Phospholipid Encapsulation Properties and Effects on Microbubble Stability and Dynamics James Jing Kwan Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Science COLUMBIA UNIVERSITY 2012 © 2011 James Kwan All rights reserved ABSTRACT Phospholipid Encapsulation Properties and Effects on Microbubble Stability and Dynamics James Jing Kwan The goal of this doctoral work was to observe and analyze the stability and dynamics of phospholipid-encapsulated microbubbles, and in particular the reaction to sudden submersion in a multi-gas medium. To accomplish this goal, first an experimental technique was developed to observe a microbubble in a single-gas environment suddenly immersed in a multi-gas environment, without perturbing the microbubble position. A modified Epstein-Plesset model was concurrently developed to account for the multiple gas species in the bulk solution. The model was used to analyze previous data for the effect of anesthesia carrier gas on microbubble ultrasound contrast agent in vivo circulation persistence. The focus of the experiments then shifted to microbubbles of different sizes encapsulated with a homologous series of saturated diacyl-chain lipid surfactants and emulsifiers. Constitutive models for the elastic and gas permeation properties of the lipid encapsulation were developed to elucidate the unique behaviors observed during the experiments. The experimental techniques employed were: (1) transmission bright field optical microscopy to obtain real-time, digital videos of microbubbles growing and dissolving in response to perturbations in the local gas environment and (2) the Langmuir trough film balance to determine the elasticity of the phospholipid monolayers during compression, expansion, and expansive relaxation. The modeling techniques employed was (1) a forward-wind finite difference method to discretize a series of non-linear differential equations and (2) a Newton- Raphson method to solve the diameter of a microbubble from the mechanical stress balance. These modeling techniques were used to determine the behavior of a microbubble a priori, whereas the fitting models implemented the iterative methods to solve for parameters without a Newton-Raphson method. Results showed that microbubbles coated with soluble surfactants and dissolving in a single gas solution could be predicted by the original Epstein-Plesset model. When subjected to a multi-gas medium, the modified Epstein-Plesset model accurately predicted microbubble growth and dissolution. The model was used to analyze the increase in microbubble circulation lifetime observed by others in anesthetized rats inhaling air rather than oxygen as the anesthesia carrier gas. The predictive capabilities of the model broke down, however, if the gas-core was encapsulated with a phospholipid monolayer. A typical, large (>40 µm diameter) lipid-coated microbubble displayed stunted growth, followed by three anomalous dissolution regimes: (1) rapid dissolution back to the initial resting diameter followed by (2) slow, steady dissolution and finally (3) stabilization, where the apparent surface tension approached a near-zero value. The model was modified to allow fitting of the radius-time curve by varying the surface tension. The analysis showed that the surface tension is dynamic, and suggested that a “break up” tension allowed for rapid expansion of the microbubble beyond the initial resting diameter. Lipid jamming was proposed as the mechanism eventually halting dissolution. Further observations of smaller microbubbles (<20 µm diameter) coated with a homologous series of saturated diacyl chain lipids gave significantly different results. Initially the microbubbles grew, but growth was severely subdued, if not eliminated, for more solid encapsulations below a threshold size (~10 µm diameter). Following growth, most microbubbles rapidly dissolved back to their original size. The microbubbles then experienced an anomalous lag time before spontaneously dissolving again. The lag times were highly variable and shown to correlate to the reduced temperature of the encapsulation, rather than the initial microbubble size. Most of the microbubbles stabilized again at a diameter of 1-2 µm, and this “stable diameter” appeared to be universal and independent of both the initial microbubble size and the rigidness of the encapsulation. Constitutive models were developed to describe these physical phenomena in the early growth and dissolution stages which were verified with independent monolayer relaxation studies. Table of Contents Abstract.............................................................................................................................................. Table of Contents..............................................................................................................................i List of Tables....................................................................................................................................v List of Figures.................................................................................................................................vi Acknowledgments............................................................................................................................ix Chapter 1: Introduction..................................................................................................................1 1.1. Natural Microbubbles..................................................................................................1 1.2. Synthetic Microbubbles and their Applications...........................................................4 1.2.1. Microbubble Production...............................................................................4 1.2.2. Microbubble Characteristics Under Ultrasound..........................................9 1.2.3. Biomedical Applications.............................................................................15 1.2.4. Applications in Biotechnology....................................................................25 1.2.5. Applications in the Food Industry...............................................................27 1.3. Experimental Techniques for Observing Microbubbles............................................29 1.3.1. Acoustic Quantification of Microbubble Properties...................................29 1.3.2. Optical Observations of Microbubble Behavior.........................................30 1.4. Microbubble Dissolution...........................................................................................33 1.4.1. Microbubble Single-Gas Dissolution..........................................................33 1.4.2. Microbubble Multi-Gas Dissolution...........................................................34 1.5. Role of the Lipid Monolayer on Microbubbles..........................................................35 1.5.1. Microbubble Shell Rheology.......................................................................35 1.5.2. Monolayer Resistance to Gas Permeation..................................................37 1.6. Specific Aims..............................................................................................................38 i Chapter 2: Multi-Gas Dissolution: Soluble Versus Insoluble Monolayers.................................40 2.1. Introduction................................................................................................................40 2.1.1. Single-Gas Microbubble System.................................................................40 2.1.2. Multi-Gas Microbubble System..................................................................40 2.2. Theoretical Aspects of Microbubble Dissolution.......................................................43 2.3. Materials and Methods..............................................................................................46 2.3.1. Langmuir Isotherms....................................................................................46 2.3.2. Preparation of SDS Microbubbles..............................................................47 2.3.3. Preparation of Lipid Microbubbles............................................................47 2.3.4. Observation and Measurement of Microbubble Growth and Dissolution...48 2.3.5. Image Processing........................................................................................50 2.3.6. Simulation...................................................................................................50 2.4. Results and Discussion..............................................................................................52 2.4.1. SDS/SF6 Microbubble in an SF6-Medium...................................................52 2.4.2. SDS/SF6 Microbubble in an Air-Medium...................................................53 2.4.3. Lipid/SF6 Microbubble in an Air-Medium..................................................56 2.5. Concluding Remarks..................................................................................................64 Chapter 3: Effect of Breathing Gas on Ultrasound Contrast Agents...........................................66 3.1. Introduction................................................................................................................66 3.1.1. Ultrasound Contrast Agent Lifetime in Rats...............................................68 3.2. Theory........................................................................................................................71