European Heart Journal Advance Access originally published online on September 15, 2005
European Heart Journal 2006 27(2):237-245; doi:10.1093/eurheartj/ehi479
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Myocardial delivery of colloid nanoparticles using ultrasound-targeted microbubble destruction
Division of Cardiology, Cliniques Universitaires St-Luc, Université catholique de Louvain, Avenue Hippocrate, 10-2881, B-1200 Brussels, Belgium
Received 25 March 2005; revised 28 July 2005; accepted 11 August 2005; online publish-ahead-of-print 15 September 2005.
* Corresponding author. Tel: +32 2 764 28 03; fax: +32 2 764 89 80. E-mail address: vanoverschelde{at}card.ucl.ac.be
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Abstract |
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Aims Ultrasound (US)-targeted microbubble destruction (UTMD) is a promising method for delivering genetic material to the heart. The aim of this study was: (i) to test whether colloid nanoparticles can be delivered to the rat myocardium using UTMD; and (ii) to determine whether tissue damage and contractile dysfunction occurs in hearts exposed to UTMD in vivo.
Methods and results Hearts from anaesthetized rats were exposed to perfluorocarbon-enhanced sonicated dextrose albumin (PESDA) (at two different microbubble concentrations) and US at peak pressures of 0.6, 1.2, or 1.8 MPa for 1, 3, or 9 min. During US, pairs of 30 and 100 nm fluorescent nanospheres were infused intravenously. Left ventricular function was assessed before and immediately after US, as well as at 24 h and 7 days. At the end of the experiments, the number of ruptured microvessels and the amount of nanospheres deposited were quantified. Rats exposed to PESDA alone or US alone showed no functional abnormalities, no capillary ruptures, and no nanosphere delivery. In contrast, rats exposed to both PESDA and US exhibited microvascular ruptures and nanosphere deposits. They also showed transient contractile dysfunction and premature ventricular contractions. All these changes were time-, US peak pressure-, and PESDA concentration-dependent.
Conclusion UTMD allows colloid nanoparticles to be delivered to the rat myocardium through microvessel rupture sites. The efficacy of delivery depends on the peak pressure applied, the duration of US exposure, and contrast concentration. UTMD also causes time- and peak pressure-dependent contractile dysfunction, and tissue alterations that are spontaneously reversible over time.
Key Words: Contrast echocardiography • Local delivery
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Introduction |
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The use of ultrasound (US)-targeted microbubble destruction (UTMD) as a tool to deliver drugs or genetic material to the heart holds considerable promise.1–6 Although, US per se can enhance vascular permeability, local delivery seems to be considerably increased in the presence of contrast microbubbles.6 Ay et al.7 and others8 have recently reported on specific bio-effects associated with the insonation of gas-filled microbubbles in microvessels of isolated perfused rabbit hearts and rat skeletal muscles. In these experiments, when microbubbles were exposed to US, their destruction created microvessel ruptures that were large enough to permit the extravasation of erythrocytes2,7,8 and the delivery of colloid particles2 deep into the pericapillary tissues. Interestingly, the data also suggested that discrete capillary and venule ruptures were mandatory to produce these effects,6,7 thus raising potential concerns about the safety of this approach.8
Accordingly, the present study was designed: (i) to test whether UTMD can facilitate the delivery of colloid particles (30–100 nm in diameter) to the interstitium of the rat myocardium in vivo; (ii) to investigate how microbubble concentration, particulate size, and factors known to affect microbubble destruction, such as US peak pressure, duration of US exposure, and pulse emission mode influence the efficacy of delivery; (iii) to determine how much tissue damage occurs in hearts exposed to UTMD in the intact animal; and (iv) to evaluate the potential long-term consequences thereof on tissue integrity and ventricular function.
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This study was approved by the Animal Research Committee at the Université catholique de Louvain and conformed to the American Heart Association Guidelines for use in animal research.
Preparation of microbubbles
Perfluorocarbon-enhanced sonicated dextrose albumin (PESDA), a second-generation contrast agent, consisting of decafluorobutane-filled albumin microbubbles with a mean diameter of 4.2±0.5 µm and a mean concentration of 0.8x109 mL–1 was used in this study.9 A slow bolus of 200 µL of PESDA was infused intravenously over 30 s, every 3 min.
