Effects of lipid saturation on bicelle to vesicle transition of a binary phospholipid mixture: a molecular dynamics simulation study

Controlling the transition from lipid bicelles to vesicles is essential for producing engineered vesicles. We perform coarse-grained molecular dynamics (CGMD) simulations of unsaturated/saturated lipid mixtures to clarify the effects of lipid unsaturation on vesiculation at the molecular scale. The results demonstrate that vesiculation depends on the concentration of unsaturated lipids and the degree of unsaturation. The probability of vesiculation increases linearly with the apparent unsaturated lipid concentration at a low degree of unsaturation. Higher degrees of unsaturation lead to phase segregation within the binary bicelles, reducing the probability of vesiculation. A comparison between CGMD simulations and the conventional theory of vesiculation shows that the theoretical predictions of binary lipid systems must explicitly include phase segregation effects. Furthermore, simulations with biased lipid distributions reveal that vesiculation is facilitated by the preconcentration of unsaturated lipids in the core region of the bicelle but is then temporally limited as the unsaturated lipids move to the bicelle edges. These findings advance theoretical and experimental studies on binary lipid systems and promote the development of tailor-made vesicles.


Changes in free energy barrier for water permeation by stretch-induced phase transitions in phospholipid/cholesterol bilayers

Water permeation through phospholipid/cholesterol bilayers is the key to understanding tension-induced rupture of biological cell membranes. We performed molecular dynamics simulations of stretched phospholipid/cholesterol bilayers to investigate changes in the free energy profile of water molecules across the bilayer and the lipid structure responsible for water permeation. We modeled stretching of the bilayer by applying areal strain. In stretched phospholipid/cholesterol bilayers, the hydrophobic tail of the phospholipids became disordered and the free energy barrier to water permeation decreased. Upon exceeding the critical areal strain, a phase transition to an interdigitated gel phase occurred before rupture, and the hydrophobic tail ordering as well as the free energy barrier were restored. In pure phospholipid bilayers, we did not observe such recoveries. These transient recoveries in the phospholipid/cholesterol bilayer suppressed water permeation and membrane rupture, followed by an increase in the critical areal strain at which the bilayer ruptured. This result agrees with experimental results and provides a reasonable molecular mechanism for the toughness of phospholipid/cholesterol bilayers under tension.Communicated by Ramaswamy H. Sarma.

Journal of Biomolecular Structure & Dynamics


Shear-flow-induced negative tension of phospholipid bilayer: Molecular dynamics simulation

Shear flow has been theoretically predicted to suppress the undulation of surfactant bilayers and generate negative tension, which is considered to be a driving force of the transition from the lamellar phase to the multilamellar vesicle phase in surfactant/water suspensions, the so-called onion transition. We performed coarse-grained molecular dynamics simulations of a single phospholipid bilayer under shear flow to clarify the relationship between the shear rate, bilayer undulation, and negative tension, providing molecular-level insight into the undulation suppression. An increasing shear rate suppressed bilayer undulation and increased negative tension; these results are consistent with theoretical predictions. The non-bonded forces between the hydrophobic tails facilitated negative tension, whereas the bonded forces within the tails suppressed it. The force components of the negative tension were anisotropic in the bilayer plane and prominently changed in the flow direction, although the resultant tension was isotropic. Our findings regarding a single bilayer will underlie further simulation studies of multilamellar bilayers, including inter-bilayer interactions and topological changes of bilayers under shear flow, which are essential for the onion transition and are unresolved in the theoretical and experimental studies.


Stretch-Induced Interdigitation of a Phospholipid/Cholesterol Bilayer.

I phase under stretching. However, there is still no conclusive experimental evidence for this process, so its existence remains controversial. In this study, to explain the transition from energy balance, we propose a free-energy model. The model consists of three energy components: the elastic deformation energy, surface energy at the bilayer-water interface, and interphase boundary energy. To determine the parameters of the model, we perform MD simulations of a stretched 1,2-dipalmitoyl- sn-glycero-3-phosphocholine/cholesterol bilayer. The phase diagrams from our model are in good agreement with those obtained from MD simulations. The energy balance among the components in the stretched bilayer quantitatively explains the stretch-induced transition. In the model, increasing the system size to that used in experiments shows that interdigitation is favorable for rigid bilayers under stretching or in alcohol solutions. These results suggest that the stretch-induced interdigitation might be observed in microscopic experiments.


