INTRODUCTION:

Ultrasound imaging has been one of the most popular medical diagnostic techniques because of its superior safety, low cost, and easy accessibility compared with other imaging modalities such as computed tomography, positron emission tomography, and magnetic resonance imaging (MRI). Ultrasound imaging can provide real-time quantitative information on the morphology and perfusion of biological tissues for evaluation a variety of diseases in cardiology, radiology, and oncology. [1]

Drug resistance is a main obstacle for curative cancer chemotherapy. Therefore, strategies need to be developed to overcome chemotherapy resistance [2]. In recent years, tumor-targeted therapy has been appearing as a promising therapeutic choice for cancer treatment. The potential approach is to develop particular carriers which can facilitate the release of the payload locally in tissue by internal or external stimuli (such as heat, light, ultrasound, etc.). Tumor imaging should be performed before and during the external stimuli or treatment. The bio distribution of drug carriers is monitored by imaging, so that the optimal timing for the application of external stimuli can be achieved [3]. Nanotechnology has the potential to in􀁌uence the detection, prevention, and treatment of cancer. Micro bubbles are commonly used as intravascular ultrasound imaging probes and are becoming increasingly popular tools for targeted drug delivery. However, the micro sized particles could only stay in blood circulation and penetrate poorly into tumor tissues, so that the wide application of the particles for in vivo tumor therapy is limited [4]. Strategies have been advised that nanoparticles can be used to deliver drug/gene to targeted tissues [5]. Nanoparticle, used as a drug/gene delivery vehicle, can not only target specific cells and tissues, but also retain the biological activity of the drug/gene during transport. Ultrasound is a noninvasive and visual theranostic modality that can be used to track drug carriers, trigger drug release, and improve local drug sediment with high spatial precision [6,7]. Therefore, the development of novel visible ultrasonic responsive nanosized drug/gene carriers is necessary.

ADVANTAGES:

Several advantages are accompanied with the use of ultrasound as a drug delivering method. First of all, ultrasound is a non-invasive technique with an external source. Second, it can be applied locally at a very small region of interest inside of the body. Third, it is able to reach deep inside of the human body, in absence of harming the tissue in the beam path. And it does not necessarily require the development of new drugs.

Physical Principles:

Ultrasonic excitation within microfluidic systems typically leads to complex fields characterized by acoustic energy gradients of various length scales. These energy gradients underlie two phenomena of significance for the manipulation of cells and drug delivery vehicles such as micro bubbles on chip: acoustic radiation forces and acoustic streaming. These are described in the following subsections prior to a discussion of the devices and typical parameters used in micro fluidic ultrasonic manipulation.

A. Radiation forces:

Cells within an ultrasonic field experience high frequency oscillatory forces due to the linear component of the acoustic field, but these forces have time-averages of zero that do not lead to translation of the cells. However there are second order terms within the field which have a finite time-average and generate acoustic radiation forces that can be used for steady state manipulation and levitation of cells and other particles. King derived an expression for the acoustic radiation forces on a spherical, rigid particle in an in in viscid fluid and this analysis was extended by Yosioka and Kawasima [33] to allow for compressible particles and bubbles.

B. Acoustic Streaming:

Gradients of acoustic energy density also lead to acoustic streaming: the acoustic generation of net fluid flows [34]. The two main sources of streaming are: i) due to acoustic energy dissipation into the bulk of a fluid, and ii) due to energy dissipation from the interaction of an acoustic field with a boundary. The first of these mechanisms, also known as “Eckart streaming”, or the “quartz wind”, is caused by attenuative loss over a number of wavelengths so is typically only an issue in micro fluidic devices when high frequency waves propagate along the length of a channel [35]. It is also less likely to be observed in standing wave systems in which the forces leading to Eckart streaming from counter-propagating waves tend to cancel each other out. The second mechanism occurs due to steep energy gradients within the acoustic boundary layer adjacent to a wall (“boundary layer driven” streaming) or near the surface of an oscillating bubble (“cavitations micro streaming”), and these forms of streaming are widely observed in ultrasonically excited micro fluidic devices.

