京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第19973号 / 薬科博第64号 / 新制||薬科||7(附属図書館) / 33069 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 橋田 充, 教授 佐治 英郎, 教授 髙倉 喜信 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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Resource-efficient technologies are essential for economically viable biodiesel production. This work focuses on conversion of microalgal lipids and vegetable oils into fatty acid alkyl esters or biodiesel. Transesterification of waste cooking oil (WCO) and extractive-transesterification of wet microalgal biomass were investigated using microwave and ultrasound irradiations through several process parametric evaluation studies to elucidate the effects of different alcohols and catalyst types (homogeneous and heterogeneous), reaction time, and reaction temperatures. First, a brief overview of process steps involved in microalgal biodiesel production and associated energy consumption and research needs were discussed. Next, energy analysis of microalgal biocrude production via extractive-transesterification under microwave and ultrasound irradiations (individually) was performed. Then, the synergistic effect of microwave and ultrasound irradiations on extractive-transesterification of microalgal lipids was evaluated through a process optimization study using response surface methodology to determine the best process conditions. For this study, a maximum biocrude conversion of 51.2% was obtained when 20 g of algal paste was treated with 30 mL methanol, 1 wt.% catalyst, 7 min reaction time, and 140 W for MW and US (280 W total). Further, biocrude yield kinetics study revealed that the activation energy for this reaction was around 17, 298 J mol-1 K-1. A series of experimental studies were conducted to understand the roles and effects of various process related conditions including the power output and power density of microwave and ultrasound irradiations in biodiesel production. The two non-conventional heating techniques were compared for their process intensification effects. Ultrasound was applied either in continuous or pulse mode. Pulse sonication was found to be more suitable for simple transesterification reaction of WCO with a 98% biodiesel yield in 2.5 min (9:1 methanol to oil ratio, 1.25% catalyst, and 150 W power output) over 82% yield for continuous sonication under the same conditions. Followed by this, a detailed study was conducted to determine optimum pulse (ON and OFF time) sonication conditions. A 99% conversion yield was obtained for a pulse ON-OFF combination of 7s-2s. Additionally, the effect of different alcohols (ethanol, methanol, and ethanol-methanol mixtures) using pulse sonication was evaluated.

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Histotripsy is a non-thermal, non-invasive, focused ultrasound ablation method that uses acoustic cavitation to mechanically break down tissues [1-8]. Histotripsy is heavily dependent on the mechanical properties of the tissue, allowing it to mechanically ablate tissues of lower mechanical stiffness while preserving the stiffer critical structures [15]. However, the mechanical properties of clinically relevant abdominal tissues and critical structures have not yet been adequately quantified under uniform testing parameters. Previous studies have tested and modeled the tissue selectivity of histotripsy, but these studies have been limited by the lack of mechanical property data available for these tissue types. In addition, there remains a need for additional experimental studies directly comparing the differential treatment doses required to induce histotripsy tissue damage in intra-abdominal tissue types. This thesis investigates the mechanical properties of intra-abdominal tissues under uniaxial tension, the effect of histotripsy treatment dose on intra-abdominal soft tissues and critical structures, and the potential of inducing damage to critical structures along the acoustic path pre-focal to the targeted histotripsy treatment. Results show that there are significant differences between the parenchymal tissues (liver, kidney) and the critical structure (stomach, gallbladder, small intestine, ducts, and vessels) elastic modulus, yield stress, yield strain, post-yield strain, energy to yield, and maximum stress and strain at yield. In general, histology analysis from the histotripsy experiments showed that there was an increase in tissue damage with increasing histotripsy pulses/point for all tissues. Critical structures with higher mechanical strength were more resistant to ablation compared to tissues with lower mechanical strength. Pre-focal studies showed damage to gallbladder and small intestine only in cases in which pre-focal cavitation was observed, while no damage occurred in skin and stomach for any samples treated at varying distances from the bubble cloud. Overall, this work improves our