Heterogeneous structure and surface tension effects on mechanical response in pulmonary acinus: A finite element analysis.

In the lungs, the heterogeneous acinar structure and surface tension induce anisotropic deformation at the acinar and alveolar scales. Further research is needed on structural variation of acini, inter-acini connectivity, or dynamic behavior to understand multiscale lung mechanics.


One (sub-)acinus for all: Fate of inhaled aerosols in heterogeneous pulmonary acinar structures.

Computational Fluid Dynamics (CFD) have offered an attractive gateway to investigate in silico respiratory flows and aerosol transport in the depths of the lungs. Yet, not only do existing models lack sufficient anatomical realism in capturing the heterogeneity and morphometry of the acinar environment, numerical simulations have been widely restricted to domains capturing a mere few percent of a single acinus. Here, we present to the best of our knowledge the most detailed and comprehensive in silico simulations to date on the fate of aerosols in the acinar depths. Our heterogeneous acinar domains represent complete sub-acinar models (i.e. 1/8th of a full acinus) based on the recent algorithm of Koshiyama & Wada (2015), capturing statistics of human acinar morphometry (Ochs et al. 2004). Our simulations deliver high-resolution, 3D spatial-temporal data on aerosol transport and deposition, emphasizing how variances in acinar heterogeneity only play a minor role in determining general deposition outcomes. With such tools at hand, we revisit whole-lung deposition predictions (i.e. ICRP) based on past 1D lung models. While our findings under quiet breathing substantiate general deposition trends obtained with past predictions in the alveolar regions, we underscore how deposition fractions are anticipated to increase, in particular during deep inhalation. For such inhalation maneuver, our simulations support the notion of significantly augmented deposition for all aerosol sizes (0.005-5.0m). Overall, our efforts not only help consolidate our mechanistic understanding of inhaled aerosol transport in the acinar depths but also continue to bridge the gap between "bottom-up" in silico models and regional deposition predictions from whole-lung models. Such quantifications provide what is deemed more accurate deposition predictions in morphometrically-faithful models and are particularly useful in assessing inhalation strategies for deep airway deposition (e.g. systemic delivery).

European Journal of Pharmaceutical Sciences


Morphological Characterization of Acinar Cluster in Mouse Lung Using a Multiscale-based Segmentation Algorithm on Synchrotron Micro-CT Images.

Understanding the three-dimensional morphology of pulmonary acini is essential when exploring the biomechanics of respiratory function. In this study, we characterized the morphology of individual acini and a cluster of acini stemming from the same terminal conducting airway using a quantitative approach based on the semi-automatic segmentation of synchrotron micro-CT images of mouse lung. The volume and surface area of five clusters of mouse acini including 50 individual acini were estimated based on the voxel and surface mesh of segmented acini at FRC. The pathway length and width were estimated for one cluster including 15 acini based on the skeleton of segmented acini. The acinar volume was 0.09 ± 0.07 mm(3) (mean ± SD), and the surface area was 6.82 ± 4.49 mm(2) , in agreement with previous studies. The volume of the acinar clusters was 0.89 ± 0.34 mm(3) , and the surface area was 68.18 ± 17.66 mm(2) . The largest volume acinus per cluster was found in the distal region of the terminal conducting airway, and apparent respiratory bronchioles were observed only in large-volume acini. The generation number of pathways per acinus was 8 ± 2 (range: 6-12). The pathway length at lower generations (generations 2-6) increased with the generation number in a single cluster, while did not significantly change at lower generations in some acinar groups. The pathway width increased with increasing generation numbers. Our approach characterized the quantitative morphology of pulmonary acinar clusters in mouse lung, and the results can be used in further biomechanical simulation studies. Anat Rec, 299:1424-1434, 2016. © 2016 Wiley Periodicals, Inc.


