Charles A. Sennogaa, Emma Kanbara, Laurent Auboirea, Paul-Armand Dujardinb, Damien Fouana,Jean-Michel Escoffrea and Ayache Bouakaza
aUMR Imagerie et Cerveau, Inserm U930, Université François Rabelais, Tours, France; bInserm CIC 1415, CHRU Tours, Tours, FranceIntroduction: Recent developments in ultrasound imaging and ultrasound contrast agents (UCAs) improved diagnostic confidence in echography and set into motion their combined use as a tool for drug delivery and therapeutic monitoring. Non-invasive, precise and targeted delivery of drug molecules to pathological tissues by employing different mechanisms of drug release is becoming feasible.Areas covered: We sought to describe: the nature and features of UCAs; outline current contrast-specific imaging modes; before describing a variety of strategies for using ultrasound and microbubbles as a drug delivery system. Our expert opinion focusses on results and prospects of using ultrasound and microbubbles as a dual modality for drug delivery and therapeutic monitoring.Expert opinion: Today, ultrasound and microbubbles present a realistic prospect as drug delivery tools that have been demonstrated in a variety of animal models and clinical indications. Besides delivering drugs, ultrasound and microbubbles have demonstrated added value through therapeutic monitoring and assessment. Successful evaluation of the sonoporation mechanism(s), ultrasound parameters, drug type and dose will need to be addressed before translating this technology for clinic use. Ultimately, the development of a strategy for monitoring targeted delivery and its implementation in clinical practice would advance therapeutic treatment to a new qualitative level.ARTICLE HISTORY Received 19 January 2016Accepted 25 November 2016KEYWORDS Contrast agents; diagnostic imaging; drug-delivery; microbubbles; sonoporation; therapeutic monitoring; ultrasoundAn important challenge of conventional therapies is the nonspecific distribution of cytotoxic drugs within the body, resulting into reduced therapeutic efficacy and systemic side effects. Strict localization of a drug’s pharmacological activity at the site of vascular pathology (targeted drug delivery) would lead to significant reduction in drug toxicity, drug dose required, and increased therapeutic efficacy [1]. To better improve therapeutic efficacy and reduce the side effects, two challenges need to be addressed. The first is concerned with how vascular extravasation of the drug in the pathological region of interest (ROI) is triggered. The second is concerned with how to efficiently induce targeted delivery with minimal side effects.Promising approaches for targeted drug delivery based on the combined use of ultrasound (US) and US contrast agents (UCAs) have been developed [2–4]. UCAs are shell encapsulated, micrometer-sized gas bubbles that are usually injected into the blood circulation of the test subject in order to enhance US image contrast and as a result, improve diagnostic confidence. Indeed, UCAs are now used routinely in cardiology and in radiology [2] with the aim of improving the detection and visualization of the blood pool and to evaluate vascular perfusion in a variety of organs (Table 1). Moreover, continued improvements in contrast-enhanced US (CEUS) and its clinical diagnostic applications are due in part to developments in US imaging modes such as pulse inversion (PI) [2,3] and power modulation (PM) imaging. Significant effort has been directed toward developing and improving US detection methods based on specific acoustic signatures from UCAs with the requirement that the resultant imaging methods be sensitive to UCAs while simultaneously discriminating echoes originating from non-perfused tissue.
Elsewhere, the combined use of US and microbubbles represents a novel and promising approach to achieving targeted therapy which will likely add a new therapeutic dimension to this modality. Specifically, drug uptake and biodistribution into target tissues can be amplified using US and UCAs, through a phenomenon termed sonoporation. It is now thought that the volumetric oscillations of microbubbles under the action of US induces a number of local acoustic phenomena which engenders the permeability of the vascular barriers (e.g. cell membrane, blood–tumor barrier or blood–brain barrier (BBB)) leading to enhanced extravasation of drugs in the targeted tissue and increasing drug bioavailability [5,6]. Accordingly, the delivery of therapeutic agents into sites of interest can be triggered on demand and controlled spatially and temporally through US focusing and directed propagation.
In this article, we describe some of the emerging therapeutic applications and discuss the potential of using US for both image guidance and therapy. Briefly, in Section 2, we illustrate the nature, composition, and types of UCAs in current use. In Section 3, we describe the main acoustic properties of UCAs and how they are exploited in newly available contrast imaging modes dedicated to the visualization of microcirculation.
