Microbubble-Mediated Delivery for Cancer Therapy

Despite an overall improvement in survival rates for cancer, certain resistant forms of the disease still impose a significant burden on patients and healthcare systems. Standard chemotherapy in these cases is often ineffective and/or gives rise to severe side effects. Targeted delivery of chemotherapeutics could improve both tumour response and patient experience. Hence, there is an urgent need to develop effective methods for this. Ultrasound is an established technique in both diagnosis and therapy. Its use in conjunction with microbubbles is being actively researched for the targeted delivery of small-molecule drugs. In this review, we cover the methods by which ultrasound and microbubbles can be used to overcome tumour barriers to cancer therapy.


Introduction
Over the last century, many treatments have been developed to combat cancer.In combination with better understanding of the risk factors and diagnostic techniques, these have led to a fall in mortality.Unfortunately, late presentation and metastasis still confound many established techniques, and cancer is currently the second most common cause of mortality in the world [1].The heterogeneity and complex microenvironment of solid tumours can make them highly resistant to treatment [2,3].Many of the currently available drugs could be effective at a sufficient dose, but the lack of effective targeting means the severity of the side effects would be intolerable for the patient.Thus, there is a pressing need to develop methods to target drugs and so reduce the dose required whilst maintaining a therapeutic level in the tumour.Ultrasound is one such modality that has been demonstrated to improve the accumulation of drugs in target areas, and is itself a well-established technique with well-understood safety and usage.This review will introduce the mechanisms by which ultrasound, and in particular the combination of ultrasound and microbubbles, can mediate delivery.

Solid Tumour Pathology
Solid tumours develop as a result of dysregulation in cellular proliferation.Progressive accumulation of genetic mutations is necessary for solid tumour development, leading to the growth of a mass of cells in a discrete location within an organ or tissue [4,5].As the cells towards the centre of the tumour become further distanced from blood vessels, they release stress signals which trigger angiogenesis from existing blood vessels to supply the tumour with oxygen and nutrients [6].The tumour then continues to grow, eventually spreading locally out of the original location or metastasising to distant sites [7].
The challenge that the solid tumour microenvironment presents to drug delivery is a topic that has been well discussed in several reviews [8][9][10].The primary issues are the poor vasculature and lack of lymphatic drainage.In particular, blood vessels in tumours are severely abnormal, having variable diameters and irregular or stagnant flow, and are prone to collapse and highly leaky (please Fluids 2018, 3, 74; doi:10.3390/fluids3040074www.mdpi.com/journal/fluidssee the recent review on tumour vasculature by Dudley et al. [11]).Without lymphatic drainage, waste materials and fluid leaking from the blood vessels remain in the tumour, resulting in a high local interstitial pressure.This is further compounded by the continued proliferation of cancer cells in a limited space, creating a highly dense mass of cells with a poorly defined extracellular matrix.The limited ability of the blood supply to carry therapeutics into the tumour, combined with the unfavourable convection gradient [12,13], results in delivery being highly heterogeneous and limited largely to the periphery of the tumour.In addition, the poor supply of nutrients to tumours changes cell activity, making them highly resistant to the effects of multiple forms of therapy.For instance, hypoxia-a state of low oxygen tension-is a common issue in large tumours (see Figure 1) which severely impacts radiotherapy [2] and chemotherapeutics [14,15].
Fluids 2018, 3, x FOR PEER REVIEW 2 of 11 variable diameters and irregular or stagnant flow, and are prone to collapse and highly leaky (please see the recent review on tumour vasculature by Dudley et al. [11]).Without lymphatic drainage, waste materials and fluid leaking from the blood vessels remain in the tumour, resulting in a high local interstitial pressure.This is further compounded by the continued proliferation of cancer cells in a limited space, creating a highly dense mass of cells with a poorly defined extracellular matrix.The limited ability of the blood supply to carry therapeutics into the tumour, combined with the unfavourable convection gradient [12,13], results in delivery being highly heterogeneous and limited largely to the periphery of the tumour.In addition, the poor supply of nutrients to tumours changes cell activity, making them highly resistant to the effects of multiple forms of therapy.For instance, hypoxia-a state of low oxygen tension-is a common issue in large tumours (see Figure 1) which severely impacts radiotherapy [2] and chemotherapeutics [14,15].