Collapse of a lipid-coated nanobubble and subsequent liposome formation.

We investigate the collapse of a lipid-coated nanobubble and subsequent formation of a lipid vesicle by coarse grained molecular dynamics simulations. A spherical nanobubble coated with a phospholipid monolayer in water is a model of an aqueous dispersion of phospholipids under negative pressure during sonication. When subjected to a positive pressure, the bubble shape deforms into an irregular spherical shape and the monolayer starts to buckle and fold locally. The local folds grow rapidly in multiple directions and forming a discoidal membrane with folds of various amplitudes. Folds of small amplitude disappear in due course and the membrane develops into a unilamellar vesicle via a bowl shape. Folds with large amplitude develop into a bowl shape and a multivesicular shape forms. The membrane shape due to bubble collapse can be an important factor governing the vesicular shape during sonication.


Effects of Stretching Speed on Mechanical Rupture of Phospholipid/Cholesterol Bilayers: Molecular Dynamics Simulation.

Rupture of biological cell membrane under mechanical stresses is critical for cell viability. It is triggered by local rearrangements of membrane molecules. We investigated the effects of stretching speed on mechanical rupture of phospholipid/cholesterol bilayers using unsteady molecular dynamics simulations. We focused on pore formation, the trigger of rupture, in a 40 mol% cholesterol-including bilayer. The unsteady stretching was modeled by proportional and temporal scaling of atom positions at stretching speeds from 0.025 to 30 m/s. The effects of the stretching speed on the critical areal strain, where the pore forms, is composed of two regimes. At low speeds (<1.0 m/s), the critical areal strain is insensitive to speed, whereas it significantly increases at higher speeds. Also, the strain is larger than that of a pure bilayer, regardless of the stretching speeds, which qualitatively agrees with available experimental data. Transient recovery of the cholesterol and phospholipid molecular orientations was evident at lower speeds, suggesting the formation of a stretch-induced interdigitated gel-like phase. However, this recovery was not confirmed at higher speeds or for the pure bilayer. The different responses of the molecular orientations may help explain the two regimes for the effect of stretching speed on pore formation.


Mathematical model of a heterogeneous pulmonary acinus structure.

The pulmonary acinus is a gas exchange unit distal to the terminal bronchioles. A model of its structure is important for the computational investigation of mechanical phenomena at the acinus level. We propose a mathematical model of a heterogeneous acinus structure composed of alveoli of irregular sizes, shapes, and locations. The alveoli coalesce into an intricately branched ductal tree, which meets the space-filling requirement of the acinus structure. Our model uses Voronoi tessellation to generate an assemblage of the alveolar or ductal airspace, and Delaunay tessellation and simulated annealing for the ductal tree structure. The modeling condition is based on average acinar and alveolar volume characteristics from published experimental information. By applying this modeling technique to the acinus of healthy mature rats, we demonstrate that the proposed acinus structure model reproduces the available experimental information. In the model, the shape and size of alveoli and the length, generation, tortuosity, and branching angle of the ductal paths are distributed in several ranges. This approach provides a platform for investigating the heterogeneous nature of the acinus structure and its relationship with mechanical phenomena at the acinus level.


Line tension of the pore edge in phospholipid/cholesterol bilayer from stretch molecular dynamics simulation

The line tension of the pore in a phospholipid bilayer is important for pore-mediated molecular transport techniques. To understand the cholesterol effects on the line tension of the pore edge at the molecular level, we perform molecular dynamics simulations of phospholipid bilayers with a pore containing cholesterol in different concentrations (0, 20, and 40 mol%). The bilayer with a pore is prepared by using an equibiaxial stretching simulation. The stretched bilayer with a pore is subsequently compressed and the pore spontaneously closes when the applied areal strain of the bilayer is below a certain value. Using the pore closure areal strain and a free energy model of a stretched bilayer with a pore, the upper and lower limits of the line tensions for the bilayers containing cholesterol at 0, 20, and 40 mol% are estimated to be 17.0-48.2, 54.5-100, and 170-261 pN, respectively. The increasing tendency of the line tension qualitatively agrees with that observed experimentally. The pores in the cholesterol-containing bilayers are lined with several cholesterol molecules, which might increase the bending rigidity of the pore edge, and result in the higher line tension of the cholesterol-containing bilayer. The considerable dependency of the line tension on the bilayer compositions might be useful to explain the large variations of the transduction efficiency observed with sonoporation treatment.