Boundary layer driven streaming was first analyzed by Rayleigh [36], describing the flow observed within a standing wave between parallel walls that leads to the four vortices within each half wavelength that characterizes Rayleigh streaming (see Figure 1). Each of these four outer vortices is driven by an inner vortex generated by viscous losses within the viscous boundary layer. For a standing wave in water with a frequency in the low MHz range the wavelength will be of the order of 1 mm, while the viscous boundary layer depth will be of the order of 1 μm [35]. Hamilton et al. [36] show that for very narrow channels, Rayleigh’s analysis ceases to be valid, and ultimately the outer streaming vortices cease to exist. More recently shows that boundary driven streaming in micro fluidic devices can occur in planes orthogonal to classical Rayleigh vortices. [37]

Streaming can disrupt the ordered manipulation of cells via acoustic radiation forces as particles are drawn along the streaming vortices by the viscous drag forces. On large particles the radiation forces tend to dominate, while smaller particles are influenced primarily by the streaming drag. Bruus [38] has shown that for typical particles in a 1 MHz standing wave in water the critical diameter below which streaming drag dominates is of the order of 2 μm. In other cases, the existence of streaming can be used to positive effect, improving mixing and mass transfer in biosensing applications [39], enhancing the interaction of cells and retroviruses [40], agglomerating cells in droplets [41] and the removal of non-specifically bound proteins from surfaces [42].

C. Devices for on-chip ultrasonic manipulation:

A variety of different types of device have been used for ultrasonic manipulation [43]. Probably the simplest approach is to use a layered, or planar, resonator [44] comprising a transducer, which is typically isolated from the fluid in which the cells are suspended by a carrier, or coupling layer. The standing wave is maintained by a passive reflecting layer at the far side of the fluid, as shown in Figure 1.

a b c

Figure 1. Schematic representation of layered resonator.

A standing wave is set up by exciting the transducer at a resonant frequency which generates a pattern of pressure gradients within the fluid which effect the cell manipulation. Uniformly distributed cells (a), first move to a pressure node (b) and are then brought together within the node (c). Such a resonator may be many wavelengths across, resulting in multiple planes into which cells are gathered [45]. Generally, the fluid layers in the micro devices considered here will be less than a wavelength thick and will gather cells into a single nodal plane [46, 47]. An alternative arrangement, the transversal resonator, has been used extensively by Laurel’s group in Lund [43, 48]. In this arrangement the standing wave is established parallel to the transducer face such that manipulation operations can be easily observed through a glass cap above the channel and also parallel to the transducer face. Variations on these approaches can use glass capillaries [49] or a combination of planar and transversal excitation [50].

A rather different approach to the excitation of the ultrasonic field uses surface acoustic waves (SAWs) coupled into a micro-channel [51, 39]. The SAWs are generated using interdigitated electrodes printed onto a piezoelectric substrate – typically lithium niobate – and generally the channel is fabricated from PDMS which is bonded on to the substrate. The use of transducer pairs allows a standing SAW to be established, which can be used to pattern particles or cells in one or two dimensions [53]. Using SAWs, significantly higher frequencies can be achieved (up to 150 MHz) allowing resolutions which can easily approach the single cell level, particularly when combined with focusing, which is typically achieved using phononic crystals . SAWs can also be used to initiate fluid flow [52] and to provide energy (thermal and mechanical) for micro cell processing including PCR and cell lysis. A useful review of recent advances in SAW devices is presented by Wang et al. [54]. Recently there has been substantial interest in building devices that allow more dexterous manipulation of particles through the use of planar arrays of transducers [55] and different configurations of opposing transducers [56, 57].

