Use of ultrasound in drug delivery systems: emphasis on experimental methodology and mechanisms

Abstract

Recent studies have shown that ultrasound energy could be applied for targeting or controlling drug release. This new concept of therapeutic ultrasound combined with drugs has induced a great amount of interest in various medical fields. In this paper, several experimental systems are cited in which ultrasound is being utilized to evaluate new application of this modality. The mechanisms of ultrasound-mediated drug delivery are discussed in addition to the review of current advances in the use of ultrasound in systems involving research in cancer therapy, gene therapy, microbubbles and other drug delivery in vitro and in vivo experiments.

• therapeutic ultrasound
• drug delivery

Introduction

Efficient delivery of a drug into target cells or tissues for therapeutic purposes has been a big challenge in medicine. There are numerous methodologies in drug delivery which entail a multitude of factors to be overcome, and involve many aspects in the therapeutic process. Nevertheless, the goal of drug delivery systems is to deploy medications intact to specifically targeted parts of the body by means of either a physiological, chemical trigger or physical energy. Therapeutic processes include drug design to maintain stability, and efficient administration and transport of the drug to the intended target in the body, while attaining the desired concentration of the drug at the particular target tissue [1] and whenever possible locally activating the drug [2–4] to minimize side-effects. These multi-faceted challenges require a multidisciplinary effort by experts in different scientific fields. Several experimental drug delivery systems show exciting signs of promise, one of them is ultrasound irradiation of tissue and cells, which is effective in enhancing drug targeting specificity, lowering systemic drug toxicity, and improving treatment absorption rates. The revival of ultrasound for use in therapy after decades of dormancy [5] has brought attention to the potential applications of ultrasound in various fields [6], [7], especially for cancer and gene therapy. Several systems by which ultrasound are being utilized to improve the overall efficiency of the therapeutic effects of drug are release of drugs from carrier agents such as micro-bubble and nano-bubbles [8], [9] and activation of ultrasound sensitive drugs at a particular lesion in the body [10]. Ultrasound can also be used to facilitate intracellular uptake of a particular drug such as antibiotics [11]. Here we aim to introduce current advances in the use of ultrasound in experimental systems involving drug delivery in vitro and in vivo. In addition, various methodologies and related mechanisms involved in these systems will be explored in the latter half of this review.

Various experimental ultrasound drug delivery systems

Basic in vitro systems

Several in vitro systems have been developed to study the effect of ultrasound in delivering drug to cells and tissues. Frequently used in vitro experimental set ups are shown in Figure 1. The most often used cell lines in these set ups are of cancer origin because they are widely available from cell banks, and excellent conditions are available for most of these cell lines to culture. Although cells in vitro may respond similarly to ultrasound exposures in vivo, it is difficult to completely replicate an in vivo environment in such a way to attain comparable level of stimulus. This difficulty is particularly true with ultrasound. Ultrasound, as a sound wave, behaves differently when travelling in a medium of different physical characteristics. The set-ups in Figure 1A and 1D, where the cell sample is in direct contact with the transducer, provide better targeting but cells in the sample container are likely to be exposed to non-uniform levels of ultrasound intensity [7], [11], [12]. Those cells close to and near the center of the transducer are likely to be exposed to a higher level of acoustic pressure. On the other hand, the sample in Figure 1B has the advantage of cells being exposed to a more uniform acoustic field [13], if the sample is purposely situated within the so called “far field” region. In addition, the use of an ultrasound absorber (UA) at the far side of the sample in Figure 1B (also in Figure 1C) further avoids standing wave formation that may result from any reflected ultrasound. It has been reported that a set up similar to that in Figure 1B has a high reproducibility in vivo [14]. The fluid-air interface in Figure 1A and 1C are likely to create standing waves in these types of set-ups [15]. In all the in vitro set ups presented in Figure 1, monolayer cells (attached cells: e.g. HeLa cells) and loosely suspended cells (e.g. U937 cells) are commonly used. This is in contrast to tumors which are likely to be composed of packed cells surrounded with connective tissues that include blood vessels. Considering all these factors, it would be logical to say that ultrasound is generally more attenuated in vivo where there is a less likelihood of standing wave formation. The general advantages and disadvantages of the different experimental set-ups are shown in Table 1.

Figure 1. In vitro set up for sonication experiments. (A) A dish containing the sample (S) is positioned directly on top of the transducer (T) after applying acoustic gel to avoid air between the transducer and the dish. (B) Ultrasound travels horizontally through degassed distilled water to irradiate the sample positioned at a certain distance. At the far end is an ultrasound absorber (UA) that prevents reflection of ultrasound. (C) The tube containing the sample is positioned a few centimeters from the transducer and could be rotated during sonication for a more uniform exposure of its content. Ultrasound is directed upwards to hit the sample. (D) For small samples (such as those using 24 or 96 well plates), small transducers (e.g. less than 10 mm in diameter) can be dipped directly into the sample for sonication.

