In vivo time-dependent gene expression of cationic lipid-based emulsion as a stable and biocompatible non-viral gene carrier

To make stable and biocompatible non-viral gene carriers for therapeutic gene therapy, we developed a cationic lipid-based emulsion (CLE) prepared by an oil-in-water (O/W) emulsion method, wherein squalene oil was used as an oil core and the cationic lipid, 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP), was employed as an emulsifier. To evaluate in vivo characteristics such as toxicity and time-dependent gene expression, a bioluminescence reporter gene in pCMV-luc plasmid DNA was simply mixed with CLE in aqueous condition, resulting in a CLE/DNA complex. The CLE/DNA complex was optimized to form a compact and stable nano-sized particle by adding different amounts of plasmid DNA, and an optimal cationic lipid-to-DNA (C/D) weight ratio of 4 was identified. Freshly prepared CLE/DNA complex, with a C/D of 4, showed a high transfection efficiency and minimal cytotoxicity in vitro, compared to controls of a liposome (DOTAP)/DNA complex and a branched poly(ethyleneimine) (Mw = 25 kDa) (bPEI)/DNA complex, respectively. The in vivo characteristics of the CLE/DNA complex were evaluated after intravenous injection into Balb/c mice. Time-dependent gene expression data in vivo were obtained using a non-invasive, whole animal bioluminescence imaging system. These data showed that the CLE/DNA complex offered prolonged high-level gene expression for 1 week, particularly in the liver and spleen. On the other hand, the controls of DOTAP/DNA complex and bPEI/DNA complex showed a relatively lower gene expression, because of the unstable and toxic properties of the control carriers. Our in vivo gene expression data demonstrate the potential of the CLE/DNA complex as a non-viral gene carrier for in vivo gene delivery.     

Subharmonic contrast intravascular ultrasound for vasa vasorum imaging

The feasibility of subharmonic contrast intravascular ultrasound (IVUS) imaging was investigated using a prototype nonlinear IVUS system and the commercial contrast agent Definity™. The system employed a mechanically scanned commercial catheter with a custom transducer element fabricated to have sensitivity at both 15 and 30 MHz. Experiments were conducted at a fundamental frequency of 30 MHz (F30; 25% bandwidth), with on-axis pressures ranging from 0.12 to 0.79 MPa, as measured with a needle hydrophone. In vitro characterization experiments demonstrated the detection of 15 MHz subharmonic signals (SH15) when pressure levels reached 360 kPa. The formation of SH15 images was shown, with tissue signals suppressed to near the noise floor and contrast to tissue ratios were improved by up to 30 dB relative to F30. In vivo experiments were performed using the atherosclerotic rabbit aorta model. Following the bolus injection of contrast agent upstream of the imaging catheter, agent was detected within the aorta, vena cava and within the perivascular space. These results provide a first in vivo demonstration of subharmonic contrast IVUS and suggest its potential as a new technique for imaging vasa vasorum. (E-mail: goertz@sri.utoronto.ca)     

DNA transfer and cell killing in epidermoid cells by diagnostic ultrasound activation of contrast agent gas bodies in vitro

DNA transfer by sonoporation and cell killing in monolayer cells were examined by contrast-aided low-power diagnostic ultrasound (US). Culture chambers with epidermoid cell monolayers were scanned at about 1 mm/s with a 1.5-MHz scan head aimed upward at the chamber in a 37 degrees C water bath. For DNA transfer tests, plasmids coding for green fluorescent protein (GFP) were added to the medium, and GFP expression was assessed by flow cytometry after 2 days. In separate tests, cell killing was determined immediately after treatment. GFP-positive cell counts were 0.4% (0.7% SD) for shams and 3.7% (1.2% SD) of cells for exposure at 2.3 MPa with 2% Optison contrast agent. The fraction of dead cells was 3.4% (1.7% SD) in shams and 28.6% (6.3% SD) in exposed chambers. Both effects increased for increasing Optison concentration and increasing peak rarefactional pressure amplitude. Contrast-aided diagnostic US has a potential therapeutic application for gene transfer, but a trade-off appears to exist with cell killing.     