Article highlights
Ultrasound contrast agents (UCAs), used in conjunction with ultrasound can play a major role in clinical decision-making
. Recent advances in the design of UCAs have opened up powerful microbubble applications such as ultrasound molecular imaging, sitespecific drug delivery and triggered-release of therapeutic payloads.
In addition to their exploitation for diagnosis, ultrasound (US) combined with UCAs today represent a new method of localised drug delivery, in which sonoporation induces a transient opening of biological barriers ultimately leading to the uptake and enhanced accumulation of drugs in the targeted region.
Drug-delivery using contrast-enhanced ultrasound (CEUS) has the potential of becoming a clinically accepted approach for locally potentiating anticancer chemotherapies; and an effective adjunct with recombinant tissue plasminogen activator (rt-PA) for clot lysis.
Careful evaluation of the choice of drugs, clinical indication, ultrasound settings, microbubble type and concentration is needed to facilitate translation of sonoporation to the clinic.
Ultimately, the overall goal of imaging, drug-delivery and monitoring response to treatment by using the same equipment would be the perfect drug delivery system, combining as it does: ease-of-use, mobility, real-time and bedside availability.
This box summarizes key points contained in the article.Therapeutic applications based on US and UCAs are described in the subsequent section, with specific focus on sonothrombolysis and sonoporation-induced drug delivery. The use of US and UCAs as a tool for evaluating therapeutic response is presented in Section 5. The article ends with a short conclusion and Expert Opinion outlining our vision of how US- and UCAs-based drug delivery methods will likely develop in the future.
2. Microbubbles as US contrast agents (UCAs)
2.1. Nature and constitution of UCAs
UCAs are dispersions of small (typically 3 µm in diameter) gaseous microbubbles that are stabilized against dissolution and coalescence by a lipid monolayer, cross-linked polymer or denatured protein (see Figure 1(a,b)). Microbubbles are usually administered into the circulation of the test subject either as an intravenously infusion or bolus injection of approximately 200 µl of gas volume, in humans. Because of their micrometer size range, microbubbles are primarily confined to the vasculature after administration, a feature that makes them excellent blood-pool tracers because they can be easily interrogated using US, as they circulate through the vasculature of the test subject. However, the circulation life-time of most microbubbles,is often short lived (typically 3–5 min [7] for SonoVue™) and this is due in part to the acoustic parameters used, that is, whetherthe US pressures used are destructive; and diffusivity of the gas from microbubble interiors and its solubility into the surrounding media. UCAs increase the intensity of US signals backscattered from blood for several minutes after their injection, and this signal enhancement can be prolonged by infusing them. Perhaps most importantly, UCAs have a stronger signal enhancement at the frequencies (1–14 MHz) employed for medical imaging. This makes UCAs extremely useful for US image enhancement, and useful for diagnostic applications such as the identification of angiogenesis in malignant tumors, left ventricular opacification (LVO), and myocardial perfusion [2]. A number of microbubbles are in current clinical use (Table 1).
2.2. Types of UCAs
Although microbubbles were originally developed to improve US imaging, recent advances have opened up powerful emerging applications including: CEUS molecular imaging [8] usingtarget-specific microbubbles [9,10], site-specific drug delivery using microbubbles as vehicles for carrying [11] and triggering the release of therapeutic payloads [12]. This has the potential of changing the way US is used in drug delivery, acoustic therapy, and treatment monitoring. Recently, it has been reported [13] that some, but not all, microbubbles are retained by the liver and spleen in a nonspecific and time-dependent manner. The retention and accumulation of microbubbles in other organs have been reported and now thought to depend on microbubble physical and chemical properties. The transit times of such microbubbles through the liver now forms the basis of a powerful new liver-specific imaging mode [4].