Animal preparation
Male Wistar rats (300–400 g) were anesthetized with sodium pentobarbital (60 mg kg–1 ip). Both femoral veins and the left femoral artery were cannulated, to allow microbubbles and fluorescent nanosphere infusion as well as the monitoring of arterial pressure. Peripheral electrodes were attached onto each leg to allow ECG-triggered US emission and to record a six-peripheral lead ECG. Echocardiography was performed in intermittent mode (1 Hz, unless otherwise stated) with a Sonos 5500 system (Philips Medical System, Andover, MA) equipped with a broadband S3 transducer that has a mean transmit frequency of 1.3 MHz, a bandwidth of about 25% centered at 1.3 MHz, and a maximal peak pressure of 1.8 MPa. The transducer was operated at a depth of 4 cm. Each frame consisted of 110 lines delivered over a period of 50 ms, forming a 90° sector. Each line was fired as a single burst of US with four cycles over 3 µs. The tip of the transducer was positioned on the chest wall to obtain a short-axis view of the heart, at the level of the papillary muscles. This position was maintained throughout the experiments. Peak acoustic pressure was calculated according to the mechanical index (MI) displayed on the screen of the US system using the equation: peak pressure=MIx(transducer centre frequency)1/2. The MIs actually delivered to the tissue were not measured.
Fluorescent nanospheres
The efficacy of particulate delivery and the relationship between particulate delivery and ventricular function were assessed by continuously infusing a solution containing a mixture of 30 nm green-fluorescent and 100 nm blue- or red-fluorescent nanospheres (Duke Scientific, Palo Alto, CA) into one of the femoral vein catheters. Infusion rate was set at 60 µL min–1.
Experimental protocol
The protocol was designed to evaluate, in separate experiments, the immediate, sub-acute (24 h) and long-term (7 days) consequences of UTMD in the heart. The combined effects of US peak pressure and the duration of US exposure on microvascular integrity and nanoparticulate delivery were evaluated in groups of rats, whose hearts were randomly exposed to a peak pressure of 0.6, 1.2, and 1.8 MPa for 1, 3, and 9 min, respectively. Two additional groups of rats randomly exposed to US alone, in the absence of PESDA or receiving PESDA alone, in the absence of US, served as control groups. The influence of the pulse emission mode was evaluated in two separate groups of rats whose hearts were randomly exposed to a peak pressure of 1.8 MPa for 9 min using either end-diastolic (ED, peak of the R-wave) or end-systolic (ES, end of the T-wave) ECG triggering (every six beats). Finally, the influence of microbubble concentration was tested in two additional groups that were randomly assigned to receive either undiluted PESDA (0.8x109 microbubbles mL–1) or diluted (0.8x108 microbubbles mL–1) PESDA. Each study group consisted of at least six animals.
Rats were euthanized at 30 min (n=144), 24 h (n=72), or 7 days (n=36) after the start of US exposure. Hearts were harvested and washed in Krebs–Henseleit buffer for 2 min at 38°C. Hearts that received the fluorescent nanospheres were then quick-frozen in liquid nitrogen and stored at –80°C. The remaining hearts were fixed in glutaraldehyde.
Left ventricular systolic function
Regional mechanical function was assessed using 2D-echocardiography. For this purpose, short-axis images of the left ventricle (LV) were obtained at the level of the papillary muscles, using a 12 MHz transducer. LV internal dimensions as well as the anterior and posterior wall thickness were measured at ED and ES and averaged over three consecutive cardiac cycles. The systolic wall thickening of the anterior and posterior walls were computed and served as indexes of regional function.
Morphological analysis
Endothelial and vascular damage was quantified on glutaraldehyde-fixed semi-thick sections of the mid-anterior and mid-posterior myocardium. Each section was stained with Toluidine Blue and examined to measure the percentage of vessels (capillaries and venules) presenting with endothelial damage or rupture. A total of 1000 vessels were examined for each sample. Morphometric assessment was performed on thick sections stained with haematoxylin–eosin–safranin (HES). Each section was examined using a special grid to measure the percentage of the sample surface covered by necrotic myocytes, inflammatory cells, fibrin deposits, or fibrosis. This procedure was repeated 20 times over different zones of the samples.