Lithotripter shock waves with cavitation nucleation agents produce tumor growth reduction and gene transfer in vivo

Cavitation nucleation agents (CNA) can greatly enhance DNA transfer and cell killing for therapeutically useful applications of nonthermal bioeffects of ultrasound (US). Renal carcinoma (RENCA) tumor cells were implanted and grown to about 400 microL tumor volumes on the hind legs of syngeneic Balb/c mice. Before treatment, mice were anesthetized, the tumor region was shaved and depilated, and a DNA plasmid coding for marker proteins was injected into the tumor. Two sets of tests were completed: the first set involved measurement of tumor growth for 4 days and use of a beta-galactosidase marker plasmid for localization of transfection, and the second set involved 2 days of growth and use of a luciferase marker plasmid for assessing overall protein expression. Either saline, Optison US contrast agent, a vaporizing perfluoropentane droplet suspension (SDS) or air bubble was also injected intratumorally at 10% of tumor volume as a CNA. In some tests, droplets or contrast agent were injected IV. Shock waves (SW) were generated from a spark-gap lithotripter at 7.4 MPa peak negative pressure amplitude. For sham exposure, tumor volume increased by a factor of 3.6 in 4 days. With 500-SW treatment, all the CNA reduced 4-day tumor growth about the same amount (to factors of 1.2 to 1.9). Marker gene expression was generally localized to the region around the needle injection path. All the agents, except saline, produced statistically significant increases of 11.8- to 14.6-fold in luciferase expression after 2 days, relative to sham exposure. IV injection of Optison or droplet nucleation agents before SW treatment reduced tumor growth to factors of 1.0 and 0.7, but did not increase transfection. These results demonstrate the efficacy of CNA in vivo and should lead to improved strategies for simultaneous SW tumor ablation and cancer gene therapy.     

Noninvasive assessment of the viscoelasticity of peripheral arteries

Currently used methods of examining the mechanical properties of blood vessel walls are either indirect or invasive, or measure vessel diameter and pressure waveforms at different sites. We developed a noninvasive technique to assess the mechanical properties and viscoelasticity of peripheral arteries. The pressure-strain elastic modulus (Ep) and the viscoelastic properties (energy dissipation ratio, EDR) of the common carotid artery (CCA), brachial artery (BA), radial artery (RA) and dorsalis pedis artery (DPA) were determined by means of palpating pressure and diameter distension waveforms extracted from high-resolution ultrasonography. The methodology was validated in vitro using an elastic tube phantom, as well as in vivo. In vivo study in 10 healthy volunteers (mean age 22 y) showed that the pressure-diameter curves were nonlinear, with an inflection at about 85–90 mmHg, and routed clockwise with slight hysteresis. The CCA (n = 5) had a mean diameter of 6.74 mm and the pulsatile diameter distension was 12.2%. The Ep calculated at the CCA was 0.44 × 10⁶ dyne/cm² with an EDR of 7.18%. The BA, RA and DPA (n = 10) had mean diameters of 3.91 mm, 2.21 mm and 2.12 mm; arterial strains of 4.60%, 4.25% and 8.91%; mean Ep of 1.39, 1.45, 0.90 × 10⁶ dyne/cm²; and mean EDRs of 6.34%, 6.15% and 5.60%, respectively. The method presented is relatively simple to implement clinically and has potential as a new diagnostic tool for detecting local vascular changes.     

Potentiometric determination of serum ionized calcium in a normal human population

Ionized calcium and total calcium were measured in sera from 101 normal individuals aged 19–96 years. Significant changes with age were observed for ionized calcium in both sexes and for total calcium in men. The relation between pH changes and the level of ionized calcium in vitro and in vivo during hyperventilation was examined. Some methodological factors that influence the results, namely preparation of the standard solutions, sample collection, and storage of the sample, have been evaluated.     

