Ultrasound-assisted microbubbles gene transfer in tendons for gene therapy

Our study aimed at evaluating the use of ultrasound-assisted microbubbles gene transfer in mice Achilles tendons. Using a plasmid encoding luciferase gene, it was found that an efficient and stable gene expression for more than two weeks was obtained when tendons were injected with 10 μg of plasmid in the presence of 5 × 10⁵ BR14 microbubbles with the following acoustic parameters: 1 MHz, 200 kPa, 40% duty cycle and 10 min of exposure time. The rate of gene expression was 100-fold higher than that obtained with naked plasmid injected alone without ultrasound or with ultrasound in absence of microbubbles. The long term expression of transgene makes ultrasound-assisted microbubble a suitable method for gene therapy in tendons.     

Real-Time Measurements and Modelling on Dynamic Behaviour of SonoVue Bubbles Based on Light Scattering Technology

The dynamic behaviour of SonoVue microbubbles, a new generation ultrasound contrast agent, is investigated in real time with light scattering method. Highly diluted SonoVue microbubbles are injected into a diluted gel made of xanthan gum and water. The responses of individual SonoVue bubbles to driven ultrasound pulses are measured. Both linear and nonlinear bubble oscillations are observed and the results suggest that SonoVue microbubbles can generate strong nonlinear responses. By fitting the experimental data of individual bubble responses with Sarkar＇s model, the shell coating parameter of the bubbles and dilatational viscosity is estimated to be 7.0 nm-s-Pa.     

Influence of nesting shell size on brightness longevity and resistance to ultrasound-induced dissolution during enhanced B-mode contrast imaging

This study aims to bridge the gap between transport mechanisms of an improved ultrasound contrast agent (UCA) and its resulting behavior in a clinical imaging study. Phospholipid-shelled microbubbles nested within the aqueous core of a polymer microcapsule are examined for their use and feasibility as an improved UCA. The nested formulation provides contrast comparable to traditional formulations, specifically an SF₆ microbubble coated by a DSPC PEG-3000 monolayer, with the advantage that contrast persists at least nine times longer in a mock clinical, in vitro setting. The effectiveness of the sample was measured using a contrast ratio in units of decibels (dB) which compares the brightness of the nested microbubbles to a reference value of a phantom tissue mimic. During a 40 min imaging study, six nesting formulations with average outer capsule diameters of 1.95, 2.53, 5.55, 9.95, 14.95, and 20.51 μm reached final contrast ratio values of 0.25, 2.35, 3.68, 4.51, 5.93, and 8.00 dB, respectively. The starting contrast ratio in each case was approximately 8 dB and accounts for the brightness attributed to the nesting shell. As compared with empty microcapsules (no microbubbles nested within), enhancement of the initial contrast ratio increased systematically with decreasing microcapsule size. The time required to reach a steady state in the temporal contrast ratio profile also varied with microcapsule diameter and was found to be 420 s for each of the four smallest shell diameters and 210 s and 150 s, respectively, for the largest two shell diameters. All nested formulations were longer-lived and gave higher final contrast ratios than a control sample comprising un-nested, but otherwise equivalent, microbubbles. Specifically, the contrast ratio of the un-nested microbubbles decreased to a negative value after 4 min of continuous ultrasound exposure with complete disappearance of the microbubbles after 15 min whereas all nested formulations maintained positive contrast ratio values for the duration of the 40 min trial. The results are consistent with two distinct stages of gas transport: in the first stage, passive diffusion occurs under ambient conditions across the microbubble monolayer within the first few minutes after formulation until the aqueous interior of the microcapsule is saturated with gas; in the second stage ultrasound drives additional gas dissolution even further due to pressure modulation. It is important to understand the chemistry and transport mechanisms of this contrast agent under the influence of ultrasound to attain better perspicacity for enhanced applications in imaging. Results from this study will facilitate future preclinical studies and clinical applications of nested microbubbles for therapeutic and diagnostic imaging.     