Fluorescent microscopy
Frozen hearts were cut into serial 2 mm-thick short-axis slices. The mid-ventricular section that encompassed the papillary muscle was used for further analysis. This section was first sliced into several consecutive 5 µm-thick slices that were subsequently examined under a fluorescent microscope. Micrographs of the sub-endocardial, mid-myocardial, and sub-epicardial layers of the anterior, posterior, lateral, and septal myocardium were digitized during green- and blue- (or red-) fluorescent illumination using a x20 objective. Image resolution was set at 1024x768 pixels. The digital images were then transferred onto a computer workstation where they were binarized to a threshold equal to 2 SD of the mean value obtained in sections of hearts that did not receive fluorescent nanospheres (Analysis Docu software, Soft Imaging System, Münster, Germany). The area occupied by the green- and the blue- (or red-)nanospheres was computed from these thresholded images.
Statistical analysis
Values are mean±1 SEM. Differences in nanoparticulate delivery, premature ventricular contraction (PVCs), and vascular rupture between groups was assessed using a two-factor analysis of variance (ANOVA), examining the effect of two fixed factors (US peak pressure and duration of US exposure). Changes in wall thickening between baseline and different time points were assessed using a repeated measurement ANOVA, using US peak pressure and duration of US exposure as fixed factors and time points as repetition factor. If the overall ANOVA was significant, individual comparisons between groups were performed post hoc using the Bonferroni test. All tests were two-sided and a P-value <0.05 was considered indicative of a statistically significant difference.
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Results |
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UTMD resulted in the rupture of small microvessels, the extravasation of erythrocytes, and the delivery of fluorescent nanospheres into the myocardial interstitium. Figure 1, which consists of composite red-fluorescent and HES-counterstained micrographs of the same section, depicts the extravasation of 100 nm red-fluorescent nanospheres deep into the pericapillary tissue.
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Effects of US peak negative pressure on nanoparticulate delivery
Figure 2 shows representative fluorescent images taken from the anterior wall of a control heart and from three hearts exposed for 9 min to a peak pressure of 0.6, 1.2, and 1.8 MPa, respectively. No deposits of the 30 or 100 nm nanospheres were seen in the control heart. In contrast, nanosphere deposition was clearly identified in the three hearts exposed to both PESDA and US. The extent of nanosphere deposition increased with US peak pressure (to 0.36±0.11, 3.75±0.99, and 4.8±0.8% in hearts exposed to 0.6, 1.2, and 1.8 MPa, respectively, P<0.001 by ANOVA). No significant differences were observed between the 30 and the 100 nm nanospheres.
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Influence of the duration of US exposure on nanoparticulate delivery
Figure 3 illustrates the influence of the duration of US exposure on nanosphere delivery to the anterior wall of three hearts exposed to a peak pressure of 1.8 MPa for 1, 3, and 9 min, respectively. The extent of nanoparticulate delivery progressively increased with the duration of US exposure (to 0.37±0.09, 1.28±0.33, and 4.8±0.8% in hearts exposed to US for 1, 3, and 9 min respectively, P<0.001 by ANOVA). No significant differences were observed between the 30 and the 100 nm nanospheres.
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Influence of pulse emission mode on nanoparticulate delivery
To test whether the pulse emission mode influenced the extent of nanoparticulate delivery, groups of rats were exposed to either non-triggered (1 Hz) or ECG-triggered (either ED or ES) 1.8 MPa US emissions. No significant differences in nanosphere deposition were noted among these three emission modes (4.8±0.8, 3.9±0.6, and 4.4±0.9%, respectively, P=0.75).
Influence of microbubble concentration on nanoparticulate delivery
To evaluate the possible influence of the dose of PESDA on the efficacy of delivery, two groups of rats received either undiluted PESDA or diluted PESDA. Whole heart nanoparticulate delivery was six times lower in hearts exposed to diluted PESDA as opposed to undiluted PESDA (0.4±0.2 vs. 2.3±0.4%, P<0.0001). In addition, two of the five hearts exposed to diluted PESDA exhibited no nanosphere deposits, whereas the remaining three hearts showed mild to modest nanosphere deposits that were confined to the sub-epicardial layer of the anterior wall.