2.3. Microbubbles as drug vehicles
US waves can potentiate therapeutic drug delivery by creating transient nonlethal perforations in cell membranes and so aid delivery of variously sized molecules that would otherwise be excluded from accessing the cell interior. While this requires substantially high acoustic power beyond levels approved for diagnostic imaging, the power needed is greatly reduced when microbubbles are present. This is because microbubbles lower the amount of energy needed for cavitation, the process in which extreme oscillations induced by US pulses lead to microbubble collapse [2,14]. Microbubbles can be formulated not only to carry therapeutic agents, but also to protect the drug payload (Figure 1 (e–h)) from enzymatic digestion during its transition to the target site or tissue. In this way, therapeutic drugs can be delivered with high precision and accuracy, although certain drug carrying approaches are preferred because of their drug loading efficiency and/or ability to protect the therapeutic payload. For example, microbubbles with shells comprising positively charged lipids are known to electrostatically bind negatively charged nucleic acids (NAs) such as siRNA, miRNA, Mrna, and plasmid DNA. Similarly, hydrophobic drugs can be encapsulated within lipid or polymeric shells that stabilize microbubbles (Figure 1). The circulation of these drug/NA-loaded microbubbles can be followed using CEUS imaging and subsequently disrupted using either purpose designed US pulses or temperature [12] to release its therapeutic payload upon reaching the target organ or tissue [15]. Using drug incorporation into UCA shells, Shohet et al. [16] showed that the delivery of a reporter gene in the mouse heart could be increased 10-fold by using microbubbles loaded with an adenoviral vector in their shell encapsulations.
2.4. Targeted microbubbles
Site-specific delivery can be aided by incorporating ligands or monoclonal antibodies onto the outer surface of drug loaded
1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC),1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid, monosodium salt (DPPA), N-(methoxypolyethylene glycol 5000 carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine,monosodium salt (MPEG5000 DPPE); Distearoylphosphatidylcholine (DSPC), Dipalmitoylphosphatidylglycerol (DPPG), hydrogenated egg phosphatidyl serine (HEPS); left ventricular opacification (LVO); endocardial border delineation (EBD).
UCAs (Figure 1(d)) and subsequently used to target receptors expressed on vascular cell membranes. For example, incorporation of a surface ligand that binds to the GPIIB/IIIA receptors expressed on activated platelets, allowed microbubbles to avidly bind to arterial thrombi [17,18]. Delivery of thrombolytic agents was then easily achieved upon disruption of the targetbound microbubbles. Similarly, microbubbles that carry anti P-selectin, E-selectin, VCAM-1, and VEGF-R2 antibodies have been widely employed for the in vivo identification of molecular expression, clearly showing that a number of receptors can be potentially used for targeted drug delivery. For a detailed review, see Supplemental Material to reference [10]).
3. US contrast imaging modes
Microbubbles scatter US waves both passively and actively when exposed to US. The earlier is due to differences in acoustic impedance between microbubble gas cores and the surrounding medium [19] while the latter is due to oscillations emanating from the high compressibility of gas cores which initiates a volume pulsation, although the shell encapsulation can dampen these effects. Because they are gas filled and smaller than the US wavelength, microbubbles undergo radial oscillation which involves sequential compression and expansion in the alternating pressures of the US field. Stable cavitation is a term that refers to oscillation without significant loss of microbubble integrity whereas inertial cavitation refers to disruption of microbubble caused by excessive oscillation. The latter event can result in the release of transient-free microbubbles. Radial oscillation of either encapsulated or free microbubbles results in strong acoustic signals that can greatly exceed conventional US backscatter signals. The degree of oscillation is in turn dependent on the density of the gas core, material properties of the surrounding medium, the nature of the encapsulating shell, and US characteristics such as frequency and pressure amplitude. These oscillations
Figure 1. Schematic representation of microbubble UCAs highlighting different functions and features. A shell encapsulation of (a) lipid monolayer, (b) cross-linked polymer or denatured protein surround the perfluorocarbon, gas composite or air bubble. Gas-filled UCAs can be designed to carry specific lipid molecules, such as (c) phosphatidylserine allowing them to non-specifically bind to certain tissues in vivo, or (d) monoclonal antibodies or antibody fragments, allowing them to bind specifically to certain tissues in vivo. Similarly, (e) hydrophilic drugs can be loaded into stealth liposomes which are in turn conjugated onto the surfaces of microbubbles; (f) negatively charged drugs or NAs can be electrostatically bound onto the surface of positively charged microbubbles; (g) drugs can be incorporated by themselves into the shell or, if insoluble in water, (h) in an oil layer inside the UCA thus allowing the drug payloads to be carried and delivered to the required site.