Ultrasound
The propagation and detection of high-frequency (>20 kHz) acoustic waves, i.e., ultrasound, in tissues is a long-established technique in diagnostic imaging [16].Ultrasound waves are reflected as a result of differences in acoustic impedance between different tissue types, and the received echoes can be processed to create two-or three-dimensional images of structures within the body [17].Depending upon the equipment used, images with millimetre spatial resolution at centimetre depths can be obtained, at frame rates capable of capturing tissue and fluid motion [18].
The attenuation of the acoustic energy in tissue can cause tissue motion, fluid streaming, and heating.For therapeutic applications, heating can be used for thermal ablation [19][20][21] or promotion of healing in physiotherapy [22,23]; whilst fluid streaming has been linked to transdermal sonophoresis [24] (see [25,26] for recent reviews).A further effect of the acoustic wave may be the production of cavitation, where a rapid fall in local pressure can cause the vaporisation and/or evolution of dissolved gases.As the acoustic pressure increases, the inertia of the surrounding fluid can cause the formed bubbles to violently collapse, and this process can cause significant damage to surrounding tissue or structures.Lithotripsy relies on this technique to fracture gallstones or kidney stones [27], although care must be taken to avoid cavitation in nontarget areas.This cavitation effect is also utilised for the sterilisation of equipment or liquids in sonication baths.At lower amplitudes, ultrasound may be used in conjunction with pre-existing bubbles or other cavitation nuclei to

Ultrasound
The propagation and detection of high-frequency (>20 kHz) acoustic waves, i.e., ultrasound, in tissues is a long-established technique in diagnostic imaging [16].Ultrasound waves are reflected as a result of differences in acoustic impedance between different tissue types, and the received echoes can be processed to create two-or three-dimensional images of structures within the body [17].Depending upon the equipment used, images with millimetre spatial resolution at centimetre depths can be obtained, at frame rates capable of capturing tissue and fluid motion [18].
The attenuation of the acoustic energy in tissue can cause tissue motion, fluid streaming, and heating.For therapeutic applications, heating can be used for thermal ablation [19][20][21] or promotion of healing in physiotherapy [22,23]; whilst fluid streaming has been linked to transdermal sonophoresis [24] (see [25,26] for recent reviews).A further effect of the acoustic wave may be the production of cavitation, where a rapid fall in local pressure can cause the vaporisation and/or evolution of dissolved gases.As the acoustic pressure increases, the inertia of the surrounding fluid can cause the formed bubbles to violently collapse, and this process can cause significant damage to surrounding tissue or structures.Lithotripsy relies on this technique to fracture gallstones or kidney stones [27], although care must be taken to avoid cavitation in nontarget areas.This cavitation effect is also utilised for the sterilisation of equipment or liquids in sonication baths.At lower amplitudes, ultrasound may be used in conjunction with pre-existing bubbles or other cavitation nuclei to produce a range of mechanical effects that can be exploited for drug delivery.These are schematically shown in Figure 2 and discussed in more detail below.produce a range of mechanical effects that can be exploited for drug delivery.These are schematically shown in Figure 2 and discussed in more detail below.

Thermal Delivery
As ultrasound propagates through tissue, energy is absorbed, causing localised heating.The use of heat as a treatment tool, thermotherapy, is a well-established field.In particular, for tumour therapy, thermal ablation of diseased tissue is a clinically accepted form of treatment for solid tumours [29].High-intensity focused ultrasound (HIFU) devices are designed to use tightly focused ultrasonic waves to thermally ablate tissue [30], and are typically combined with magnetic resonance imaging (MRI) for targeting and treatment monitoring.This combination has been highly effective for treating prostate cancers and uterine fibroids and is being trialled in a number of other cancers [30,31].The tightly focused ultrasound enables lethal hyperthermia in the focal zone, whilst minimising destruction of surrounding healthy tissue.However, the focal zone is typically much smaller than the target tumour and treatment requires translation of the focus throughout the tumour, resulting in long treatment times.As the technique is noninvasive, discerning the boundary between tumour and healthy tissues can also be challenging [32].
Besides ablation, HIFU can be used to cause mild hyperthermia of tumours.Hyperthermia has a number of effects on tumours which can improve the effect and delivery of systemically delivered