脉冲超声激励下SonoVue微泡的瞬态空化特性

将SonoVue微泡从临床疾病诊断拓展至治疗引起了诸多研究人员的兴趣。为了平衡治疗效率和生物安全性,深入理解声学参数和SonoVue微泡瞬态空化的关系至关重要。本研究自行制备仿体容器放置SonoVue微泡,使用1 MHz发射换能器激励其产生空化效应,另一个7.5 MHz的聚焦换能器接收声信号,经放大及高速数据采集后送上位机处理。通过深入分析信号的时频域特征,我们提出以宽带信号的能量及其随时间变化曲线的半高宽来表征瞬态空化的剂量(ICD)和相对持续时间(ICP),并确定:瞬态空化的发生和ICD依赖于峰值负声压,但ICP随峰值负声压的增加而减小;脉冲重复频率和脉冲持续时间都和ICD及ICP正相关;且脉冲持续时间的影响较大。这些结果有望为SonoVue微泡的治疗应用提供理论支持。      

A preliminary in vitro assessment of polymer-shelled microbubbles in contrast-enhanced ultrasound imaging

This paper focuses on the use of poly (vinyl alcohol)-shelled microbubbles as a contrast agent in ultrasound medical imaging. The objective was an in vitro assessment of the different working conditions and signal processing methods for the visual detection (especially in small vessels) of such microbubbles, while avoiding their destruction. Polymer-shelled microbubbles have recently been proposed as ultrasound contrast agents with some important advantages. The major drawback is a shell that is less elastic than that of the traditional lipidic microbubbles. Weaker echoes are expected, and their detection at low concentrations may be critical. In vitro experiments were performed with a commercial ultrasound scanner equipped with a dedicated acquisition board. A concentration of 100 bubbles/mm³, excitation pressure amplitudes from 120 kPa to 320 kPa, and a central frequency of 3 MHz or 4.5 MHz were used. Three multi-pulse techniques (i.e., pulse inversion, contrast pulse sequence based on three transmitted signals, and contrast pulse sequence in combination with the chirp pulse) were compared. The results confirmed that these microbubbles produce a weaker ultrasound response than lipidic bubbles with a reduced second-order nonlinear component. Nevertheless, these microbubbles can be detected by the contrast pulse sequence technique, especially when the chirp pulse is adopted. The best value of the contrast-to-tissue ratio was obtained at an excitation pressure amplitude of 230 kPa: although this pressure amplitude is higher than what is typically used for lipidic microbubbles, it does not cause the rupture of the polymeric contrast agent.     

Resonance frequencies of lipid-shelled microbubbles in the regime of nonlinear oscillations

Knowledge of resonant frequencies of contrast microbubbles is important for the optimization of ultrasound contrast imaging and therapeutic techniques. To date, however, there are estimates of resonance frequencies of contrast microbubbles only for the regime of linear oscillation. The present paper proposes an approach for evaluating resonance frequencies of contrast agent microbubbles in the regime of nonlinear oscillation. The approach is based on the calculation of the time-averaged oscillation power of the radial bubble oscillation. The proposed procedure was verified for free bubbles in the frequency range 1-4 MHz and then applied to lipid-shelled microbubbles insonified with a single 20-cycle acoustic pulse at two values of the acoustic pressure amplitude, 100 kPa and 200 kPa, and at four frequencies: 1.5, 2.0, 2.5, and 3.0 MHz. It is shown that, as the acoustic pressure amplitude is increased, the resonance frequency of a lipid-shelled microbubble tends to decrease in comparison with its linear resonance frequency. Analysis of existing shell models reveals that models that treat the lipid shell as a linear viscoelastic solid appear may be challenged to provide the observed tendency in the behavior of the resonance frequency at increasing acoustic pressure. The conclusion is drawn that the further development of shell models could be improved by the consideration of nonlinear rheological laws.     