are governed by variations in pressure of the US wave, constituted of a compression phase (leading to microbubble compression) and rarefaction phase (microbubble expansion).For higher US amplitudes, the scattering of the microbubble becomes nonlinear and as a result, the US echoes contain harmonic components in addition to the main fundamental frequency. In fact, the interaction of US and microbubbles can be described as a function of the applied acoustic pressure, as described by mechanical index (MI) in clinical US scanners. MI is defined as the ratio of the peak negative pressure (PNP) in MPa, that is, the maximum amplitude of the peak rarefaction pressure, to the square root of the driving frequency (f) in MHz [14].MI can be used to predict the degree of linear and nonlinear bioeffects that a given set of US parameters will induce. When exposed to MI<0.1, microbubbles exhibit a weak nonlinear and nondestructive response. At MI = (0.1–0.3), microbubble responses are nondestructive but exhibit a significant nonlinear response. Finally, at MI > 0.3, most microbubbles exhibit nonlinear response and are easily destructed [20].
Based on the nonlinear acoustic signatures of UCAs and the advanced understanding of their dynamics in an US field, a number of imaging modes directed to the detection and imaging of microbubbles have been proposed. CEUS imaging modes are generally aimed at detecting US echoes emanating from microbubbles while eliminating or reducing echoes issued from non-perfused tissues. Below we describe the main CEUS imaging modes currently used on clinical US scanners.
3.1. Pulse inversion
PI is widely exploited in clinical applications for medical diagnosis. This imaging mode provides contrast enhancement between the microbubbles injected intravenously and the surrounding tissues by detecting the nonlinear backscattered signal components from microbubbles [2,3,21–24]. PI involves a successive transmission of two US pulses; the second pulse being an inverted replica of the first pulse. The echoes received from the two pulses are collected and summed. Because tissue responds mostly linearly, the sum of its echoes will thus be canceled out. Conversely, microbubbles exhibit asymmetric oscillations resulting into nonlinear echoes and their sum will be cumulative instead of suppressed. In other words, with PI linear components cancel while nonlinear components do not cancel. PI is known to preserve all even harmonic components and to cancel out the uneven ones (including the fundamental component). Accordingly, PI allows the detection of the second harmonic component arising from the microbubbles. PI offers greater advantages over conventional harmonic imaging which requires the transmission of long pulses (narrow bandwidth signals) to avoid the overlapping of the fundamental frequency and the second harmonic frequency bands. PI overcomes this technical limitation by providing a better compromise between axial resolution and sensitivity. However, because it is a multi-pulse technique, PI suffers from substantial frame rate reduction (down by a factor of 2) when two pulses are transmitted. Another limitation of the PI is due to the fact that two firings are required to create one beam. As a result, relative motion of the tissue can occur during these firings. Under these conditions, the fundamental signal component is not completely canceled, but instead results into motion artifacts [3].Like PI imaging mode, PM also called amplitude modulation is a multi-pulse sequence which enables the detection of the nonlinear backscattered signals of the microbubbles with cancelation of the linear response from non-perfused tissue [25,26]. Here, two US pulses are transmitted, where the second pulse has twice the amplitude of the first pulse. The scattered echo from the first pulse (E1) is scaled by a factor of two and then subtracted from the second echo (E2). As a result, linear echoes are canceled by this simple operation (E2-2E1) while echoes from microbubble nonlinear scatter, is enhanced. As with PI, the frame rate in PM mode is similarly reduced by afactor of 2.3.3. Contrast pulse sequenceContrast pulse sequencing (CPS) has been developed to further increase the specificity of microbubble detection. CPS is based on the combination of PI and PM sequences [27–29].CPS uses a sequence of more than two pulses which can reduce motion artifacts [30] leading to better sensitivity and specificity for microbubble detection (see also Figure 2).CEUS is still an active research area where acoustic microbubble-specific signatures are carefully exploited in order to develop more sensitive microbubble detection methods. Other novel approaches have been proposed based on coded excitations such as Chirp Reversal Imaging [31] as well as specific nonlinear behavior of microbubbles such as subharmonic imaging [32,33] and super-harmonic imaging[34,35].