Regional variations in nanoparticulate delivery
In hearts exposed to a peak pressure of 1.8 MPa for 9 min, nanosphere delivery was always more prominent in segments directly exposed to US, like the anterior walls as opposed to the lateral, posterior, and septal (4.8±0.8, 2.6±0.8, 1.4±0.6, 0.3±0.1%, respectively P<0.001 by ANOVA) walls or the epicardial layer of the anterior wall when compared with the mid-myocardial and endocardial layers of the same wall (7.6±1.8, 3.8±0.6, and 2.9±0.6%, respectively, P<0.001 by ANOVA). This phenomenon was also observed at the lower peak pressures or during shorter US exposures (data not shown).
Effects of UTMD on tissue integrity
Thirty minutes after the start of UTMD, areas of intramural haemorrhage were identified in most hearts (Figure 4). Whenever tissue haemorrhage was present, contraction bands necrosis were also evidenced (Figure 5A). Tissue haemorrhage was more extensive in hearts exposed to a peak pressure of 1.8 MPa than in hearts exposed to a lower peak pressure. In the light microscope, these haemorrhagic zones corresponded to areas of microvascular ruptures and erythrocyte extravasation. The number of vascular rupture sites increased with both the peak pressure applied (0.02±0.02, 1.08±0.25, and 1.64±0.33% in hearts exposed for 9 min to peak pressures of 0.6, 1.2, and 1.8 MPa, respectively, P<0.001 by ANOVA) and the duration of US exposure (0.32±0.07, 0.47±0.14, and 1.64±0.33% in hearts exposed to 1.8 MPa US for 1, 3, and 9 min, respectively, P<0.001 by ANOVA).
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At 24 h, erythrocyte extravasation could no longer be seen. The space previously occupied by haemorrhage was now filled with fibrin deposits and mononuclear cells (Figure 5B).
At 7 days, no more structural alterations could be seen, except in the group exposed to the 1.8 MPa US for 9 min, in which significant increases in connective tissue were noted (Figure 5C, 12±3%, P=0.0016 vs. the other groups).
Effects of UTMD on LV systolic function
Figure 6 shows representative ED and ES short-axis still frames obtained at 30 min, 24 h, and 7 days after US exposure in a heart exposed to 1.8 MPa US for 9 min. Definite anterior wall dysfunction was evidenced immediately after US exposure and at 24 h. By 7 days, however, regional function had completely recovered. As illustrated in Figure 7, UTMD resulted in a time- and peak pressure-dependent decrease in anterior wall thickening. Although these functional abnormalities persisted at 24 h, they were no longer present at 7 days, except in the group exposed to 1.8 MPa US for 9 min. No dysfunction was seen in the posterior wall (data not shown).
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P<0.05, 
P<0.01 vs. PESDA alone and US alone.As shown in Figure 8, anterior wall thickening and nanosphere deposits were inversely correlated. Wall thickening remained normal (45±2%) in hearts in which no nanosphere deposits could be seen. In contrast, in every single heart in which fluorescent nanospheres could be delivered, regional dysfunction was observed. In addition, the severity of dysfunction was more pronounced in the hearts in which the delivery was most successful.
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Effects of UTMD on cardiac rhythm
As expected, PVCs occurred during UTMD. Their frequency increased with peak pressure (none in controls, 0.4±0.2, 6.2±2, and 18.1±3 min–1 in hearts exposed, for 9 min, to peak pressures of 0.6, 1.2, and 1.8 MPa, respectively, P<0.001 by ANOVA). No lethal arrhythmias were noted.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionConclusionsAcknowledgementsReferences |
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The aim of this study was: (i) to investigate if colloid nanoparticles, whose size closely approximates that of the viral vectors used for gene therapy, can be delivered to the rat myocardium; and (ii) to determine whether tissue damage and contractile dysfunction occurs in hearts exposed to UTMD in vivo. Our results indicate that effective myocardial delivery of these nanoparticles can be indeed achieved using this approach provided that the hearts are being exposed for a prolonged period of time to both high US peak pressure and a high concentration of microbubbles. However, the data also show that UTMD induces significant bio-effects, which include transient tissue damage, microvascular ruptures, PVCs, and LV dysfunction.
Effects of UTMD on microvascular integrity
Although the occurrence of tissue haemorrhage and endothelial cell damage after US exposure of organs containing air, such as the lungs or the intestine, has been known for several years, the observation that similar bio-effects can develop in tissues exposed to US and manufactured microbubbles is quite recent. Skyba et al.8 were among the first to observe microvessel ruptures in a solid organ exposed in vivo to contrast microbubbles and US. Subsequently, we made very similar observations in the ex vivo setting of an isolated perfused rabbit heart preparation.
