in the modelling of laser ablation, nanoparticles assisted magnetic hyperthermia and non- invasive RFA. The application of RFA in pain relief has been extensively reviewed from modelling point of view. Additionally, future directions have also been provided to improve these models for their successful translation and integration into the hospital work flow

Design and development of magnetic resonance imaging (MRI) compatible tissue mimicking phantoms for evaluating focused ultrasound thermal protocols

Animal models are often used to test the efficacy and safety of clinical applications employing focused ultrasound that range in various stages of research, development and commercialization. The animals are usually subjected to conditions that cause pain, distress and euthanasia. Access to cadaveric models is not easy and affordable for all research institutions, whereas conservation and changes of their physical properties over time can be a delimiting factor for translational research. The above set the motivation for this project, which its primary objective is to design and develop appropriate tissue mimicking phantoms using a simplistic and cost effective methodology. These phantoms are expected to contribute in reducing the need for animal testing and allow researchers to get hands experience with tools that will promote and accelerate testing in focused ultrasound thermal protocols. The main requirements for these phantoms are to be geometrically accurate, compatible with magnetic resonance imaging (MRI) and to be composed of materials that approximate the acoustic and thermal properties of the replicated tissues. Throughout the duration of the project three ultrasonic composite phantoms (head, femur bone-muscle and breast-rib) were developed. The acoustic properties of candidate materials were assessed using pulse-echo immersion and through transmission techniques. The thermal properties were estimated by observing the rate of heat diffusion following a sonication in the soft tissue parts with MR thermometry. Acrylonitrile butadiene styrene (ABS) was used to replicate bone tissue, where its acoustic attenuation coefficient was found to be 16.01 ± 6.18 dB/cm at 1 MHz and the speed of sound at 2048 ± 79 m/s. Soft tissue parts consisted out of agar-based gels doped with varying concentrations of additives that controlled the relative contribution of acoustic absorption (evaporated milk) and scatter (silica dioxide) to total attenuation independently. Brain tissue phantom (2 % w/v agar - 1.2 % w/v SiO2 - 25 % v/v evaporated milk) matched an attenuation coefficient of 0.59 ± 0.05 dB/cm-MHz whereas muscle and breast mimicking phantom (2 % w/v agar - 2 % w/v SiO2 - 40 % v/v evaporated milk) were estimated of inducing an attenuation coefficient of the order of 0.99 ±0.08 dB/cm-MHz. The speed of sound for the brain and muscle/breast recipe were estimated at 1485 ± 12 m/s and 1529 ± 13 m/s respectively. The thermal conductivity of the brain phantom was estimated to be 0.52 ± 0.06 W/mº-C and 0.57 ± 0.10 W/mº-C for the muscle/breast phantom. The acoustic and thermal properties of candidate materials were within range of the replicated tissues extracted from literature, except the speed of sound in ABS compared which was lower compared to bone (~3000 m/s). Three dimensional models of bone parts (skull, femur, rib) were reconstructed in Standard Tessellation Language (STL) format by segmenting bony tissue of interest from adult human computed tomography (CT) images. The STL bone models were 3D printed in ABS using a fused deposition modelling (FDM) machine. The final composite phantoms were fabricated by molding the agar based soft tissue phantoms inside/around the ABS bone phantoms. The functionality of all three composite phantoms was assessed with focused ultrasound sonications applied by a 1 MHz single element transducer while temperature was monitored with 1.5 Tesla MRI scanner. A spoiled gradient recalled (SPGR) pulse sequence was used to produce phase images that were analyzed using a custom coded software developed in Matlab that employed proton-resonance frequency shift (PRFS) thermometry

Doctor of Philosophy

dissertationIt is estimated that in 2016, more than 848,000 new cases of cancer will be diagnosed in men with more than a quarter being prostate cancer and more than 26,000 deaths attributed to this disease. Prostate cancer poses a limited risk when detected at an early stage and treatment of stages II-III has a 5-year survival rate of almost 100%. However, these early-stage cancers can eventually progress and develop into stage IV, dramatically dropping the 5-year survival rate to 28%. Thus, development of a new therapy is needed to fully eliminate these tumors. Combination of heat and chemotherapy improves therapeutic efficacy while allowing for reduced dosing of drugs and limiting side effects. Localized hyperthermia has been used to enhance the delivery of polymer therapeutics to prostate tumors through increased blood flow, vascular permeability, and incorporation of heat shock targeting. This strategy has been shown to increase the delivery and retention of polymer-drug conjugates leading to enhanced efficacy. Although much work has been done using this strategy, the effects of different thermal dosing on polymer accumulation are unknown. The first aim of this research is to examine how altering heating parameters influences polymer tumor accumulation. The hypothesis for this aim is that there is an optimal thermal treatment that leads to the maximal amount of polymer accumulation in the tumors. Additionally, the previously used heating method of plasmonic photothermal therapy (PPTT) can result in long-term accumulation of gold nanoparticles in healthy organs, potentially limiting clinical applicability. The second aim of this proposal will be focused on investigating the alternative method of high intensity focused ultrasound (HIFU) for selective heating of tumors and enhancing macromolecular delivery. HIFU has shown the capability for precise, noninvasive heating of specific regions within the prostate through magnetic resonance imaging (MRI) guidance. The hypothesis to be tested in this aim is that mild hyperthermia produced with HIFU will have the same effect as that produced by PPTT in improving the delivery of macromolecular systems to solid tumors. Finally, in the third aim, the enhanced delivery of targeted polymer therapeutics to prostate tumors in mice models will be investigated using mild hyperthermia produced with HIFU. In the long term, it is anticipated that HIFU can be used in conjunction with delivery of polymer-drug conjugates for enhanced efficacy and reduced toxicity of chemotherapy to produce a clinically relevant treatment of advanced prostate cancer