Journal of Biomechanical Science and Engineering


Computational studies on strain transmission from a collagen gel construct to a cell and its internal cytoskeletal filaments.

We developed a mechanical tissue model containing a cell with cytoskeletal filaments inside to investigate how tissue deformation is reflected in the deformation of a cell and its internal cytoskeletal filaments. Tissue that assumes a collagen gel construct was depicted as an isotropic linear elastic material, and the cell was modeled as an assembly of discrete elements including a cell membrane, nuclear envelope, and cytoskeletal filaments. Mechanical behaviors were calculated based on the minimum energy principle. The results demonstrated the effects of the type of tissue deformation on deformations of cytoskeletal filaments. The distribution of strains of cytoskeletal filaments was skewed toward compression when a tissue was stretched, toward stretch when the tissue was compressed, and almost normal when the tissue was sheared. The results also addressed the dependency of deformations of a cell and cytoskeletal filaments on the ratio of the Young's modulus of a tissue to that of a cell. Upon tissue stretching, cell strain increased and the distribution of strains of cytoskeletal filaments broadened on both stretch and compression sides with an increase in the Young's modulus ratio. This suggested that the manner of tissue deformation and the tissue/cell Young's modulus ratio are reflected in the distribution pattern of strains of cytoskeletal filaments. The present model is valuable to understanding the mechanisms of cellular responses in a tissue.


Molecular dynamics simulations of pore formation in stretched phospholipid/cholesterol bilayers.

Molecular dynamics (MD) simulations of pore formation in stretched dipalmitoylphosphatidylcholine (DPPC) bilayers containing different concentrations of cholesterol (0, 20, 40, and 60 mol%) are presented. The stretched bilayers were simulated by constant NPZA||T MD simulations with various constant areas. The effects of the cholesterol concentration on pore formation are examined in terms of the critical areal strain where the pore is formed, the processes of pore formation, and the change in molecular orientation of the DPPC molecules by analyzing the order parameters and radial distribution functions of the DPPC molecules. With increasing cholesterol concentration, the critical areal strain initially increases, peaks at 40 mol%, and then decreases, which agrees well with the available experimental data. For the bilayers containing cholesterol, DPPC molecules become disordered at low areal strains, whereas the order slightly increases when the areal strain exceeds a certain value depending on the cholesterol concentration. For 40 mol% cholesterol, the two monolayers in the bilayer interpenetrate under high areal strains, inducing an increase of the order parameters and the peak positions of the radial distribution function compared with their states at low areal strains, indicating the formation of an interdigitated gel-phase-like structure. The transient increasing of the order of the molecular orientations may inhibit water penetration into the bilayer, resulting in increased critical areal strain in the phospholipid/cholesterol bilayers.


A semiautomatic segmentation algorithm for extracting the complete structure of acini from synchrotron micro-CT images.

Pulmonary acinus is the largest airway unit provided with alveoli where blood/gas exchange takes place. Understanding the complete structure of acinus is necessary to measure the pathway of gas exchange and to simulate various mechanical phenomena in the lungs. The usual manual segmentation of a complete acinus structure from their experimentally obtained images is difficult and extremely time-consuming, which hampers the statistical analysis. In this study, we develop a semiautomatic segmentation algorithm for extracting the complete structure of acinus from synchrotron micro-CT images of the closed chest of mouse lungs. The algorithm uses a combination of conventional binary image processing techniques based on the multiscale and hierarchical nature of lung structures. Specifically, larger structures are removed, while smaller structures are isolated from the image by repeatedly applying erosion and dilation operators in order, adjusting the parameter referencing to previously obtained morphometric data. A cluster of isolated acini belonging to the same terminal bronchiole is obtained without floating voxels. The extracted acinar models above 98% agree well with those extracted manually. The run time is drastically shortened compared with manual methods. These findings suggest that our method may be useful for taking samples used in the statistical analysis of acinus.