Figure 2. Detection and delineation of a patient liver lesion in contrast ultrasound image with CPS imaging mode showing an early uptake of the contrast microbubbles compared to adjacent hepatic tissue (courtesy, A. Bleuzen and F. Tranquart CHRU Tours).4. Microbubbles as drug delivery and therapeuticagents4.1. Drug delivery using microbubble-assisted USResearch into in vivo drug delivery using CEUS has developed rapidly throughout the past decade and the potential of this modality is clearly demonstrated by the increasing number of publications [36] on the subject. The exposure of biological tissues to US in the presence of microbubbles, induces nonlinear microbubble oscillations which mediate permeabilization of cells and tissues, through a process termed sonoporation [37]. Here, microbubbles are co-injected [38] with the therapeutic drug or directly administered into the targeted superficial tissues [39]. It is thought that in the former, oscillation of microbubbles in the tumor microvasculature promotes permeabilization of the tumor endothelium only, and enhances drug extravasation. The permeabilization of endothelial cells is thought to augment drug uptake into the cells, thus leading to the destruction of tumor vasculature and reduction of nutrient supply. In the latter, oscillating microbubbles close to the target cells probably result in the permeabilization of the cell plasma membrane, which is followed by increased intracellular uptake of drugs.Although the exact mechanism(s) underlying CEUS drug delivery are still not fully understood, it is now recognized that cell/tissue permeabilization is correlated to the acoustic response of the microbubbles. Since, US-driven microbubble response depends on the acoustic parameters of the US wave (i.e. excitation frequency, acoustic pressure, exposure time, etc.) used [40], when exposed to low acoustic pressure, microbubbles will undergo stable oscillation through a process termed stable cavitation. In this regime, microbubble oscillations produce fluid flows around the microbubbles, termed acoustic microstreaming, which result in shear stress on the cell membrane (Figure 3) [41]. At higher acoustic pressures, microbubbles oscillate more vigorously, leading to their violent collapse and destruction, through a process termedIS, described as death of neuronal cells due to reduction/ interruption of regional cerebral blood flow by a clot, is the second leading cause of death worldwide [71]. Besides mechanical clot retrieval, alternative treatment for IS commonly involve the use of recombinant tissue-type plasminogen activator (rt-PA) [72]. However, rt-PA is presently only applicable in 3–5% of acute IS patients. This is due in part to strict treatment initiation times of ≤4.5 h and associated hemorrhagic side effects [73]. Today, transcranial US used in conjunction with microbubbles, which can be coadministrated
Figure 4. (a):Images of a control clot (without insonification nor microbubbles) after one hour in a plasma flow circuit (10 cm/s, tube of 1.6 mm inner diameter), the clot is full of red blood cells. (b): Images of a clot after one hour in a plasma flow circuit (10 cm/s, tube of 1.6 mm inner diameter) after ultrasound insonation at 500 KHz (Peak negative pressure of 500 KPa and duty cycle of 0.08%) in the presence of BR14® microbubbles (4 × 105 MB/min): No red blood cells are identified, the fibrin network is still present. A-B: Scale of 20 µm. Adapted with permission from [79].with other drugs or used as a drug carrier, are under investigation as an alternative thrombolysis treatment (sonothrombolysis).4.2.1. Preclinical studies of sonothrombolysisTwo different therapeutic strategies are used to deliver medication to lyse thrombus (sonothrombolysis). The first, based on the coadministration of UCAs and fibrinolytic drugs showed that US combined with UCAs potentiates the effect of fibrinolytic drugs. Tachibana et al. [74] showed that the use of UCAs led to significant enhancement of clot fibrinolysis rates and clot destructions equating to: 26.6% for clots treated with urokinase alone; 33.3% for urokinase and US; and, 51.3% for UCAs, urokinase, and US, thus giving the first clear demonstration of the potential role of UCAs as suitable adjuncts for sonothrombolysis. Indeed, the ability of UCAs to enhance fibrinolysis has been extensively studied and the findings (also described 4.1), have led to the accepted view that during stable cavitation, microstreaming promotes contact between fibrinolytic drugs and fibrin, leading to better access to the enzymatic sites. This assumption is supported by the work of Lauer et al. [75] who showed that flow between the fibrin fibers is potentiated by US radiation forces. In