A novel method to measure acoustic speed of bone tissue

Acoustic speed is an important parameter that can be used to characterize bone tissue for evaluation of osteoporosis. Traditional approaches for measuring acoustic speed require knowledge of either the specimen thickness, which is sometimes difficult to obtain from biological tissues, or the reference fluid velocity that, moreover, is an unknown parameter for internal tissue in in vivo measurement. In this paper, a new method is proposed to measure acoustic speed from dual reflected ultrasound signals. This technique utilizes two transducers placed on the same side of the test object; one for transmitter and receiver, and the other one for receiver only. The acoustic speed of the test object is based on the information of time-of-flight from the signals received by both transducers and the separation distance between the transducer pair. The technique developed here not only eliminates the requirement of knowledge of specimen thickness, but also shows a feasibility for clinical applications. The results of different porcine and bovine bone samples measured in vitro by this technique are in good agreement with those measured by other published methods. In vivo measurement results of 10 healthy young volunteers' tibias are also reported.     

Tumor growth reduction and DNA transfer by cavitation-enhanced high-intensity focused ultrasound in vivo

The potential application of high-intensity focused ultrasound (US), HIFU, was investigated for nonthermal gene transfer and tumor ablation. Renal carcinoma (RENCA) tumors were implanted on the hind leg of BALB/c mice and injected with a marker plasmid. Optison US contrast agent was also injected into the tumor (IT) or into the venous (IV) circulation. HIFU at 1.55 MHz was applied to the tumors with guidance from diagnostic US images. One test of transfection was also performed with lithotripter shock waves. In one set of exposures, tumor volume was followed for 4 days and a beta-galactosidase marker plasmid was used for localization of transfected cells. A second set of exposures employed a luciferase marker plasmid for assessing overall transfection after 2 days. Use of 100-ms bursts at 8-MPa peak rarefactional pressure amplitude stopped tumor growth during the 4-day period, compared to a 2.8-fold growth in shams and yielded luciferase expression 34-fold greater than in shams. Longer bursts or higher pressure amplitudes led to decreases in tumor growth, but did not yield increases in transfection. The HIFU results were similar to those of shock waves for cavitation enhanced by IT Optison. These results should aid in optimizing the application of HIFU for nonthermal tumor treatment.     

Bragg diffraction scanner for ultrasonic tissue characterisation in vivo

An ultrasonic scanner, which exploits the concept of Bragg diffraction of ultrasound for tissue characterisation, has been constructed for in vivo operation. A Grey-Scale B-scan is produced, and the ultrasonic transducer can be mechanically constrained to point at one specific volume of tissue, first identified on the B-scan, throughout an angular movement of 50°. Compensation for variations in the attenuation and time delay of echoes from the interrogated volume due to the changing ultrasonic path length is performed electronically. Qualitative results have been obtained in vivo from human calf muscle, liver parenchyma, and a metastatic deposit in liver, and are similar in many respects to results from in vitro measurements. The ultrasonic exposure is identical to that normally delivered in conventional ultrasonic examinations.     

Cell damage by lithotripter shock waves at high pressure to preclude cavitation

Acoustic cavitation has been implicated as a cause of cell damage by lithotripter shock waves, particularly under in vitro conditions. When red blood cells were exposed to shock waves (from an electrohydraulic lithotripter) while under high hydrostatic pressure (> 80 atm), cell lysis was dramatically reduced over that seen at atmospheric pressure, which is consistent with damage due to acoustic cavitation. However, even at > 120 atm of pressure, lysis was still 97% above that of cells not exposed to shock waves, revealing significant damage that apparently was due to mechanisms other than cavitation. Hydrostatic pressure alone did not cause cell lysis, and shock-wave–dependent damage occurred when the cells were in fluid suspension, or when they were centrifuged to the end of the vial. Shock-wave damage at high pressure increased with increasing shock-wave number, and was seen at 24 and 20 kV, but not at 16 kV. This shock-wave damage at high pressure makes up a noteworthy portion of the total cell injury seen at atmospheric pressure (about 10% at 24 kV), suggesting significant noncavitational injury to cells in vitro. Because cavitation occurs far more readily in vitro than in vivo, the noncavitational damage seen in the present study could represent a substantial portion of cell injury seen in vivo with shock-wave lithotripsy.     