Computational and Mathematical Methods in Medicine


Effects of Cytoskeletal Orientations on Deformation of a Cell Residing in a Collagen Gel Construct

The effects of cytoskeleton orientation angle on strain transmission from a tissue to a cell and its internal cytoskeletons were investigated by using a model where a cell was integrated in a tissue that assumes a collagen gel construct. A cell with uni-directionally or randomly aligned cytoskeletons was embedded in a tissue, which was stretched to a strain of 0.1. When the initial orientation angle of the cytoskeleton was zero, which corresponded to the stretch direction of the tissue, cell strain was minimal and mean cytoskeletal strain was maximal. As the initial cytoskeleton orientation angle increased, mean cytoskeletal strain values decreased, while cell strain increased. Cell strain decreased at an initial cytoskeleton orientation angle of 60°. At this angle, the mean cytoskeletal strain value was nearly zero, which was absolutely minimal. Subsequent increases in the initial cytoskeleton orientation angle resulted in further decreases in cell and cytoskeletal strain. The present multi-scale model may help in achieving structural integration of the biomechanical organization and provide valuable information for understanding the mechanisms underlying cellular remodeling.


Molecular dynamics simulations of pore formation dynamics during the rupture process of a phospholipid bilayer caused by high-speed equibiaxial stretching.

Rupture of a phospholipid bilayer under mechanical stresses is triggered by pore formation in an intact bilayer. To understand the molecular details of the dynamics of pore formation we perform molecular dynamics simulations of a phospholipid bilayer under two different equibiaxial stretching conditions: first, unsteady stretching with various stretching speeds in the range of 0.1-1.0m/s, and second, quasistatic stretching. We analyze (i) patterns of pore formation, (ii) the critical area where a pore forms, (iii) the deformation of the bilayer, and (iv) the apparent breaking force. With stretching, the bilayer deforms anisotropically due to lipid chain packing and water penetrating into the hydrophilic region of the bilayer, and when the area exceeds a critical value, water filled pore structure penetrating the bilayer forms and develops into a large pore, resulting in rupture. For a high stretching speed, small pores (multipore) can temporarily form in a small area. It has been statistically determined that the probability of the multipore formation, the critical areal strain, and the apparent breaking force increase with the stretching speed in the range of 0-50%, 0.8-2.0, and 250-400 pN, respectively. The results qualitatively agree with the experimental and other simulation results, and rationalize the leakage of hemoglobin from erythrocytes in shock wave experiments.


Modeling of Lung Microstructure Using Phase-Field Method

フェーズフィールド法を用いた肺微細構造のモデリング

We have developed a novel technique for modeling the realistic lung microstructure using phase-field method. In the phase-field method for the lung microstructure, the state (air and tissue) of a system is measured with an order parameter and the time-evolutions of the order parameters in the system, a tissue initially including seeds of the air, are obtained by solving the time-dependent bistable reaction-diffusion equations modified from the Allen-Cahn equation. The field of the order parameters is reconstructed as the 3D lung microstructure model by using the binalized slice images with a certain threshold of the order parameter between the two states (air and tissue) on the binalization. We found that irrespective of the number of the initial seeds, the results demonstrate isotropic evolution of alveolar regions (air) from the initial seeds, and the alveolar regions came closer with evolution, but were never merged because of the presence of alveolar wall (tissue). A further evolution of alveolar regions develops into spatially compartmentalized pore structure, which appeared to be similar to a natural lung. Variations in the number of, and the shape of the initial seeds, and the threshold of the order parameter for binalization result in various patterns of the ductal porous structure (i. e., alveolar duct or alveolar sac), satisfying the experimentally-obtained mean alveolar volume and mean alveolar wall thickness. Furthermore, the radial distribution function calculated from the centroids of alveoli were saturated to one without any significant peaks when the inter-alveolar distance exceeds a certain value, indicating that the alveoli in our model are disorderly distributed and repel each other with a certain distance. In conclusion, those results demonstrate that the method developed here is the promising method for parametric control and anatomically realistic production of lung microstructure model.