D. Limitations and typical parameters for on-chip manipulation:

Given the flexibility noted previously, and its potential for use in both lab-on-chip systems and in vivo, ultrasound has many useful potential capabilities. However, it also has several limitations. The first of these, shared with several other manipulation techniques, is the limited contrast between a fluid medium and cells or the particles (drug delivery vehicles for example) of interest in drug delivery. In the case of ultrasound, this contrast is expressed in terms of the densities and compressibility’s of the fluid and the particle, as discussed in section 2.1 above. Cells, micelles, and loaded drug delivery vehicles such as liposomes have parameters similar to those of water and tissue, limiting the efficiency of the ultrasound intervention. Relatively high ultrasound intensities are thus sometimes required to exert sufficient force, making secondary problems more likely. These include: acoustic streaming, which increases with increasing ultrasound intensity [34]; the need to control excessive heating of the lab-on-chip device through high drive signals applied to the piezoelectric source of ultrasound [58] and other materials in the structure; and cavitations, which occurs above a threshold depending on the level of the impurities in the fluid which act as cavitations nucleation sites and which may be expected to be high in lab-on-chip media. All these problems may affect cell viability [59]. On the other hand, gas-filled micro bubbles have high ultrasound contrast because of their high compressibility [33]. This offers the possibility to label cells, to implement effective manipulation of drug delivery vehicles, and to manipulate various functional particles.

Another fundamental limitation of ultrasound is the difficulty to isolate it to specific regions within a substrate. Acoustic absorber materials are available commercially (e.g. Precision Acoustics, Dorchester, UK) but these may be difficult to integrate with other lab-on-chip fabrication processes. Ultrasound does not penetrate gaseous media effectively, so it may be possible to configure physical gaps in the device to contain propagation, but it is likely that ultrasound will propagate within the substrate. Thus, reports of arrays of ultrasound-actuated lab-on-chip devices on a common substrate focus on multiwall configurations with ultrasound generated in a remote position on the substrate for application in the micro fluidic components of a device, for example in transversal resonance [48] and SAW devices [51].

Acoustic isolation is a complicating factor in the fabrication of ultrasound-actuated lab-on-chip systems and the need for a piezoelectric material to generate ultrasound is another one. For SAW devices, the substrate is typically made of lithium niobate and for bulk wave resonators, piezoelectric ceramic is used. However, both of these are relatively expensive materials so they point towards the possibility to use a separate sterile micro fluidic component, such as a capillary, to reduce the cost of disposal and replacement for each biological sample [43]. It is also possible to integrate piezoelectric materials in thick or thin film form, or indeed bonded bulk material with other components. Requiring an additional level of fabrication sophistication, typically including photolithography this approach may be very useful when the number of devices is high and may be a key issue in differentiating acoustic manipulation from optical manipulation where integration is still much harder.

As noted previously, another important issue is temperature. Ultrasound, in its nature as a wave phenomenon, involves propagation of local motion of material within the host solid or liquid media. (It does not propagate effectively in gas at the frequencies required for lab-on-chip use.) This motion is ultimately dissipated as heat, particularly in the solid components of the lab-on-chip system. Ingeniously, this is being harnessed in systems in which it is taken into account in the need to maintain the optimum temperature for cells [58]. In that case, it simply allows less external heating to be applied. However, care must be taken to avoid local hot-spots which may occur near the transducer because of excess heating from the ultrasound generation process or in relatively lossy media. For example, the acoustic attenuation (approximately equivalent to heating) in PDMS and other plastics is much higher than in glass and silicon [60].

On the other hand, ultrasound frequency typically determines at least one dimension of the device used to generate it, for example thickness of piezoelectric material in bulk wave devices . Thus, higher frequency operation almost inevitably complicates fabrication of thickness and transversal resonance devices [62]. It also increases attenuation and thus heating, and acoustic streaming (see Section 2.2). The former may be beneficial in reducing leakage of ultrasound into unwanted regions in the device, but it is also likely to hinder scale-up. Thus, in general, where high frequency is easy to achieve, in SAW devices, it is used and where it is difficult and high resolution is desirable in bulk wave devices, the highest practical frequency is used. Nevertheless, there are many situations in which relatively low frequency bulk waves are used, even in relatively complicated devices; in this case, they are simply matched to the micro fluidic channel dimensions [55].