understanding of tissue selectivity of histotripsy and provides mechanical properties measurements for clinically relevant tissues that can be used to improve predictive models of tissue-selective histotripsy treatments. This work can be used in the planning of histotripsy treatments to establish proper margins of safety for treating intra-abdominal tumors. / Master of Science / Histotripsy is a non-invasive cancer treatment that mechanically breaks down tissues by rapidly forming and bursting bubbles within the tumor [1-8]. Histotripsy is heavily dependent on the mechanical properties of the tissue, allowing it to destroy weaker tissues while preserving the stiffer tissues in the surrounding area [15]. The mechanical properties of clinically relevant intra-abdominal tissues have not been quantified under uniform testing parameters. Previous studies have tested and modeled the tissue selectivity of histotripsy, but these studies have been limited by the mechanical property data available. This thesis investigates the mechanical properties of intra-abdominal tissues under tension, the effect of histotripsy treatment dose on intra-abdominal tissue damage, and the damage to critical structures from histotripsy treatment at varying distances from the tissue. Results show that there are significant differences between the liver and kidney mechanical stiffness and strength compared to the other tissues. In general, histology analysis showed that there is an increase in tissue damage with increasing histotripsy dose. Tissues with higher mechanical strength were more resistant to damage at lower doses compared to tissues with lower mechanical strength. Histotripsy damage to critical structures that are along the beam path, set distances in front of the focal point of the cavitation bubble cloud was studied. This study showed damage to gallbladder and small intestine only in cases in which pre-focal cavitation, cavitation bubbles that are not within the focal point of the cloud but are in contact with the tissue, was observed, while no damage occurred in skin and stomach for any samples treated at varying distances from the bubble cloud. Overall, this work improves our understanding of tissue selectivity of histotripsy and provides mechanical properties for clinically relevant tissues that can be used to improve predictive models of tissue-selective histotripsy treatments. This work can be used in the planning of histotripsy treatments to establish proper margins of safety for treating intra-abdominal tumors.

This dissertation explores the potential of using perfluorocarbon emulsion droplets to add an ultrasound-sensitive element to drug delivery systems. These emulsion droplets may be induced to vaporize with ultrasound; during the rarefactional phase of an ultrasound wave, the pressure around the droplets may fall below the vapor pressure of the liquid forming the emulsion, providing a thermodynamic potential for vaporization. This ultrasound-induced phase change of the emulsion droplet could release therapeutics attached to the droplet surface or aid in drug delivery due to mechanical effects associated with vaporization and expansion, similar to the ability of cavitating bubbles to aid in drug delivery. In contrast to bubbles, stable emulsions can be formed at nano-scale sizes, allowing them to extravasate into tissues and potentially be endocytosed into cells. Perfluorohexane and perfluoropentane were selected to form the emulsions due to their relatively high vapor pressure, low water solubility, and biocompatibility. Acoustic droplet vaporization was explored for its potential to increase ultrasound-induced drug release from liposomes. Liposomes have proven to be versatile and effective drug carriers, but are not inherently responsive to ultrasound. eLiposomes, defined as a liposome with encapsulated emulsion droplets, were developed due to the potential of the expanding vapor phase to disrupt bilayer membranes. The resulting vesicle retains the advantages of liposomes for drug delivery, while adding an ultrasound-sensitive element. eLiposomes were loaded with calcein, a fluorescent molecule, as a model drug in order to quantify ultrasound-mediated drug release compared to release from conventional liposomes. Upon exposure to ultrasound, eLiposomes typically released 3 to 5 times as much of the encapsulated load compared to conventional liposomes, with some eLiposome samples approaching 100% release. Emulsion droplets were also added to the outside of conventional liposomes, but resulted in little to no increase compared to control samples without emulsions. Lastly, in vitro experiments were performed with HeLa cells to explore the ability of emulsion droplets and eLiposomes to deliver calcein inside of cells. Calcein delivery to the cytosol was accomplished, and the emulsion-containing samples demonstrated the ability to aid in endosomal escape.