addition, Dattaet al. [76] showed a deeper penetration of rt-PA within clots, when UCAs were coadministrated with rt-PA and subsequent treatment with US. In related studies, Rooney [77] had shown that hemolysis was achievable using US alone and more recently with the combined use of US and UCAs (see Figure 4) [78,79].The second therapeutic strategy involves the incorporation of therapeutic agents within microbubbles in order to locally deliver drugs at the ROI. The ability to maximize the concentration of medication where therapeutic action is needed and to lower the overall dose used, remains a key goal for achieving better therapeutic benefit-to-risk ratio. Luan et al. [80]showed that stable and inertial cavitation-induced lipid shedding from the shell of the microbubbles, thus giving a clear demonstration of how drugs can be released under sonothrombolytic conditions. While the amount of lipid shedding is significant for inertial cavitation, there remains concern that using this acoustic regime in the ischemic brain, might lead to dramatic intracerebral hemorrhages [81,82]. To illustrate the potential of drug-loaded microbubbles, Hua et al. [83], have successfully recanalized femoral arteries in rabbits by using rtPA loaded microbubbles. In addition, these authors showed that microbubbles loaded with rt-PA at a concentration 15- times lower than the recommended dose, achieved similar rates of recanalization as non-loaded microbubbles coadministered with a normal rt-PA dose.
4.2.2. Clinical studies of sonothrombolysis
The broadly positive in vitro and in vivo results led to the initiation of clinical trials. However, two trials, MUST and CLOTBUST [84,85], have been interrupted after significant levels of intracerebral hemorrhages were recorded, without prognostic improvement at 3 months. Other clinical trials have been conducted, but the significant loss (60%) of subjects at 3 months [85] and/or absence of study randomization [86,87] were notable concerns. A more recent clinical trial, NOR-SASS [88], has been prematurely ended for lack of funding. Today, the safety of sonothrombolysis remains a concern and will require careful evaluation of preclinical studies using clinical end points (e.g. hemorrhage rates, volume of infarcted brain, and functional recovery), and well-defined protocols of microbubbles administration and US parameters employed. Finally, implementation of recent technologies such as MRI guidance [66] should increase the safety of sonothrombolysis as this would allow for adjustment of US treatment parameters to the skull on a personalized level.5. US contrast imaging for therapeutic monitoringA number of objectively measured and evaluated biomarkers are implicated in normal biological, pharmacologic responses to therapeutic intervention or pathogenic processes, including angiogenesis in tumor progression. Angiogenesis, the process by which tumors increase their blood and nutrients supplies, is an essential feature of tumor growth and invasion; and its continued expansion is thought to be associated with poor prognosis [89]. The goal of anti-angiogenic therapies is to interrupt tumor supplies by inhibiting vessel formation [90].Unfortunately, the classical approach recommended for monitoring therapeutic response, response evaluation criteria in solid tumors (RECIST) [91], have proved to be of limited value in assessing tumor response to anti-angiogenic therapies because early necrosis is often observed prior to any reduction in tumor size [92]. As a result, CEUS, an imaging modality based on vascularity assessment has been adopted for the early monitoring of tumor therapeutic efficacies.CEUS has the advantage of being low-cost, nonionizing and presents a better tolerance compared to other imaging modalities such as CT or PET. Because microbubbles are incapable of extravasation from the vasculature, today CEUS is under evaluation as a tool for quantifying tumor perfusion before and after treatment. CEUS quantification involves the selection of an ROI bordering the lesion and plotting the US enhancement intensity in the ROI to obtain a time-intensity curve (TIC). The resultant TICs are then fitted to a mathematical model reflecting the kinetics of UCA tumor uptake [93]. Amplitude parameters of the TIC, for example, peak enhancement (PE), wash-in rate (WiR), wash-out rate (WoR), wash-in area underthe-curve (WiAUC), wash-out area under-the-curve (WoAUC), or total area under-the-curve (AUC), and temporal parameters, for example, time-to-peak (TTP) or mean transit-time (mTT) are then analyzed, to assess blood volume or blood flow in the lesion. A similarly sized ROI, in the healthy tissue is usually employed as a background reference (Figure 5) [94].