Transactions of the Japanese Society for Medical and Biological Engineering


Self-organization of a stable pore structure in a phospholipid bilayer.

We demonstrate the self-organization process of a stable pore structure in a phospholipid bilayer by unsteady and nonequilibrium molecular dynamics simulations. The simulation is started from an initial state including some amount of water molecules in its hydrophobic region, which is a model of a cell membrane stimulated by ultrasound radiation for the membrane permeabilization (sonoporation). We show that, in several nanoseconds, the bilayer-water system can spontaneously develop into a water-filled pore structure without any mechanical and electrical forcing from outside, when the initial number of water molecules in the hydrophobic region exceeds a critical value. The increase in the initial number of water molecules enhances the probability of pore formation, and sometimes induces the formation of transient micellelike structures of phospholipid molecules.


Cavitation Bubbles Mediated Molecular Delivery During Sonoporation

Molecular delivery using ultrasound (US) and nano/microbubbles (NBs), i.e., sonoporation, has applications in gene therapy and anticancer drug delivery. When NBs are destructed by ultrasound, the surrounding cells are exposed to mechanical impulsive forces generated by collapse of either the NBs or the cavitation bubbles created by the collapse of NBs. In the present study, experimental, theoretical and numerical analyses were performed to investigate cavitation bubbles mediated molecular delivery during sonoporation. Experimental observation using lipid NBs indicated that increasing US pressure increased uptake of fluorescent molecules, calcein (molecular weight: 622), into 293T human, and decreased survival fraction. Confocal microscopy revealed that calcein molecules were uniformly distributed throughout the some treated cells. Next, the cavitation bubble behavior was analyzed theoretically based on a spherical gas bubble dynamics. The impulse of the shock wave (i.e., the pressure integrated over time) generated by the collapse of a cavitation bubble was a dominant factor for exogenous molecules to enter into the cell membrane rather than bubble expansion. Molecular dynamics simulation revealed that the number of exogenous molecules delivered into the cell membrane increased with increasing the shock wave impulse. We concluded that the impulse of the shock wave generated by cavitation bubbles was one of important parameters for causing exogenous molecular uptake into living cells during sonoporation.

Journal of Biomechanical Science and Engineering


Molecular dynamics simulation of structural changes of lipid bilayers induced by shock waves: Effects of incident angles.

Unsteady and nonequilibrium molecular dynamics simulations of the response of dipalmitoylphosphatidylcholine (DPPC) bilayers to the shock waves of various incident angles are presented. The action of an incident shock wave is modeled by adding a momentum in an oblique direction to water molecules adjacent to a bilayer. We thereby elucidate the effects of incident shock angles on (i) collapse and rebound of the bilayer, (ii) lateral displacement of headgroups, (iii) tilts of lipid molecules, (iv) water penetration into the hydrophobic region of the bilayer, and (v) momentum transfer across the bilayer. The number of water molecules delivered into the hydrophobic region is found to be insensitive to incident shock angles. The most important structural changes are the lateral displacement of headgroups and tilts of lipid molecules, which are observed only in the half of the bilayer directly exposed to a shock wave for all incident shock angles studied here. As a result, only the normal component of the added oblique momentum is substantially transferred across the bilayer. This also suggests that the irradiation by shock waves may induce a jet-like streaming of the cytoplasm toward the nucleus.

Biochimica et Biophysica Acta (BBA) - Biomembranes


Structural change in lipid bilayers and water penetration induced by shock waves: molecular dynamics simulations.