The present study thus confirms and extends these previous results by demonstrating that, in vivo as well, the simultaneous exposure of rat hearts to US and contrast microbubbles causes microvessel ruptures and erythrocyte extravasation. In addition, our data shows for the first time that it is possible to deliver colloid particles directly into the cardiac interstitium through these microvessel ruptures. Interestingly, the effectiveness of UTMD-assisted delivery seems to be less in our experiments than previously reported in skeletal muscle.2,8 Indeed, by our calculations, each 1.8 MPa US emission caused the rupture of only 0.003% of all microvessels examined and allowed the delivery of colloid particles to only 0.009% of the myocardial mass. This is in contrast with the estimations made by previous investigators2,8 in the externalized rat spinotrapezius muscle. These authors indeed calculated that each 1.5 MPa US exposure caused the rupture of 0.015% of all microvessels and the delivery of colloid particles to 0.5% of the muscle mass, which is 50 times more than in our experiments. Theoretically, several factors could account for these discrepancies, including differences in US peak pressure, transmit frequency, microbubble shell, experimental design, or organ susceptibility. If one takes US attenuation into account (which was negligible in Price et al.2 and Skyba et al.8 studies, but around of 1.1 dB cm–1 MHz–1 in our study10), it becomes unlikely that US peak energy significantly contributed. Indeed, attenuation corrected-US peak pressures were quite similar among the three studies. Significant contributions of US transmit frequency and microbubble shell is also unlikely. We have indeed previously shown that exposure to higher US transmit frequencies results in less rather than more capillary ruptures. We have also shown that Optison and PESDA caused similar amount of capillary ruptures. Altogether, the data would thus suggest that the relative effectiveness of UTMD-assisted delivery may be organ specific, which should not be surprising as the resistance of the tissue interstitium, which depends on extracellular matrix density and composition, and the pressure gradient across the rupture sites, which determines the extent to which particulate material can be driven into the interstitium, are known to differ widely in different organs.
Influence of US peak pressure and duration of US exposure
As in previous studies, we found that the amount of microvessel ruptures and the extent of particulate delivery increased with the US peak pressure applied and the duration of US exposure.7,8,11,12,16 On the basis of the estimates of tissue attenuation, we have calculated that the myocardium needs to be exposed to a minimum of 1.0 MPa (at the tissue level) to observe significant delivery. This has important implications for UTMD-assisted drug or gene delivery to the human heart. In the clinical setting, where tissue attenuation is responsible for a decrease in transmitted energy of 0.5–1.0 dB cm–1 MHz–1,13 the transmit US peak pressure needed to obtain 1.0 MPa at 5 cm from a 1.3 MHz transducer would be 1.5–2.1 MPa. Transthoracic transducers emitting at a higher US peak pressures will probably be needed to achieve local delivery with UTMD in human.
Dose of contrast
One additional important finding in our study relates to the crucial role played by the microbubbles blood concentration in determining the extent of nanoparticulate delivery to the heart. To investigate this, we randomly assigned two groups of rats to receive either undiluted (0.8x109 microbubbles mL–1) or diluted (0.8x108 microbubbles mL–1) PESDA, 600 µL of which were infused over a period of 9 min. Given to 350 g rats, this corresponded to a dose of 1.5x108 and 1.5x107 microbubbles min–1 kg–1, respectively. In clinical practice, these doses would be respectively, 20 and 2 times higher than the dose given to a 70-kg adult undergoing routine dipyridamole stress perfusion imaging. Quite interestingly, nanoparticulate delivery was small and even inexistent in two of the five animals when using the lowest dose of PESDA, whereas significant delivery was seen in every single animal exposed to the highest dose of PESDA. Our data thus indicate that sufficient amount of contrast must be given to achieve significant and reproducible UTMD-assisted local delivery. It is therefore not surprising if most previous author's interested in local delivery used large amount of contrast in their delivery experiments (Table 1).