The Mechanisms of Ultrasound-Mediated Drug:

The exact mechanisms of ultrasound-mediated drug/gene delivery with nanocarriers are still uncertain. According to the reports, they may be related to non thermal effect (such as cavitations and mechanical effect) and thermal effect.

A. Nonthermal Effects:

Nonthermal effects can be divided into cavitation and other mechanical effects [9]. Studies have shown that the combination of ultrasound and bubbles can increase the targeted delivery efficacy in vivo. The bioeffect may be attributed to the acoustic cavitation [10, 11]. Cavitation refers to the bubble activities induced by ultrasound, which can occur in liquid, liquid-like material containing bubbles and pockets containing gas or vapor. Under the action of adequately high ultrasonic pressure levels, the bubble oscillates and finally collapses. Cavitation can induce temperature rise, mechanical stress, and free radical production, thus influencing the biological function. The behavior of bubbles in low-intensity ultrasound field is different from high-intensity ultrasound field. Low-intensity ultrasound produces stable cavitation state, which can lead to intense friction and shear stress on the surrounding structures. When bubbles encounter high-intensity ultrasound (>1 MPa, 1 MHz), the amplitude of bubble oscillation rises instantly. The transient cavitation is produced, which can result in shockwaves and microjets [12]. Microjets can be described as a powerful stream of liquid caused by asymmetric implosion of micro bubbles [13]. The micro streams give rise to temporary pores on surrounding vessel walls and cell membranes, promoting gene and drug targeted delivery [13–14]. Indeed, sonoporation (transient hole), induced by acoustic cavitation near the cell surface, has been shown to enhance the intracellular delivery of both small molecules and macromolecules [15–16]. Husseini and Pitt [6] reported that ultrasonic drug delivery from micelles usually employs polyether block copolymers and has been found effective in vivo for treating tumors. Ultrasound releases drug from micelles, most probably via shear stress and shock waves from the collapse of cavitations bubbles. It is also supposed that the release originates from acoustic streaming produced by radiation force. The collision of carriers may lead to shear stress, which results in reversible destabilization of the carrier and release of compounds. With the help of HIFU, drug releases from polymer micelles, which is most likely due to the effect of shear stress and/or shock waves produced by the collapse of a larger number of cavitations bubbles [17].

B. Thermal Effect:

Another potential mechanism for ultrasound-mediated drug/gene delivery is a localized temperature rise in tissue. The temperature rise affects the liquidity of phospholipids bilayer, which directly results in changed membrane permeability. Ultrasound is used to trigger the collapse of cavitations bubbles, and the amplitude of the wave can produce high local temperatures. The main mechanism in the current therapeutic applications of ultrasound is creation of a controlled, localized temperature increase in situ [8,9]. This can cause hyperthermia, which is also known to increase the cellular uptake of anticancer drugs [18]. The possibility to achieve hyperthermia in situ through HIFU presents distinct improvements over conventional methods of heat generation in tissue. HIFU-induced hyperthermia has already been shown to produce significant enhancement of delivery of anticancer agents into tumor sites in vivo, with targeted release from thermo sensitive liposomes [19, 20]. The combination of MR-guided focused ultrasound and drug-encapsulated nanocarriers could increase cellular uptake of agents [21].