Thermal Delivery
As ultrasound propagates through tissue, energy is absorbed, causing localised heating.The use of heat as a treatment tool, thermotherapy, is a well-established field.In particular, for tumour therapy, thermal ablation of diseased tissue is a clinically accepted form of treatment for solid tumours [29].High-intensity focused ultrasound (HIFU) devices are designed to use tightly focused ultrasonic waves to thermally ablate tissue [30], and are typically combined with magnetic resonance imaging (MRI) for targeting and treatment monitoring.This combination has been highly effective for treating prostate cancers and uterine fibroids and is being trialled in a number of other cancers [30,31].The tightly focused ultrasound enables lethal hyperthermia in the focal zone, whilst minimising destruction of surrounding healthy tissue.However, the focal zone is typically much smaller than the target tumour and treatment requires translation of the focus throughout the tumour, resulting in long treatment times.As the technique is noninvasive, discerning the boundary between tumour and healthy tissues can also be challenging [32].
Besides ablation, HIFU can be used to cause mild hyperthermia of tumours.Hyperthermia has a number of effects on tumours which can improve the effect and delivery of systemically delivered drugs.Increased temperatures lead to increased permeability of blood vessels and cell membranes in tumours [44,45] and increased diffusivity of drug molecules [46], ensuring greater penetration and uptake [47,48].Additionally, drug delivery and effect may be improved as increased tumour temperatures reduce interstitial pressures and hypoxia [49,50].A further factor is the immune system, which has a strong influence on tumour progression.Mild hyperthermia of tumours has been shown to improve immune cell infiltration [51], and the application of thermal energy is under consideration for enhancing both immune responses to traditional therapy and specific immune therapies (recently reviewed in [52]).
One popular use of mild hyperthermia by HIFU is the release of drugs from loaded nanoparticles.Numerous nanoparticles have been designed to take advantage of locally applied heat to a tumour location, and the topic has been reviewed extensively elsewhere [53][54][55].As an alternative to drug delivery, acoustically responsive nanoparticles have also been used to enhance the thermal ablation effect to reduce the intensities and durations required for standard ablation.Nanoscale phase change agents encased in block copolymers have been used to increase damage in a tumour during HIFU [56,57].Nanoparticles can passively localised in tumour regions by the enhanced permeability and retention effect or be purposefully trapped by the use of vascular obstructive agents, such as Lipodol.Subsequent vaporisation of the particles to generation micrometre-sized gas bubbles results in mechanical damage to the vasculature and blood supply, which could be further amplified by cavitation of the formed gas bubbles.Additionally, both iron and gold nanoparticles loaded on to polymer-based agents have been shown to enhance HIFU ablation damage [58,59].

Mechanical Delivery
Besides heat, ultrasound can also transfer momentum to the tumour due to the pressure gradient formed along the acoustic path.In fluids, this results in acoustic streaming [60], which has been postulated as a possible mechanism for delivery [26,61,62].The effect of momentum transfer on solid tissues, i.e., acoustic radiation force (ARF), generates additional bioeffects of potential use in tumour delivery.It has been demonstrated that the motion induced by ARF can cause the disruption of tight junctions in endothelial tissues [63][64][65][66][67], resulting in increased uptake.Additionally, the macroscale tissue motion acts in a similar manner to physical massage, increasing permeability in deeply situated tissue [68][69][70].The sonophoresis effect of ARF is also commonly utilised in transdermal drug delivery applications [71,72].ARF has been shown to improve the tumoral uptake and effect of small-drug molecules [33,[73][74][75][76].However, as ARF is dependent upon energy absorption, it is best achieved with high intensities and/or frequencies.As such, it is necessary to consider the required treatment depth and pulse design to avoid excessive heating and tissue damage.
As indicated above, cavitation nucleating agents are also commonly used in drug delivery applications.Typically systemically administered, these reduce the intensity required for cavitation and other mechanical effects.Whilst many molecules and objects can act as ultrasound cavitation nuclei, the best studied and most clinically relevant types are microbubbles [77][78][79][80][81][82][83].As microbubbles are highly echogenic, concurrent imaging can also be performed, allowing better target site recognition [84][85][86][87].
Typically, microbubbles consist of a high-molecular-weight gas core on the order of 1 to 10 µm in diameter, encapsulated in a lipid, protein or, less commonly, a polymer shell [88].In diagnostic imaging, their high compressibility and resonant behaviour causes significant linear and nonlinear scattering of incident acoustic waves [89], facilitating a number of imaging techniques [90][91][92].It should be noted that microbubbles are vascular, or "blood pool", agents only.More recently, ultrasound-responsive nanodroplets of liquefied perfluorocarbons (PFCs) for drug delivery have shown an ability to extravasate in leaky tumours [93][94][95].In addition, solid nanoparticles with entrained gas pockets are being used for similar purposes [96,97].
Fluids 2018, 3, x FOR PEER REVIEW 5 of 11 potential severity of blood vessel disruption [112] is undesirable in certain locations in the body, for instance, the brain [113,114] or kidneys [115].In these areas, stable oscillation generates sufficient shear stress to disrupt cell membranes [102] and increase vascular permeability [116]; particularly in the highly selective blood brain barrier [117] (see Figure 3), recently reviewed in [118,119].Endocytotic pathways are also known to be activated by the acoustically driven shear stresses, and these are also implicated in delivery [120][121][122][123].
In addition to coadministration, cavitation agents can be modified to load the drug of interest [124][125][126][127] or nanoparticle forms of the therapeutic (extensively reviewed in the literature [128][129][130]).In general, as permeability is only temporarily altered, colocalising the drug or nanocarrier to the site of microbubble activity promotes the delivery [131], effect [132], and release [133,134].Finally, appropriate characterisation of the cavitation agent and drug loading is of vital importance to any drug delivery strategy (recently reviewed in [135]).