The structural change of a phospholipid bilayer in water under the action of a shock wave is numerically studied with unsteady nonequilibrium molecular dynamics simulations. The action of shock waves is modeled by the momentum change of water molecules, and thereby we demonstrate that the resulting collapse and rebound of the bilayer are followed by the penetration of water molecules into the hydrophobic region of the bilayer. The high-speed phenomenon that occurs during the collapse and rebound of the bilayer is analyzed in detail, particularly focusing on the change of bilayer thickness, the acyl chain bend angles, the lateral fluidity of lipid molecules, and the penetration rate of water molecules. The result shows that the high-speed phenomenon can be divided into two stages: in the first stage the thickness of bilayer and the order parameter are rapidly reduced, and then in the second stage they are recovered relatively slowly. It is in the second stage that water molecules are steadily introduced into the hydrophobic region. The penetration of water molecules is enhanced by the shock wave impulse and this qualitatively agrees with a recent experimental result.


Transfection effect of microbubbles on cells in superposed ultrasound waves and behavior of cavitation bubble.

The combination of ultrasound and ultrasound contrast agents (UCAs) is able to induce transient membrane permeability leading to direct delivery of exogenous molecules into cells. Cavitation bubbles are believed to be involved in the membrane permeability; however, the detailed mechanism is still unknown. In the present study, the effects of ultrasound and the UCAs, Optison on transfection in vitro for different medium heights and the related dynamic behaviors of cavitation bubbles were investigated. Cultured CHO-E cells mixed with reporter genes (luciferase or beta-gal plasmid DNA) and UCAs were exposed to 1 MHz ultrasound in 24-well plates. Ultrasound was applied from the bottom of the well and reflected at the free surface of the medium, resulting in the superposition of ultrasound waves within the well. Cells cultured on the bottom of 24-well plates were located near the first node (displacement node) of the incident ultrasound downstream. Transfection activity was a function determined with the height of the medium (wave traveling distance), as well as the concentration of UCAs and the exposure time was also determined with the concentration of UCAs and the exposure duration. Survival fraction was determined by MTT assay, also changes with these values in the reverse pattern compared with luciferase activity. With shallow medium height, high transfection efficacy and high survival fraction were obtained at a low concentration of UCAs. In addition, capillary waves and subsequent atomized particles became significant as the medium height decreased. These phenomena suggested cavitation bubbles were being generated in the medium. To determine the effect of UCAs on bubble generation, we repeated the experiments using crushed heat-treated Optison solution instead of the standard microbubble preparation. The transfection ratio and survival fraction showed no additional benefit when ultrasound was used. These results suggested that cavitation bubbles created by the collapse of UCAs were a key factor for transfection, and their intensities were enhanced by the interaction of the superpose ultrasound with the decreasing the height of the medium. Hypothesizing that free cavitation bubbles were generated from cavitation nuclei created by fragmented UCA shells, we carried out numerical analysis of a free spherical bubble motion in the field of ultrasound. Analyzing the interaction of the shock wave generated by a cavitation bubble and a cell membrane, we estimated the shock wave propagation distance that would induce cell membrane damage from the center of the cavitation bubble.


A non-invasive tissue-specific molecular delivery method of cancer gene therapy.

A Japanese word, monozukuri (literally translated "making things") is the philosophy of first having the idea and then the faith in the technical expertise and experience to accomplish the result. We believe that the concept of engineering is monozukuri. Through the process of monozukuri, engineered natural science based on mathematics and physics has been developed. Medicine is the field of study which has been developed for maintaining daily healthy life with diagnosis, treatment, examination, and protection. Biomedical engineering is the interdisciplinary study of engineering and medicine, and should be developed based on monozukuri. In this particular research, we have developed a physical molecular delivery method for cancer gene therapy using nano/microbubbles and ultrasound. First, the behavior of cavitation bubbles and subsequent shock wave phenomena involved in the mechanism of molecular delivery were analyzed, combining theory and computer simulation. In a second step, the methodology was optimized in vitro and in vivo. Finally, the therapeutic potential of the method in pre-clinical models was evaluated using transgenes relevant to cancer gene therapy instead of reporter genes, and whole body, non-invasive imaging using single photon emission computed tomography (SPECT/CT) was used to evaluate the selectivity of gene delivery in vivo.

Minimally Invasive Therapy & Allied Technologies