C. Other Mechanisms:

In fact, the mechanisms of ultrasound-mediated drug/gene delivery with nanocarriers may be associated with many other factors, such as endocytosis and active membrane transport. Targeted nanocarriers may change or fuse the phospholipids bilayer, so that lipid carriers release the payload contents directly into the cells [22]. Compared with equivalent thermal dose, pulsed-HIFU treatment leads to much enhancement in distribution of nanoparticles. Additional studies also proved that the effects enhanced by pulsed-HIFU sustained longer time than that of cavitations effect and heat, which offered another possible mechanism for ultrasound-mediated delivery [21]. Duvshani-Eshet et al. [23] suggested that therapeutic ultrasound by itself operated as a mechanical force which could drive the gene through the cell membrane and traversed from the cytoplasmic network to the nucleus, rather than by increasing membrane permeability. Transfect ion studies and confocal analyses showed that the act in fibers impeded transfection by ultrasound in BHK cells, but not in a fibroblasts. A unique mechanism of drug delivery is supposed based on a so-called contact facilitated delivery, by which the phospholipids membranes of nanodroplets are merged into cell membranes of target cells, thus directly releasing their payload into the cytoplasm.

Ultrasonic drug release at targeted sites:

Drug-delivery with ultrasound relies on the interaction between a biocompatible carrier and an acoustic wave. The spatial specificity of the release is established by focusing the waves in the zone to be treated using physical principles and technologies developed in the past for diagnostic and therapeutic ultrasound [such as high intensity focused ultrasound (HIFU) or lithotripsy]. The main challenge in ultrasound triggered therapy is the design of carriers that are both responsive to ultrasound and biologically active. These agents should be able to carry large payloads and have access, or even accumulate preferentially, within the tumor. These challenges have been addressed by early researchers, such as Tacker and Anderson (24), along with wide and recent international collaborations such as Sonodrugs (25-31). In this section, we will first highlight the mechanisms by which ultrasound can release a payload and then describe various drugs, agents or nucleic acids that have been released with ultrasound in pre-clinical studies.

Drug delivery:

The first challenge is to overcome dose limiting factors that are caused by the systemic toxicity of the used drug. This is mostly due to the non-specific nature and spread of the drug throughout the blood circulation of the body. This causes the drug to also accumulate in healthy tissue. The dose in the target tissue has not the desired concentration and it is not possible to increase the overall dose. This limits the effectiveness of the drug in the diseased tissue and treatment is far from optimal. It is well known that local administration of drugs is a promising strategy, so several solutions have been proposed to increase the target concentration. These methods can be divided into three groups; ‘active targeting drug delivery’, ‘passive targeting drug delivery’ and ‘physical targeting drug delivery’.[32]

Active targeting is usually achieved by combining the drug particle with a targeting moiety, like antigen–antibody and ligand–receptor binding this result in preferred accumulation of the drug in the targeted region. Passive targeting takes advantage of the differences in permeability between tissues, allowing the drug to accumulate at regions with higher permeability. Passive targeting also includes the administration of drugs exactly at the desired place, for example invasively into an organ artery.

Physical targeting makes use of an external trigger, such as ultrasound or magnetic fields to release the drug at a desired region. In the past most research was performed on the active targeting of drugs to the target region, but in de past few decades increasing numbers of studies are dedicated to passive and physical targeting of drugs, because of the huge improvement of concentrating drug in a very small region. Ultrasound mediated local drug delivery utilizes a form of passive targeting and/or physical targeting for drug delivery. The second challenge is to make sure the drug can enter the diseased tissue efficiently, in other words, specific barriers that inhibit the drug to pass, have to be opened or lifted. In most cases the particles are too large to cross barriers, such as vascular tissue and the blood-brain barrier.

To overcome these barriers a modification of the target environment is required. The interaction of ultrasound with tissue causes increased permeability in several ways. The vascular wall can be ruptured and as a result drug particles can pass through. Next to that the vascular wall can have increased permeability without being ruptured. A big problem with drug molecules and particles is their inability to distribute homogeneously in a adequate concentration in the diseased cells. This is due to the fact that it has to pass several barriers, before reaching the diseased cells. The transport of the drug will here be explained for tumours, as they are widely investigated and play a big role in drug delivery. When a drug is administered intra venous, Ultrasound will enter blood circulation of the tumour after a certain period. Then it first distributes through the vascular space of the tumour. [8]