Scaling of the viscoelastic shell properties of phospholipid encapsulated microbubbles with ultrasound frequency

Phospholipid encapsulated microbubbles are widely employed as clinical diagnostic ultrasound contrast agents in the 1–5 MHz range, and are increasingly employed at higher ultrasound transmit frequencies. The stiffness and viscosity of the encapsulating “shells” have been shown to play a central role in determining both the linear and nonlinear response of microbubbles to ultrasound. At lower frequencies, recent studies have suggested that shell properties can be frequency dependent. At present, there is only limited knowledge of how the viscoelastic properties of phospholipid shells scale at higher frequencies. In this study, four batches of in-house phospholipid encapsulated microbubbles were fabricated with decreasing volume-weighted mean diameters of 3.20, 2.07, 1.82 and 1.61 μm. Attenuation experiments were conducted in order to assess the frequency-dependent response of each batch, resulting in resonant peaks in response at 4.2, 8.9, 12.6 and 19.5 MHz, respectively. With knowledge of the size measurements, the attenuation spectra were then fitted with a standard linearized bubble model in order to estimate the microbubble shell stiffness S_p and shell viscosity S_f, resulting in a slight increase in S_p (1.53–1.76 N/m) and a substantial decrease in S_f (0.29 × 10⁻⁶–0.08 × 10⁻⁶ kg/s) with increasing frequency. These results performed on a single phospholipid agent show that frequency dependent shell properties persist at high frequencies (up to 19.5 MHz).     

In vitro contrast-enhanced ultrasound measurements of capillary microcirculation: Comparison between polymer- and phospholipid-shelled microbubbles

The focus of contrast-enhanced ultrasound research has developed beyond visualizing the blood pool and its flow to new areas such as perfusion imaging, drug and gene therapy, and targeted imaging. In this work comparison between the application of polymer- and phospholipid-shelled ultrasound contrast agents (UCAs) for characterization of the capillary microcirculation is reported. All experiments are carried out using a microtube as a vessel phantom. The first set of experiments evaluates the optimal concentration level where backscattered signal from microbubbles depends on concentration linearly. For the polymer-shelled UCAs the optimal concentration level is reached at a value of about 2 × 10⁴ MB/ml, whereas for the phospholipid-shelled UCAs the optimal level is found at about 1 × 10⁵ MB/ml.Despite the fact that the polymer shell occupies 30% of the radius of microbubble, compared to 0.2% of the phospholipid-shelled bubble, approximately 5-fold lower concentration of the polymer UCA is needed for investigation compared to phospholipid-shelled analogues. In the second set of experiments, destruction/replenishment method with varied time intervals ranging from 2 ms to 3 s between destructive and monitoring pulses is employed. The dependence of the peak-to-peak amplitude of backscattered wave versus pulse interval is fitted with an exponential function of the time γ = A(1 − exp(−βt)) where A represents capillary volume and the time constant β represents velocity of the flow. Taking into account that backscattered signal is linearly proportional to the microbubble concentration, for both types of the UCAs it is observed that capillary volume is linearly proportional to the concentration of the microbubbles, but the estimation of the flow velocity is not affected by the change of the concentration. Using the single capillary model, for the phospholipid-shelled UCA a delay of about 0.2-0.3 s in evaluation of the perfusion characteristics is found while polymer-shelled UCA provide response immediately. The latter at the concentration lower than 3.6 × 10⁵ MB/ml have no statistically significant delay (p < 0.01), do not cause any attenuation of the backscattered signal or saturation of the receiving part of the system. In conclusion, these results suggest that the novel polymer-shelled microbubbles have a potential to be used for perfusion evaluation.     