A number of studies have investigated the utility of CEUS in monitoring treatment response to anti-angiogenic therapies. Williams et al. evaluated the impact of sunitinib on renal cell carcinomas [95] and reported a significant reduction of PE, WiAUC, and TTP after the first cycle of treatment. Lassau et al. assessed the efficacy of bevacizumab in patients with advanced hepatocellular carcinoma [96]. The study showed significant correlation between a decrease in PE and progression-free survival, for tumor perfusion before treatment and 3 days after treatment. More recently, a large multicenter study reported the effects of several anti-angiogenic treatments on different types of tumors, comprising >2330 CEUS examinations from 539 patients [97]. The study demonstrated that the ratio of AUC at day 30 as compared to baseline (before treatment) of less than 0.6, corresponding to a decrease of >40% of AUC, is an indicator of lesions not responding to treatment. Together, these results support the development of a patient-specific adaptation of treatment regimens, which will allow early decision-making on the efficacy of anti-angiogenic treatment based on the evolution of CEUS quantification results [98]. However, additional investigations need to be undertaken to further improve the efficacy of CEUS quantification techniques. In particular, 3-dimensional acquisition would be more informative for the monitoring of whole lesion instead of single plane acquisitions. Post-processing tools, such as parametric imaging which allows displayingwith a color map, a group of pixels according to a parameter’s mean value at a given location, could provide an additional way of enhancing diagnostic confidence [99].
The exploitation of US modality for drug delivery and therapeutic guidance has evolved with the introduction of microbubbles as a tool for the therapeutic delivery of drugs and the realization that the selected acoustic behavior of microbubblecan noninvasively drive clot lysis. The main advantages of USare its wide availability, portability, real-time character, easeof-use, and low cost as compared to other imaging modalities.In particular, the use of real-time imaging can lead to novelinsights and facilitate US-triggered drug delivery.Currently available reports indicate that CEUS can play a major role in monitoring and evaluating response to antiangiogenic therapies. Large multicenter studies carried out on different types of tumors in large cohorts of patients have demonstrated the correlation between quantitative parameters and response to therapy. These results support the development of a patient-specific adaptation of treatmentregimens, which will allow early decision-making on the efficacy of anti-angiogenic treatment based on the evolution of CEUS quantification resultsUS and microbubbles can indeed induce the permeabilization of tumor barrier and potentiate the extravasation of chemotherapies and therapeutic antibodies into the targeted lesion. Today, a number of clinical studies have demonstratedFigure 5. (a) Parameters extracted from a Time-Intensity Curve (TIC): Peak Enhancement (PE), Wash-in Rate (WiR), Wash-out Rate (WoR), Time-to Peak (TTP), Rise Time (RT), Fall Time (FT) and mean Transit Time (mTT). (b) B-mode and contrast-enhanced ultrasonography of the liver in late phase. Two ROIs are drawn: one outlining a metastasis (green) and one placed in the healthy parenchyma (yellow). (c) Two TICs before treatment (red) and two months after treatment (blue) of a metastasis from a responder patient. Amplitude parameters are shortened and temporal parameters are increased. More particularly, total Area Under the Curve (AUC) decreased of 87%. The analysis was carried out using Bracco software VueBox™. Full color available online.the ability of sonoporation to induce enhanced uptake of chemotherapies for both glioblastoma and pancreatic cancer. Nevertheless, a number of evaluation and clinical validation issues need to be tackled including standardization of US parameters, selection of appropriate microbubbles, identification of optimal clinical indications and drugs before further translation to the clinic. In addition, there is evidence to show that the increased throughput of free radicals resulting fromHIFU and inertial cavitation in the presence of some chemotherapeutic drugs carry sufficient capacity to weaken the therapeutic potency of certain drug molecules [100].
The ultimate goal is to realize image-guided/monitored drug delivery into the desired tissues within a clinical setting as demonstrated elsewhere [101] for other modalities. Various reports in this journal [102–104] closely relate to the present topic and interested readers are encouraged to examine thesereports.