Medical ultrasonography:

Diagnostic sonography (ultrasonography) is an ultrasound-based diagnostic imaging technique used for visualizing subcutaneous body structures including tendons, muscles, joints, vessels and internal organs for possible pathology or lesions. Obstetric sonography is commonly used during pregnancy and is widely recognized by the public[61]

Diagnostic applications:

Typical diagnostic sonographic scanners operate in the frequency range of 2 to 18 megahertz, though frequencies up to 50-100 megahertz has been used experimentally in a technique known as biomicroscopy in special regions, such as the anterior chamber of eye. The choice of frequency is a trade-off between spatial resolution of the image and imaging depth: lower frequencies produce less resolution but image deeper into the body. Higher frequency sound waves have a smaller wavelength and thus are capable of reflecting or scattering from smaller structures. Higher frequency sound waves also have a larger attenuation coefficient and thus are more readily absorbed in tissue, limiting the depth of penetration of the sound wave into the body[63].

Therapeutic applications:[73,74]

Therapeutic applications use ultrasound to bringheat or agitation into the body. Therefore much higher energies are used than in diagnostic ultrasound. In many cases the range of frequencies used are also very different.

 Ultrasound is sometimes used to clean teeth in dental hygiene.

 Ultrasound sources may be used to generate regional heating and mechanical changes in biological tissue, e.g. in occupational therapy, physical therapy and cancer treatment. However the use of ultrasound in the treatment of musculoskeletal conditions has fallen out of favor.

 Focused ultrasound may be used to generate highly localized heating to treat cysts and tumors (benign or malignant), This is known as Focused Ultrasound Surgery (FUS) or High Intensity Focused Ultrasound (HIFU). These procedures generally use lower frequencies than medical diagnostic ultrasound (from 250 kHz to 2000 kHz), but significantly higher energies. HIFU treatment is often guided by MRI.

 Focused ultrasound may be used to break up kidney stones by lithotripsy.

 Ultrasound may be used for cataract treatment by phacoemulsification.

 Additional physiological effects of lowintensity ultrasound have recently been discovered, e.g. its ability to stimulate bonegrowth and its potential to disrupt the blood brain barrier for drug delivery.

 Procoagulant at 5-12 MHz.

Linear contrast-enhanced ultrasound (CEUS) imaging:

The strategies for contrast imaging are currently based on either the linear or nonlinear properties of micro bubbles. The linear scheme simply utilizes the echo enhancement of micro bubbles. One example is from our pervious study of a colon cancer model in a BALB/c mouse. The imaging was conducted using a homemade ultrasound imaging system with a single element 25-MHz focused transducer.[64] After the administration of micro bubbles, the contrast of tumor tissue was clearly enhanced for up to 6 min, as shown in Fig. 2A–F. Owing to the entrances and exits of circulating micro bubbles to the imaging plane, the regional brightness varied over a period of time. These variations served as the signature of the micro bubbles, and they were extracted using a high pass filter for interframe filtering. The obtained information was demonstrated as a color-coded overlay on a co localized B-mode image, as shown in Fig. 2G, which clearly showed the distribution of the tumor microcirculation. The color pixel values were proportional to the micro vascular blood flow volume and velocity. Further, a destruction-replenishment technique can be used to accurately assess the flow velocity of the microcirculation. In this technique, a destructive ultrasound pulse was first used to destroy most of the micro bubbles in the region of interest. Blood flow velocity can be measured based on the refill rate of micro bubbles indicated by the recovery of contrast enhancement. The concept of this technique was first described by Wei et al. in 1998 for use in echo cardiology.[65] An adaptation has been made by our group to improve the sensitivity of this technique for use in micro perfusion.[66-67]

Nonlinear CEUS imaging (micro bubble-specific contrast imaging):