Impact of acoustic pressure on ambient pressure estimation using ultrasound contrast agent

Local blood pressure measurements provide important information on the state of health of organs in the body and can be used to diagnose diseases in the heart, lungs, and kidneys. This paper presents an approach for investigating the ambient pressure sensitivity of a contrast agent using diagnostic ultrasound. The experimental setup resembles a realistic clinical setup utilizing a single array transducer for transmit and receive. The ambient pressure sensitivity of SonoVue (Bracco, Milano, Italy) was measured twice using two different acoustic driving pressures, which were selected based on a preliminary experiment. To compensate for variations in bubble response and to make the estimates more robust, the relation between the energy of the subharmonic and the fundamental component was chosen as a measure over the subharmonic peak amplitude. The preliminary study revealed the growth stage of the subharmonic component to occur at acoustic driving pressures between 300 and 500 kPa. Based on this, the pressure sensitivity was investigated using a driving pressure of 485 and 500 kPa. At 485 kPa, a linear pressure sensitivity of 0.42 dB/kPa was found having a linear correlation coefficient of 0.94. The second measurement series at 485 kPa showed a sensitivity of 0.41 dB/kPa with a correlation coefficient of 0.89. Based on the measurements at 500 kPa, this acoustic driving pressure was concluded to be too high causing the bubbles to be destroyed. The pressure sensitivity for these two measurement series were 0.42 and 0.25 dB/kPa with linear correlation coefficients of 0.98 and 0.93, respectively.     

Harmonic responses and cavitation activity of encapsulated microbubbles coupled with magnetic nanoparticles

Encapsulated microbubbles coupled with magnetic nanoparticles, one kind of hybrid agents that can integrate both ultrasound and magnetic resonance imaging/therapy functions, have attracted increasing interests in both research and clinic communities. However, there is a lack of comprehensive understanding of their dynamic behaviors generated in diagnostic and therapeutic applications. In the present work, a hybrid agent was synthesized by integrating superparamagnetic iron oxide nanoparticles (SPIOs) into albumin-shelled microbubbles (named as SPIO-albumin microbubbles). Then, both the stable and inertial cavitation thresholds of this hybrid agent were measured at varied SPIO concentrations and ultrasound parameters (e.g., frequency, pressure amplitude, and pulse length). The results show that, at a fixed acoustic driving frequency, both the stable and inertial cavitation thresholds of SPIO-albumin microbubble should decrease with the increasing SPIO concentration and acoustic driving pulse length. The inertial cavitation threshold of SPIO-albumin microbubbles also decreases with the raised driving frequency, while the minimum sub- and ultra-harmonic thresholds appear at twice and two thirds resonance frequency, respectively. It is also noticed that both the stable and inertial cavitation thresholds of SonoVue microbubbles are similar to those measured for hybrid microbubbles with a SPIO concentration of 114.7 μg/ml. The current work could provide better understanding on the impact of the integrated SPIOs on the dynamic responses (especially the cavitation activities) of hybrid microbubbles, and suggest the shell composition of hybrid agents should be appropriately designed to improve their clinical diagnostic and therapeutic performances of hybrid microbubble agents.     

Factors affecting sound exposure from firing an SA80 high-velocity rifle

The effect of distance on the peak sound pressure level and sound exposure level from an SA80 rifle has been investigated. Sound pressure waveforms were measured in two directions from the gun: downrange, from 50 m to 300 m, and to the left-hand side, from 0.3 m to 32 m. Some additional measurements were made to the right of the gun. Measurements made downrange showed three distinct features of the waveform; the shock wave from the supersonic bullet, the reflection from the ground, and the muzzle blast. The time elapsed between the shock wave and the muzzle blast increased with increasing distance: 94 ms for a distance of 50 m, and 507 ms for a distance of 300 m. The highest peak sound level downrange from a single round was between 151 dB(C) and 148 dB(C) at distances from 50 m to 300 m, and varied little if at all with distance. To the left of the gun, the peak sound pressure level of 161 dB(C) at 0.3 m reduced to 128 dB(C) at 32 m. The peak sound pressure level was estimated to be 137 dB(C) at a distance of approximately 20 m to the left-hand side. Hearing protection must therefore be worn by anyone closer than 20 m to a person firing. The peak sound pressure level was estimated to be 135 dB(C) at a distance of approximately 25 m and therefore hearing protection is recommended at distances of up to 25 m. The sound exposure level of 98 dB(A) at 20 m indicated that an observer at this distance could hear about 1440 rounds without hearing protection before the noise exposure reached the upper exposure action value specified in the Control of Noise at Work Regulations 2005. Peak sound pressure levels were on average 2.4 dB higher at the left ear compared with the right ear.     