Transcranial ultrasound-improved thrombolysis: diagnostic vs. therapeutic ultrasound

Success of stroke treatment with rt-PA depends on rapid vessel recanalization. Enzymatic thrombolysis may be enhanced by additional transcranial application of ultrasound (US). We investigated this novel technique using a 185-kHz probe and compared it to standard diagnostic US. In vitro studies were performed in a continuous pressure tubing system. Clots were placed in a postmortem skull and treated with rt-PA together with or without transtemporal 185-kHz US insonation (2W/cm²) and in comparison to 1-MHz diagnostic US (0.5 W/cm²). Recanalization time was significantly (p < 0.01) shorter in the 185-kHz (14.1 min) and 1-MHz (17.1 min) US rt-PA treatment group compared to rt-PA treatment alone (29.3 min.). Flow rate was significantly higher (p < 0.025) and increased faster in the combined treatment group with rt-PA + 185-kHz US compared to rt-PA + 1-MHz US. We investigated the blood-brain barrier in rats after 90-min exposure time of the brain with 185-kHz US, but no damage was observed. Results suggest efficacy and safety of the 185-kHz transducer, which is superior to diagnostic US. Such a novel US probe may be able to optimize thrombolytic stroke treatment. (E-mail: hennerici@neuro.ma.uni-heidelberg.de)     

On the IVUS plaque volume error in coronary arteries when neglecting curvature

Plaque volume determined by common linear 3-D IVUS analysis systems will show under- or overestimation in curved vessel segments because these systems approximate the true 3-D transducer pull-back trajectory by a straight line. We developed a mathematical model that showed that the error is primarily dependent on the curvature of the pull-back trajectory and not on vessel tortuosity. Furthermore, we measured this error in vivo in the coronary arteries of 15 patients, comparing the plaque volume using a true 3-D reconstruction method with that of the linear approach. The in vivo plaque volume error ranged from 2.3% to −1.2% for 15 coronary segments with lengths ranging from 38.8 to 89.1 mm (62.2 ± 13 mm). The volume error introduced by linear 3-D IVUS analysis systems is dependent on the curvature of the pull-back trajectory. The error measured in vivo was small and inversely related to segment length.     

A comparison of shock wave and sinusoidal-focused ultrasound-induced localized transfection of HeLa cells

Both shock waves and sinusoidal continuous wave ultrasound can mediate DNA transfer into cells. The relative transfection efficiencies of different ultrasound modalities are unclear. The purpose of this paper is to compare the transfection efficiency of lithotripter shock waves and focused sinusoidal ultrasound in vitro. HeLa cells were transfected with β-galactosidase and luciferase plasmid DNA reporter. Shock waves were generated by an electromagnetic sound source. Sixty to 360 pulses at 1 Hz pulse frequency were administered at 13, 16 or 19 kV capacitor voltage. Sinusoidal focused ultrasound was generated by a single focus piezoceramic air-backed disk transducer at a carrier frequency of 1.18 MHz operated in a pulsed mode. Compared to cells mixed with DNA only, shock waves induced up to eightfold more transfected cells at a cell viability of 5%, while sinusoidal-focused ultrasound induced up to 80-fold more transfected cells at a cell viability of 45%. The corresponding transfection efficiencies of the HeLa cells were 0.08% for shock waves and 3% for focused ultrasound. These results may contribute to the selection of the ultrasound modality as a localized, noninvasive and safe tool to mediate gene transfer.     

A high-frequency continuous-wave Doppler ultrasound system for the detection of blood flow in the microcirculation

Basic ultrasound physics and several clinical and experimental observations suggest that high-frequency Doppler ultrasound (HFD) operating in the frequency range 20–100 MHz holds the promise of detecting blood flow in the microcirculation. This article describes a directional, continuous-wave (CW), 1- to 200-MHz Doppler ultrasound system. The system electronics have a dynamic range of 100 dB, a noise floor of 10 nV and a directional isolation of 50 dB. The development of a 40-MHz Doppler transducer composed of two, 81-μm-thick, lithium niobate crystals that have been air-backed and transmission-line tuned for maximum sensitivity is described. This device is used to test the CW Doppler system using string and capillary phantoms and in vivo tissue. We show that HFD can detect and measure velocities on the order of the blood velocities found in the capillaries (1 mm/s) and arterioles (5 mm/s) with suitable velocity (50–500 μm/s) and temporal (20–250 ms) resolutions. In vivo measurements demonstrate that HFD is sensitive to the detection of blood flow in small vessels.     