The performance of conventional CEUS imaging can deteriorate with low bubble concentration, tissue motion, and slow perfusion. Several studies have shown the utilization of micro bubble nonlinear emissions in micro bubble-specific contrast imaging. [68-69]The second harmonics are not used because of interference from tissues at the same frequency.[70] However, inducing the resonance of micro bubbles requires the application of sufficiently long ultrasound pulses. Since the resonance frequencies of commercial agents are 2–10 MHz, imaging at a half of these frequencies with long pulses can lead to low spatial resolution. To overcome this limitation, a phase inversion technique that utilizes the sum of a pair of images obtained from 2 inverted short ultrasound pulses was proposed.[71] The oscillation of micro bubbles results in nonlinear distortion of reflected echoes that cannot be clearly cancelled in paired images, thereby leaving the signature of micro bubbles in the summed image. Nonetheless, this technique has to be operated at half the maximum frame rate, and its performance may still be susceptible to movement. Our group has demonstrated two techniques to perform nonlinear contrast imaging with improved spatial resolution. One is amplitude modulation chirp imaging, which utilizes 2 different ultrasound pulses transmitted from separate ultrasound transducers.[72] A low-frequency pumping pulse is transmitted to induce resonance of the micro bubbles. A high-frequency chirp pulse for imaging is then transmitted to simultaneously act on the same group of micro bubbles. Periodic changes in the backscattering cross sections of the micro bubbles in response to the pumping pulse can modulate the amplitude of the backscattered echoes of the chirp pulse, producing modulated components in the frequency spectrum.

usefulness of micro bubble cavitations:

The therapeutic usefulness of micro bubbles has gained much attention in recent years. Both inertial cavitations and destruction of micro bubbles are capable of producing strong mechanical stress to enhance the permeability of the surrounding tissues and further increase the extravasations of drugs into the cytoplasm or interstitium. This may involve several mechanisms. High-energy micro streaming and liquid jets arising from the collapse of micro bubbles can locally produce transient holes for direct passage of drugs. They may also cause a transient increase in temperature (reportedly up to 5000 K) to alter the fluidity of the cell membrane.[56] Local deposition of such high energy may result in the production of free radicals, which probably cause cell damage that enhances the permeability of endothelial cell layers.[76,77] In vivo applications haven focused, for instance, on disrupting the blood–brain barrier (BBB), a layer of tightly-packed endothelial cells surrounding all capillaries in the brain.[78] Many groups have reported the use of commercial micro bubbles with low-frequency ultrasound (0.4–5 MHz) to increase the permeability of the BBB, allowing the therapeutic or diagnostic agents to leak into the affected regions.[79-81] The opening can be temporary and recoverable, and does not damage the neural cells. However, micro bubbles exposed to low-frequency ultrasound have been shown to cause rupture of micro vessels with extravasations of red blood cells, even at a pressure under the FDA regulatory limit for diagnostic ultrasound equipment. The clinical safety of BBB disruption with low-frequency ultrasound still carries great concern about the risk of intracerebral hemorrhage. Given that concern, our group has developed a high-frequency-based technique (> 10 MHz) to disrupt the BBB by using stable cavitation of micro bubbles. It has been shown that stable cavitations may also mildly increase the tissue permeability by induced acoustic streaming. A noteworthy advance that we have made in this technique was the use of homemade micro bubbles that resonate at > 10 MHz. Sprague–Dawley rats were used in these experiments. The presence of BBB disruption was evaluated by the extravasations of a model drug, Evans blue, into the brain tissue. Stable cavitations at a high frequency enhanced by the resonance of small micro bubbles was able to produce effective BBB disruption, which was comparable to that with the low-frequency technique but without any damage even at pressures of up to 2.5 MPa. Interestingly, the amount of drug extravasations was found to highly correlate with the enhancement of subharmonic emission (i.e., the signature of stable cavitations of micro bubbles). Remarkable safety with the possibility of monitoring the extent of BBB disruption in real time suggests this technique has great promise in clinical use.