Cardiac magnetic resonance imaging in arrhythmogenic right ventricular cardiomyopathy: correlation to the QRS dispersion

The aim of the study was to evaluate the relationship between the presence of right ventricular abnormalities detected by cardiac magnetic resonance (CMR) and QRS dispersion, the strongest independent predictor of sudden death in ARVC. A consecutive series of 40 patients from a single institution were recruited with a clinical diagnosis of ARVC based on the diagnostic criteria. All patients underwent systematic clinical evaluation, including history and examination, electrocardiography, 24-h Holter monitor, chest radiography, echocardiography and CMR examination and were divided into two groups according to the QRS dispersion: group I, QRS dispersion ≥ 40 ms; group II, QRS dispersion < 40 ms. The relationship between the characteristic parameters of CMR image and QRS dispersion were analyzed in two groups. There were significant differences in QRS dispersion (57±14 ms vs. 26±11 ms), right ventricular end-diastolic diameter (57±10 mm vs. 48±11 mm, P=.012), right ventricular end-systolic diameter (52±10 mm vs. 44±11 mm, P=.010), right ventricular end-diastolic volume (260±105 ml vs. 180±66 ml, P=.006), right ventricular end-systolic volume (222±98 ml vs. 148±61 ml, P=.006) and myocardial fibrosis detection rate (74% vs. 38%, P=.024) between two groups. For all patients with ARVC, QRS dispersion and right ventricular end-diastolic volume (r= 0.66, P<.001), right ventricular end-systolic volume (r= 0.67, P<.001), right ventricular outflow tract area (r= 0.68, P<.001) showed a moderate positive correlation. Right ventricular outflow tract area, right ventricular end-diastolic volume and end-systolic volume detected by CMR in patients with ARVC were positively correlated to the extent of QRS dispersion (≥ 40 ms), the strongest independent predictor of sudden cardiac death.     

All-optical synchronization of a 40 GHz self-pulsating distributed Bragg reflector laser to return-to-zero 10, 20 and 40 Gbit/s data streams

This paper presents experimental investigations of the all-optical synchronization of a distributed Bragg reflector (DBR) laser self-pulsating at 40 GHz on various injected bit-rate signals. Even though there is no modulation applied to this laser, it exhibits a modulation of its output emission, measured at 39.7 GHz with a linewidth of 30 MHz. Such performance is exploited in all-optical clock recovery for a return-to-zero data stream at 40 Gbit/s. The SP-DBR laser wavelength and the injected signal wavelength are 10 nm apart. All-optical synchronization is demonstrated at 40 Gbit/s with a linewidth of less than 20 MHz for injected signals at 10 and 20 Gbit/s, respectively. Thus the SP-DBR laser proves to be very versatile and can be synchronized on various bit-rate data signals.     

Dual-high-frequency ultrasound excitation on microbubble destruction volume

Objective and motivation

The goal of this work was to test experimentally that exposing air bubbles or ultrasound contrast agents in water to amplitude modulated wave allows control of inertial cavitation affected volume and hence could limit the undesirable bioeffects.

Methods

Focused transducer operating at the center frequency of 10 MHz and having about 65% fractional bandwidth was excited by 3 μs 8.5 and 11.5 MHz tone-bursts to produce 3 MHz envelope signal. The 3 MHz frequency was selected because it corresponds to the resonance frequency of the microbubbles used in the experiment. Another 5 MHz transducer was used as a receiver to produce B-mode image. Peak negative acoustic pressure was adjusted in the range from 0.5 to 3.5 MPa. The spectrum amplitudes obtained from the imaging of SonoVue^TM contrast agent when using the envelope and a separate 3 MHz transducer were compared to determine their cross-section at the - 6 dB level.