Therapeutic ultrasound is a deep heating modality often used to produce vigorous heating (≥4°C Δ) in tissues. The vigorous heating effects of 3 MHz therapeutic ultrasound have only been tested to a 2.5 cm depth, but its maximal depth of producing vigorous heating has yet to be established. Objective: To investigate the tissue temperature change produced by a 3 MHz ultrasound treatment at depths of 3 and 3.5 cm in the human triceps surae muscle group. Design: Randomized control design. Setting: Therapeutic modalities research laboratory. Patients or Other Participants: Twenty healthy college-aged participants (male = 13, female = 7; age = 23.4 ± 1.31; calf subcutaneous fat thickness= 0.6 cm ± 0.2 cm). Participants were randomized into treatment (n = 15) and sham (n = 5) groups. Participants were blinded to their group assignment. Interventions: Two MT-26/6 needle thermocouples were inserted into the left posterior triceps surae at depths of 3.0 ± 0.1cm and 3.5 ± 0.1cm from the skin's surface. Participants in the treatment group received a continuous 3 MHz ultrasound treatment at 1.4 W/cm2 for 8 minutes with 10mL of 100% ultrasound gel as a coupling medium. Participants in the sham group received the same treatment parameters, but the ultrasound device was not turned on. The Omnisound 3000 ultrasound device (ERA = 4.2cm2, BNR = 3.0:1) was used for all treatments. A 15 cm2 template was used to ensure a constant and proper treatment size. Baseline temperature (TB) was established by taking a mean of intramuscular tissue temperature (TIM)for five minutes before the treatment and TIM were recorded every 10 seconds throughout the experiment session. Participants marked a visual analog scale (VAS) indicating heat sensation at pre-treatment and post-treatment. Main Outcome Measures: A 2 x 2 x 2 (probe depth x condition x time) ANCOVA with TB used as a covariate analyzed the difference in TIM. We only used the time points of baseline and final TIM for our analysis as we are only interested in the change in TIM from beginning to end of the ultrasound treatment. Descriptive statistics for TIM and VAS for heat sensation were computed as post-treatment minus pre-treatment for each condition and probe depth. Results: There was a significant difference in TIM between the conditions at the different probe depths from the beginning and end of the ultrasound treatment (F1,15 = 7.35, p = 0.016). The mean changes in TIM for each condition at each probe depth were: sham 3cm = -0.4 ± 0.3°C, sham, 3.5cm = -0.2 ± 0.3°C, treatment, 3cm = 4.4 ± 0.2°C, treatment, 3.5cm = 3.5 ± 0.2°C. Mean VAS scores for each group were: sham = 0 ± 0mm and treatment = 71.8 ± 11.8mm. Conclusions: At 3cm deep into the posterior calf, the Omnisound 3000 using a 3 MHz treatment produced vigorous heating (≥4°C Δ). Moderate heating (2-3°C Δ) occurred at 3.5cm deep into the calf. Three MHz ultrasound may be used to heat tissues deeper than previously theorized, but it does, however, create a moderately high level of heat sensation for the patient.

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Clinical ultrasound imaging techniques can be greatly improved by the use of ultrasound contrast agents (UCAs). While microbubbles (MBs) without shell are unstable and cannot be used for practical applications,a shell produced from biocompatible polyvinylalcohol (PVA) significantly improves chemical versatility and stability. The oscillation characteristics of a UCA are strongly dependent on concentration, applied pressure and viscoelastic parameters of the shell. Modifications in the shell as incorporation of antibodies or targeted molecules affect the bubble oscillation and resonance frequency of the MB suspension. In this presented work a tool for systematic characterization of UCAs is developed. Linear acoustic behaviour of PVA shelled MBs is examined. The acoustic driving pressure is kept below 100 kPa. The MB concentration is 1·10^{6} ml^{-1}. Attenuation and phase velocity profiles of ultrasound waves propagating through the UCA are measured using six narrow-band single crystal transducers that cover a frequency range between 1 and 15 MHz. The oscillation of a single bubble is modeled as a linear oscillator adapting HOFF’s model suitable for allshell thicknesses. The suspension is modeled through superposition of single bubbles. Knowing all parameters the resonance frequency of a MB suspension can be predicted. The model is fitted to experimental data to determine the viscoelastic shell parameters. The shell thickness is challenging to determine exactly and assumed to be either proportional to the outer shell radius or constant. Assuming a proportional shell thickness the calculated resulting shell parameters were shear modulus G_s = 14.5 MPa, shear viscosity η_s = 0.322 Pa·s and shell thickness d_s = 16 % of the outer radius. When instead assuming a constant shell thickness the determined parameters were in similar order of magnitude. Resonance frequency of the suspension was determined to 11.6 MHz. The developed tool can be used to characterize MBs with a modified shell independently of shell thickness and to predict resonance frequency of gas or air filled UCAs with known shell parameters.