Characterization of extracorporeal ablation of normal and tumor-bearing liver tissue by high intensity focused ultrasound

Treatment parameters of extracorporeal high intensity focused ultrasound (HIFU) were analysed in normal and tumor-bearing rabbit liver. HIFU was generated with a 1 MHz transducer and energy was provided by a 7.5 kW power amplifier. In vivo experiments were conducted on 74 New Zealand rabbits. Normal rabbits and rabbits bearing an intrahepatic VX2 tumor were used. In group 1, spatial peak temporal peak (SPTP) intensities ranging from 1470 to 5500 W cm⁻² and exposure times from 0.5 to 5 s were tested at a constant depth in the liver; in group 2, the output power was adjusted as a function of the target depth in order to keep constant the focal in situ intensity in the liver; in group 3 (liver tumors), the focal in situ intensity was 1365 W cm⁻² in eight rabbits and 500 W cm⁻² in nine. In groups 1, 2 and 3, rabbits were sacrificed 48 h after the treatment. Groups 4 and 5 were designated for analysis of the lesion in the normal liver 4 weeks after treatment at 1000 W cm⁻² and 3000 W cm⁻² SPTP intensities, respectively. In normal rabbits, the lesion volume increased with exposure time at constant intensity; there was a negative correlation between intensity and exposure time (group 1). When the output power was adjusted as a function of the path length, the lesion size was nearly constant (group 2). In VX2 rabbits, tumor destruction rates were significantly higher in rabbits treated at 500 W cm⁻² than in rabbits treated at 1365 W cm⁻² (p < 0.05; group 3). As in the normal liver, the lesion volume increased with the exposure time at constant intensity. HIFU lesions treated at 1000 W cm⁻² (SPTP) healed as thin fibrous scars, and no severe complication occurred (group 4); at 3000 W cm⁻² (SPTP), scars were larger and perforation of a neighboring organ was seen in 7 of 11 rabbits (group 5).     

An endoscopic micromanipulator for multiplanar transesophageal imaging

We have developed an esophageal probe with a precision micromanipulator and a transversely oriented 32 element ultrasonic array which operates at 3.5 MHz. The probe allows us to obtain multiple two-dimensional images of the heart with known angular relationships between them over a series of cardiac cycles. Our ultimate purpose is to acquire images for left ventricular volume estimation with a three-dimensional reconstruction method. Technical details of the probe design are given. In vitro tests have shown that the imaging plane can be angulated within 1.5° root mean square error. In vivo results with dogs have demonstrated its ability to obtain multiplanar short axis images of the heart.     

Application of Newcastle disease virus in the treatment of colorectal cancer

Colorectal cancer (CRC) is one of the main reasons of tumor-related deaths worldwide. At present, the main treatment is surgery, but the results are unsatisfactory, and the prognosis is poor. The majority of patients die due to liver or lung metastasis or recurrence. In recent years, great progress has been made in the field of tumor gene therapy, providing a new treatment for combating CRC. As oncolytic viruses selectively replicate almost exclusively in the cytoplasm of tumor cells and do not require integration into the host genome, they are safer, more effective and more attractive as oncolytic agents. Newcastle disease virus (NDV) is a natural RNA oncolytic virus. After NDV selectively infects tumor cells, the immune response induced by NDV’s envelope protein and intracellular factors can effectively kill the tumor without affecting normal cells. Reverse genetic techniques make NDV a vector for gene therapy. Arming the virus by inserting various exogenous genes or using NDV in combination with immunotherapy can also improve the anti-CRC capacity of NDV, and good results have been achieved in animal models and clinical treatment trials. This article reviews the molecular biological characteristics and oncolytic mechanism of NDV and discusses in vitro and in vivo experiments on NDV anti-CRC capacity and clinical treatment. In conclusion, NDV is an excellent candidate for cancer treatment, but more preclinical studies and clinical trials are needed to ensure its safety and efficacy.     