7. Expert opinion
UCAs can have a role in clinical decision-making. This is characterized by demonstrable success in a number of clinical indications aimed at vascular diagnosis and therapy in various organs. For example, CEUS has emerged as a new functional imaging approach for assessing tumor perfusion. Using this approach, it is now feasible to predict with accuracy and well in advance, the outcome of anti-angiogenic therapy using CEUS. This is a highly effective treatment monitoring approach compared with current therapeutic evaluations [91].Accordingly, the use of CEUS for the monitoring of anti-angiogenic treatment has been added to the clinical practice guidelines by both the European Federation of Societies for US in Medicine and Biology and World Federation for US inMedicine and Biology.Beyond their exploitation for diagnosis, UCAs and US, today represent a new method of localized drug delivery. Recent research shows that under the action of US waves, microbubbles transiently perforate biological barriers (e.g. cell membrane, endothelial layer) thus leading to the uptake and enhanced accumulation of drugs in the targeted region. In this way, the bioavailability of therapeutic agents is site-specifically augmented only in the zone where US waves are focused with millimetric precision.
Significant progress has been made in the field of CEUS drug delivery. Whereas CEUS drug delivery has been demonstrated in proof-of-concept studies in various organs as well as clinical indications in a number of small animal models, only a few studies report on the evaluation and validation of this therapeutic strategy in large animals, with the majority focusing mainly on the opening of the BBB [105–109]. To facilitate CEUS drug delivery to the clinic, a number of questions need to be addressed such as: which organ should be targeted? Which drugs are suitable for sonoporation drug delivery? If cancer is the clinical target, which lesions (solid tumors or metastases) should be considered? Since microbubbles need to be localized within the target organ or zone for better drug extravasation, should considerations concerning the perfusion of the target lesion take precedence? What US parameters (frequency, pressure/MI, and duty cycle) should be used to activate the microbubbles and induce drug uptake? A number of studies show that sonoporation efficiency is strongly correlated with the concentration of UCAs in the target zone, suggesting that UCA concentration is a crucial sonoporation parameter. Accordingly, what UCA concentrations should be used? Similarly, the appropriate method of UCA administration, that is, bolus injection or continuous infusion delivered intra-arterially or intravenously needs careful consideration. Similarly, the treatment schedule is of significant importance, since administration of UCAs and drugs should be performed in a prescribed manner and US insonation timing (duty cycle) should be synchronized to coincide with the arrival of elevated UCA concentrations. The type of UCAs employed is also of significant importance because UCA response to US activation depends on its physical properties. Pragmatically, it should be easier and faster to evaluate drug delivery using existing commercial agents, as that will facilitate quick translation to clinic. Whether US scanners operated in specific modes (such as Doppler mode) can deliver US sequences capable of inducing drug delivery; are just some of the questions that also need addressing for the effective translation of this therapeutic technology to the clinic.
Another essential aspect is the mechanism behind the sonoporation process. Despite the proof-of-concept achieved in preclinical studies, there is no consensus, neither with regard to the acoustic phenomena involved, nor the underlying US-induced permeabilization mechanism(s). Although an increasing amount of data on sonoporation now exist, several challenges still need addressing before bringing a number of US-based therapeutic strategies, including sonoporation and sonothrombolysis to clinical use. The main translational challenges include a clear understanding of the underlying mechanism(s) of permeabilization; and fully tested US protocols that will ensure precise, efficient, and localized drugdelivery.
US is now experiencing a paradigm shift in both technology and applications. In addition to its intrinsic advantages of real time, portability and low cost; US is being intensively investigated as a drug delivery tool. The goal therefore is to realize image-guided and monitored drug delivery into the desired tissues. It is anticipated that the combined use of CEUS in therapy may eventually become even more important than its diagnostic use. Ultimately, image guidance, drug delivery, and the ability to monitor treatment response if successfully implemented on the same equipment, would be the perfect drug delivery system; providing combined ease-of-use, mobility, real time, and bedside availability. We anticipate that in the future,the combined use of US and microbubbles in therapy will be planned, applied, and monitored in a rapid sequence with high spatial and temporal resolutions. Moreover, this would alleviatethe need for image coregistration since the imaging equipment would also be used to induce local therapy ensuring a perfect collocation. Finally, striking a balance between safety and efficacy will remain a key challenge for the successful clinical application of this methodology, which combines real-time imaging guidance with drug delivery and therapeutic monitoring.
References
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