Results

The conventional 3 MHz tone-burst excitation resulted in the region of interest (ROI) cross-section of 2.47 mm while amplitude modulated, dual-frequency excitation with difference frequency of 3 MHz produced cross-section equal to 1.2 mm.

Conclusion

These results corroborate our hypothesis that, in addition to the considerably higher penetration depth of dual-frequency excitation due to the lower attenuation at 3 MHz than that at 8.5 and 11.5 MHz, the sample volume of dual-frequency excitation is also smaller than that of linear 3-MHz method for more spatially confined destruction of microbubbles.     

Anisotropic powder from sintered NdFeB magnets by the HDDR processing route

Sintered NdFeB-based scrap magnets were recovered and processed using the HD and HDDR routes. The effects of varying the HDDR processing temperature were investigated (over the range 835-930 °C). The disproportion was carried out with a pressure ramp to a maximum of 1000 mbar hydrogen pressure with a 1 h hold time at each step and the optimum recombination conditions were set at 100 mbar with a 20 min hold time. Anisotropic NdFeB powder was produced in all cases with the best magnetic properties achieved at a processing temperature of 880 °C, producing powder with a remanence of 1.10(±0.02) T and an intrinsic coercivity of 800 (±16) kA m⁻¹ and giving a (BH)_max of 129(±2.5) kJ m⁻³.     

超声脉冲长度与重复频率对流动微泡群稳态空化特性的影响

为了深入理解超声脉冲长度(PL)与重复频率(PRF)对流动微泡群稳态空化特性的影响,本研究自行制备仿体血管,利用注射泵推动微泡群在其中恒速流动,使用1 MHz的聚焦换能器激励微泡群产生稳态空化,另一个7.5 MHz的平面换能器接收声信号,经前置放大及高速采集后送至上位机存储。在定义临界重复频率(Critical PRF,CPRF)的基础上,通过深入的时频域分析,我们发现PL和PRF共同决定流动微泡群的稳态空化特性:当PRF低于CPRF时,流动微泡群的稳态空化剂量(Stable Cavitation Dose,SCD)与PL呈正相关,且时域分布均匀;而当PRF大于CPRF时,其稳态空化特性由PL决定,在长PL (＞100μs)下,SCD随着PL增加逐渐减小,且时域分布不均匀;在短PL (＜50μs)下,SCD和PRF无显著相关关系,且时域分布均匀。本研究结果深入阐明了流动微泡群的稳态空化特性,期望可用其获取可预测可控的生物效应,并应用于相关临床治疗。      

Nanometric laser trapping of microbubbles based on nanostructured substrates

Laser trapping near the surface of a nanostructured substrate is demonstrated. Stable microbubbles with radii of 1-20 μm have been created and manipulated with sub-micron precision by a focused laser beam in an immersion oil covering arrays of pairs of gold nanopillars deposited on a glass substrate. The threshold for bubble creation and trapping characteristics depended on near-field coupling of nanopillars. The nanometric laser tweezers showed giant trapping efficiency of Q ∼ 50 for the trapped microbubbles.     

Platelet-targeted microbubbles inhibit re-occlusion after thrombolysis with transcutaneous ultrasound and microbubbles