Targeted tissue transfection with ultrasound destruction of plasmid-bearing cationic microbubbles

The aim of this study was to assess the relative efficacy and mechanism of gene transfection by ultrasound (US) destruction of plasmid-bearing microbubbles. Luciferase reporter plasmid was charge-coupled to cationic lipid microbubbles. Rat hindlimb skeletal muscle was exposed to intermittent high-power US during dose-adjusted intra-arterial (IA) or IV administration of plasmid-bearing microbubbles via the carotid artery or jugular vein, respectively. At 4 days, luciferase activity in US-exposed skeletal muscle was 200-fold greater with IA than with IV administration of plasmid-bearing microbubbles, and was similar to transfection achieved by IM injection of plasmid (positive control). No transfection occurred with US and IA injection of plasmid alone. Intravital microscopy of the cremaster muscle in mice following administration of microbubbles and US exposure demonstrated perivascular deposition of fluorescent plasmid, the extent of which was twofold greater for IA compared to IV injection. Electron microscopy demonstrated a greater extent of myocellular microporations in US-exposed muscle after IA injection of microbubbles. We conclude that muscle transfection by US destruction of plasmid-bearing cationic microbubbles is amplified by IA, rather than IV, injection of microbubbles due to greater extravascular deposition of plasmid and to greater extent of myocellular microporation. (E-mail: jlindner@virginia.edu)     

Induction of apoptosis in sonoporation and ultrasonic gene transfer

The role of apoptosis in sonoporation and ultrasound-enhanced gene transfection of cell suspensions was examined in vitro. Suspensions of HL-60 and of CHO-K1 cells were exposed to 2.25-MHz continuous ultrasound for 1 min in a 60-rpm rotating-tube exposure system, with ultrasound contrast media added to ensure nucleation of cavitation. Cell necrosis was measured by trypan blue dye exclusion (using a hemacytometer) and by propidium iodide nuclear staining (using flow cytometry). Apoptosis was detected by the annexin V method with Alexa Fluor 350 as the fluorescent label, and confirmed by Hoechst 33342 nuclear staining. Sonoporation cell loading was assessed by uptake of large fluorescent-dextran molecules from the medium. Transfection was demonstrated by expression of green fluorescent protein (GFP) from plasmids transferred into the cells by the treatment. Cell scoring was performed by flow cytometry, with necrotic cell events excluded. For HL-60 cells at 0.4 MPa, cell loading and transfection was significantly increased relative to shams at 2, 6 and 24 h post exposure, peaking at 19.0 +/- 5.5% and 9.6 +/- 4.2% of non-necrotic cells, respectively, at 6 h. However, about one third of the treatment-positive cells were identified as apoptotic. The cell loading and gene transfer effects increased for increasing peak rarefactional pressure amplitude, reaching 24.4 +/- 7.7% and 12.7 +/- 5.1% of non-necrotic cells, respectively, for 0.6-MPa exposure. However, the lethal cellular injury caused by cavitation in the rotating tube system reduced the overall apparent efficacy of cell loading and gene transfer to 5.1 +/- 2.1% and 2.1 +/- 0.9%, respectively, after accounting for necrosis and apoptosis. Similar tests with CHO cells showed increased sonoporation but mostly cell death by necrosis, rather than apoptosis. The induction of apoptosis by cavitation treatments should be considered as a possible confounding factor, in addition to necrosis, in sonoporation and ultrasonic gene transfer research.     

The search for cavitation in vivo

Until the mid 1970s, it was generally assumed that, with the short pulses of ultrasound (US) used in medical diagnosis, there was little need for concern about the possibility of inertial cavitation in vivo. This assumption came into question when experimental evidence indicated that killing of fruit fly larvae by diagnostically relevant US was associated with the presence of gas in the respiratory apparatus of the organisms. Independent theoretical contributions by Flynn and Apfel in the early 1980s made it clear that complacency in regard to cavitation was not warranted. Later, the mammalian lung, as with larva, was shown to be particularly vulnerable when it contained air. Yet, overall evidence suggests that lung hemorrhage is not consistent with the classical picture of inertial cavitation. Most recently, however, hemolysis and hemorrhage associated with the use of contrast agents have provided nearly incontrovertible evidence of the occurrence of cavitation in vivo.