Drug-loaded micro bubbles and ultrasound-controlled release:

Micro bubbles have been proposed as a new vehicle for carrying drugs and genes. Lipophilic chemotherapeutic drugs such as doxorubicin, paclitaxel, and docetaxel can be incorporated into the lipid layer of micro bubbles.[66-68] It has been shown that the in vivo toxicity of paclitaxel-loaded micro bubbles is about tenfold lower than that of unencapsulated paclitaxel.[82] To increase the loading capacity, oil that dissolves lipophilic drugs can be introduced into the micro bubbles.[83-84] Drug-loaded particles such as micelles or liposomes can be conjugated to the surface of micro bubbles using ligand–receptor interaction.[85] Genetic materials (e.g., plasmid DNA) can be electrostatic ally attached to the surface of positively charged micro bubbles that bear cationic lipids.[86] Unlike liposomes, drug loaded micro bubbles are acoustically active and are able to exhibit stable or inertial cavitations in response to ultrasound. The payload of drugs or DNA can be locally released by the destruction of micro bubbles within the ultrasound-treated region, with a simultaneous increase in the permeability of the tissues. This suggests the potential of micro bubble technology in aiding drug or gene therapy, with reduced side effects to normal tissues.

Our group has developed the loading of micro bubbles with 1, 3-bis (2-chloroethyl)-1- nitrosourea (BCNU), a chemotherapeutic agent commonly used in the treatment of brain tumors.[75] The loading efficiency achieved was 75%. Sprague–Dawley rats were used to test the delivery of BCNU by these micro bubbles into brain tissues. The deposition of BCNU in the left hemispheres treated by ultrasound increased with the number of sonication sites, and was higher than that in the right hemispheres which had no ultrasound treatment, as shown in Fig. 5B. This indicates that the destruction of BCNU-loaded micro bubbles released the BCNU payload and simultaneously induced the disruption of the BBB for passage of BCNU into the brain tissues. The therapeutic efficacy of delivered BCNU was further validated on a rat brain glioma model. The results of MRI monitoring show that the tumor volume on day 13 had reduced to 37% of its original volume on day 6.

Ultrasound targeted contrast agents and imaging:-

Over the past decade, the development of bimolecular science has extended traditional morphology and perfusion imaging to functional imaging in the assessment of the presence and extent of diseases at a molecular scale. Ultrasound targeted imaging relies on micro bubble contrast agents conjugated with targeting ligands that specifically bind to the molecular signatures of diseases or physiological systems. Various ligands, typically antibodies and peptides, have been conjugated to the surface of micro bubbles.[87] Site-specific accumulation of image contrast from targeted micro bubbles provides great opportunities to noninvasively visualize physiology or pathology that is difficult to distinguish simply based on conventional morphological information. Preclinical applications have been reported in the assessment of atherosclerosis by the expression of intercellular adhesion molecule-1, and vascular thrombi by the expression of glycoprotein IIb/IIIa receptors.[88-89]The extent of tissue inflammation can also be evaluated after incorporating phosphatidylserine lipids into micro bubbles to attach to leukocytes. Note that the conjugation can be conducted by either noncovalent linkage, such as biotin–avidin interaction, or covalent linkage, such as maleimide thiol tethering. Although the biotin–avidin interaction has been extensively used in preclinical studies, the covalent linkage is generally preferable in clinical settings because it is more stable and has lower immunogenicity.[87]

CONCLUSIONS:

Ultrasonic drug delivery has been limited to in vitro experiments for decades. Promising in vivo results have accumulated in the past ten years and this field is now nearing clinical trials. Ultimately, FUS-enhanced drug delivery is one tool in the armamentarium for optimal treatment of cancer. It may be enough on its own in some cases, but in other more complex cases, a combination therapy approach may be more effective.

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Received on 14.01.2016 Modified on 01.02.2016

Res. J. Pharm. Dosage Form. & Tech. 8(1): Jan.-Mar. 2016; Page 55-65

DOI: 10.5958/0975-4377.2016.00008.2