Microbubbles (MBs) can augment the acoustic cavitation’ (US), thereby facilitating the thrombolysis of external ultrasound. But we observed re-thrombosis after successful thrombolysis by MBs and transcutaneous ultrasound in an endothelium injury model. This study was designed to explore whether platelet-targeted MBs can prevent the reformation of thrombi. Arterial injury was induced in canine femoral arteries with balloon, and the arteries were completely thrombotically occluded. The arteries were treated with intra-arterial MBs or platelet-targeted MBs (TMB) and transcutaneous low frequency ultrasound (LFUS) to achieve complete thrombolysis. The arterial flow was monitored with angiogram for 4 h following treatment. Results showed that both MBs and TMBs produced successful dissolution of clots in the presence of ultrasound. The re-occlusion began to occur 1 h after thrombolysis in MB/LFUS treatment, and 7 of 8 arteries were re-occluded within 3 h. Most of the arteries (7 of 8) in the TMB/LFUS group remained patent for 4 h following treatment. The flow tended to decrease after thrombolysis in MB/LFUS treatment. These results indicated that platelet-targeted microbubbles were beneficial in preventing re-thrombosis in vivo and microbubbles served as good carrier of thrombolytic and anticoagulation drugs.     

Parametric harmonic-to-fundamental ratio contrast echocardiography: a novel approach to identification and accurate measurement of left ventricular area under variable levels of ultrasound signal attenuation

Objectives

We introduced a harmonic-to-fundamental ratio (HFR) of the radiofrequency (RF) signals that reduces confounding effects of attenuation. We studied whether HFR analysis of RF signals received from contrast microbubbles allows accurate measurement of the left ventricular (LV) cavity area under varying levels of attenuation.

Background

Attenuation is a fundamental problem in ultrasound imaging and limits the use of clinical echocardiography.

Methods

RF data from short axis systolic and diastolic scans were obtained from 14 open-chest dogs following left-atrial bolus of Optison. Attenuation was induced by interposed silicone pads calibrated to induce 7 dB or 14 dB reductions of the backscattered RF signal. RF images were reconstructed from the RF signals, HFR values calculated for each image pixel for 0 dB, 7 dB and 14 dB attenuation conditions, and LV area obtained by summation of “LV cavity pixels”. A reference LV cavity area was obtained from endocardial border tracings in enhanced scans by experts.

Results

Correlation of the HFR-defined and reference areas at systole was R = 0.95, R = 0.94, and R = 0.91 for 0 dB, 7 dB and 14 dB levels of attenuation, respectively, and at diastole was R = 0.95 for 0 dB, 7 dB and 14 dB levels of attenuation. The mean difference from both systolic and diastolic values was <1.45 cm² (i.e. negligible) in all attenuation settings.

Conclusion

Our novel HFR method supports precise measurement of the LV cavity area in contrast images with simulated high attenuation of ultrasound signals.     

Preparation and in vitro evaluation of an ultrasound-triggered drug delivery system: 10-hydroxycamptothecin loaded PLA microbubbles

10-Hydroxycamptothecin (HCPT) loaded PLA microbubbles, used as an ultrasound-triggered drug delivery system, were fabricated by a double emulsion-solvent evaporation method. The obtained microbubbles were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and confocal laser scanning microscope (CLSM). In addition, the effect of diagnostic ultrasound exposure on BEL-7402 cells combined with HCPT-loaded PLA microbubbles was evaluated using cytotoxicity assay, CLSM and flow cytometry (FCM). It was found that the HCPT-loaded PLA microbubbles showed smooth surface and spherical shape, and the drug was amorphously dispersed within the shell and the drug loading content reached up to 1.69%. Nearly 20% of HCPT was released upon exposure to diagnostic ultrasound at frequency of 3.5 MHz for 10 min. Moreover, HCPT fluorescence in the cells treated only with the HCPT-loaded PLA microbubbles was discernible, but less intense, while those treated with the microbubbles in conjunction with ultrasound exposure was evident and intense, indicating an increased cellular uptake of HCPT by ultrasound exposure. Cytotoxicity test on BEL-7402 cells indicated that the HCPT-loaded PLA microbubbles combined with ultrasound exposure were more cytotoxic than the microbubbles alone. The results suggest that the combination of drug loaded PLA microbubbles and diagnostic ultrasound exposure exhibit an effective intracellular drug uptake by tumor cells, indicating their great potential for antitumor therapy.