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UTMD-induced myocardial damage
We and others have previously demonstrated that the combined exposure of isolated rabbit hearts7 and rat spinotrapezius muscles2,8,12 to US and US-contrast agents causes capillary ruptures, in an amount proportionate to the US peak pressure applied. The present study confirms and extends these previous observations. As in those previous reports, we indeed observed significant vascular damage in the hearts exposed to both PESDA and US. In contrast, no significant changes were noted in hearts exposed to either PESDA alone or US alone.
Tissue damage was not confined to microvessels, but also involved cardiomyocytes. Immediately after insonation, areas of contraction band necrosis could be clearly identified. At 24 h, significant albeit small amounts of cell necrosis were present, together with the infiltration of the insonated zone by macrophages and other mononucleated cells. These abnormalities were more prominent in hearts exposed to the highest US peak pressure for the longest time and in segments directly exposed to US, like for instance the anterior wall. Conversely, vascular ruptures, contraction band necrosis, and cell death were uncommon in hearts exposed to a peak pressure of 0.6 MPa, in those insonated for short periods of time and in areas that were less likely to be exposed to US, like the posterior wall (because of the presence of contrast in the LV cavity and the resulting far field attenuation).
UTMD-induced tissue damage has been reported before. In externalized rat spinotrapezius muscles exposed to Optison and a single sweep of US, Skyba et al.8 have also observed the occurrence of skeletal muscle cell death and already noticed the dependence of this phenomenon on the US peak pressure applied. Although the authors did not elaborate much on these findings, they reported a strong 1:1 relationship between the number of microvessels ruptured and the number of necrotic myocytes, which somehow suggests that each microvessel rupture may have resulted in the necrosis of its supplied myocyte. Other studies in rats,14 and in humans,15 have demonstrated that high peak pressure UTMD could also cause the release of troponin in the coronary sinus, which also suggests that some cardiomyocytes were suffering from the high peak pressure US exposure.
UTMD-induced LV dysfunction
One of the salient findings of this study is probably the observation that UTMD can induce LV dysfunction. As for the other bio-effects noted in this study, LV dysfunction was mostly seen in rats exposed to high peak pressure, for a prolonged period of time. It is noteworthy that the occurrence of LV dysfunction required the hearts to be simultaneously exposed to PESDA and US. It was indeed never observed in animals exposed to either PESDA alone or US alone. Our data also suggests that UTMD-induced microvascular injury may somehow have contributed to regional LV dysfunction. We indeed observed a high degree of covariance between both the extent and amount of vascular ruptures and the severity of regional dysfunction. It is thus tempting to speculate that UTMD-induced vascular ruptures somehow interfered with nutritive perfusion.
In view of the close relationship found between the extent and magnitude of local delivery and the severity of UTMD bio-effects, our observations clearly raise the concern that the potential benefits of UTMD-assisted local delivery could be offset by the occurrence of untoward side effects, whenever high peak pressure is used. Further studies are needed to address this issue and to investigate how US settings can be optimized to maximize local delivery and at the same time minimizing tissue damage. Our data suggest that short exposure times to high peak pressure or more prolonged exposures to lower peak pressure might provide a reasonable compromise.
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Conclusions |
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TopAbstractIntroductionMethodsResultsDiscussionConclusionsAcknowledgementsReferences |
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UTMD allows for colloid nanoparticles to be delivered to the rat myocardium through microvessel rupture sites. The efficacy of UTMD-assisted local delivery depends on the peak pressure applied, the duration of US exposure, and contrast concentration. UTMD also induces significant bio-effects, which include transient LV dysfunction, PVCs, microvascular, and morphological tissue damage, all of which are both time- and peak pressure-dependent. For this technique to be effective, large amounts of contrast must be given and the hearts must be exposed to high peak pressure, the benefits of delivering therapy through capillary ruptures must be carefully weighed against the costs of damaging tissue.
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Acknowledgements |
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This work is supported in part by Grant nos. 3-4563-98 and 3-4504-03 from the ‘Fonds National de la Recherche Scientifique et Médicale’ and by the ‘Action de Recherche Concertée’ no. 01/06-271. D.V. is supported by the ‘St-Luc’ and the ‘Damman’ Foundations, Louvain-la-Neuve, Belgium. P.R. is from Philips Medical Systems, Andover, MA. L.B. is Research Associate of the Fonds National de la Recherche Scientifique, Belgium.
Conflict of interest: none declared.
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