Conclusions
In the last 30 years, cancer survival rates have increased due to the better understanding of environmental risk factors, better diagnostic techniques, and improved therapies.However, drugresistant cancers and toxic side-effects still represent significant barriers to successful treatment.Techniques to better localise existing chemotherapy drugs could vastly improve outcomes in these difficult cases.The combination of ultrasound and microbubbles has considerable potential as such a technique.In addition to coadministration, cavitation agents can be modified to load the drug of interest [124][125][126][127] or nanoparticle forms of the therapeutic (extensively reviewed in the literature [128][129][130]).In general, as permeability is only temporarily altered, colocalising the drug or nanocarrier to the site of microbubble activity promotes the delivery [131], effect [132], and release [133,134].Finally, appropriate characterisation of the cavitation agent and drug loading is of vital importance to any drug delivery strategy (recently reviewed in [135]).

Conclusions
In the last 30 years, cancer survival rates have increased due to the better understanding of environmental risk factors, better diagnostic techniques, and improved therapies.However, drug-resistant cancers and toxic side-effects still represent significant barriers to successful treatment.Techniques to better localise existing chemotherapy drugs could vastly improve outcomes in these difficult cases.The combination of ultrasound and microbubbles has considerable potential as such a technique.

Figure 1 .
Figure 1.Schematic of the solid tumour environment.As distance from a blood vessel increases, the supply of oxygen, nutrients, and drug concentration becomes limited, leading to hypoxic, starved, and drug-resistant populations of tumour cells (ECM-extracellular matrix).Reprinted by permission from Springer Nature, Nature Reviews Cancer [10].

Figure 1 .
Figure 1.Schematic of the solid tumour environment.As distance from a blood vessel increases, the supply of oxygen, nutrients, and drug concentration becomes limited, leading to hypoxic, starved, and drug-resistant populations of tumour cells (ECM-extracellular matrix).Reprinted by permission from Springer Nature, Nature Reviews Cancer [10].

Figure 2 .
Figure 2. Illustration of mechanisms involved in ultrasound-mediated delivery.These can be divided into the mechanical effects of acoustic cavitation and acoustic radiation force, and thermal effects.Reprinted by permission from John Wiley and Sons, Journal of Labelled Compounds and Radionuclides [28].

Figure 2 .
Figure 2. Illustration of mechanisms involved in ultrasound-mediated delivery.These can be divided into the mechanical effects of acoustic cavitation and acoustic radiation force, and thermal effects.Reprinted by permission from John Wiley and Sons, Journal of Labelled Compounds and Radionuclides [28].

Figure 3 .
Figure 3.Opening the blood brain barrier (BBB) by focused ultrasound (FUS) and microbubbles in mice.The (a) setup utilises a coregistered magnetic resonance imaging (MRI) to identify targets in the brain.The targets in the mouse hippocampus are (b) marked on the image as red dots and FUS is applied after an injection of Definity® microbubbles.After treatment, a gadolinium-based MRI contrast agent is injected.Typically unable to cross the blood brain barrier, after treatment (c), an enhancement of contrast is seen at the target sites in the mouse hippocampus.Reprinted with permission from the Radiological Society of North America, Radiology[117].

Figure 3 .
Figure 3.Opening the blood brain barrier (BBB) by focused ultrasound (FUS) and microbubbles in mice.The (a) setup utilises a coregistered magnetic resonance imaging (MRI) to identify targets in the brain.The targets in the mouse hippocampus are (b) marked on the image as red dots and FUS is applied after an injection of Definity® microbubbles.After treatment, a gadolinium-based MRI contrast agent is injected.Typically unable to cross the blood brain barrier, after treatment (c), an enhancement of contrast is seen at the target sites in the mouse hippocampus.Reprinted with permission from the Radiological Society of North America, Radiology[117].