Table 1.  Advantages and disadvantages among experimental systems

Experimental system	Advantages	Disadvantages
Basic in vitro (See Figure 1 for sample set-ups)	• Generally easy to design • Economical • Suitable for processing large number of samples • Easy to do assay for drug delivery experiments on cultured cells	• Less reproducible in vivo • Set-ups are usually varied • Acoustic factors may be difficult to control
Ex vivo	• Less expensive than in vivo • In vitro or in vivo-simulated set-ups used for cell culture experiments may be applied in this system	• Some surgical skills may be required • Assays for cells experiments may not be suitable • Drug delivery may be difficult to quantify
In vivo-simulated (See Figure 2 for an example set-up)	• Greater reproducibility in vivo • Less expensive than in vivo • May use cultured cells for drug delivery experiments	• Generally difficult to design • May not be suited for large number of samples
In vivo	• Experimental design and results could be useful to design protocol for eventual clinical trials	• Can be expensive • More time may be needed before getting the end results • Limited options for assays to evaluate drug delivery experiments

As there are many physical, structural, and chemical factors to consider, replicating an in vivo environment in vitro can never be perfect. However, putting efforts in simulating an in vivo conditions in vitro will certainly improve the reproducibility of the results for subsequent in vivo experiments.

In vitro experiments with anticancer agents

The combination of therapeutic ultrasound and other therapeutic modalities (e.g. chemotherapy and radiation therapy) in the treatment of cancer is being investigated. In particular the ability of ultrasound to facilitate cellular uptake of the therapeutic agents [16–18] shows exciting signs of promise. The most common end results observed were enhanced cancer cell killing and enhanced cellular uptake [18].

Previous reports have suggested that the cell membrane structure is the major obstacle for cellular uptake of anticancer agents [19]. Molecular uptake and cell viability are often dependent on acoustic factors such as negative acoustic pressure, exposure time, and the presence of microbubbles. Actual physical and structural changes in cell membranes induced by sonodynamic treatments are of importance focus in investigating the mechanism involved in cancer cell killing. It has been reported that enhanced cell killing was observed when sonicated under a non-lethal hyposmotic environment [20]. Modifying cellular structure before sonication may alter cellular response to the mechanical effects of ultrasound on the cells; conversely, ultrasound itself may alter the cellular integrity of the cell membrane [21], thus affecting its ability to take up extracellular materials. Lidocaine, an anesthetic agent known to increase cell membrane fluidity, was also shown to enhanced cellular uptake of DNA thus modifying sonotransfection [22]. In addition to the physical modification of the cell membrane to attain the desired effect, biomolecular mediators involved in cell membrane repair and eventual cell death must be considered. One important component is the role of calcium ions in cell membrane repair and eventual apoptosis induction. While it has been shown that calcium ions play a vital role in the initiation of cell membrane repair [23], it is also responsible for the eventual cell death if the membrane repair fails. Hutcheson et al. [19] showed that chelating calcium ions significantly prevented ultrasound-induced apoptosis immediately after a membrane-damaging sonication. This finding is particularly important in sonotransfection wherein the success is determined by the survival of sonotransfected cells and for these cells to be able to produce the protein as directed by the therapeutic gene.

In vitro experiments with antibiotics

A study has shown that some antibiotic treatments of Pseudomonas aeruginosa or Escherichia coli coupled with ultrasound enhance the bactericidal activity of these antibiotics [24]. Involving a wide array of antibiotics (especially with aminoglycosides), a more recent study found that similar synergistic effects with ultrasound treatment can be observed in both Gram-positive and Gram-negative bacteria [25], [26]. In another study, it was reported that Staphylococcus spp. in biofilms respond well to vancomycin when combined with ultrasound [27]. This finding might have strong clinical significance considering that it is generally considered that bacteria growing with biofilm [28] phenotypes are highly resistant to traditional antibiotic chemotherapy [29], [30]

In order to evaluate the mechanism of how ultrasound facilitates antimicrobial action, one must understand how conventional antibiotics work. There are at least three steps necessary for an antibiotic to kill a bacterium. First, the antibiotic must be transported to the surface of the bacterium. Second, the antibiotic must be transported through the outer membrane into the cytoplasmic membrane of bacteria. Third, the antibiotic must bind to its biological target in the cell (e.g. ribosome for gentamicin) and interfere with a pathogenic characteristic of the bacteria. During this process, the most important step is that the antibiotics are taken into the bacteria by passive transport through the small pores (porins) in the outer membrane of the bacteria or energy-dependent active transport through the cytoplasmic membrane. However, there is low permeability of the membrane to some antibiotics such as gentamicin [30]. Many bacterial species show a significant relationship between the outer membrane permeability to antibiotics and the minimum inhibitory concentration (MIC) of antibiotics. In this regard, ultrasound may provide an additional mechanism for an increased intracellular uptake of the antibiotics through a process called sonopermeabilization. In the case of mammalian cells, the direct mechanism of augmentation of drug action by ultrasound exposure could be that acoustic cavitation is related to the cell killing [31], [32]. This cavitation could chemically activate antibiotics that are specifically bound to the cell, and this could result in enhanced cytotoxic effects.

Although increased uptake of the antimicrobial agents is regarded as a major mechanism in the enhanced effects, several other factors such as free radical formation induced by ultrasound may also play a role [33]. Another possible mechanism is that the low-intensity ultrasound may affect the bacteria at the ribosomal level. Some antibiotics act on the bacteria by binding to specific sites on the bacterial ribosome and interrupting protein transcription [25]. Ultrasound might destabilize the binding of mRNA or the growing peptide chain to the respective grooves in the ribosome, or it might prevent the attachment of amino acids to the growing peptide chain because of physical disruption. Taken together, ultrasound destabilizes the ribosome in such a way that protein synthesis is hampered entirely. These events coupled with an increased concentration of antibiotics through sonopermeabilization could explain for the augmented killing of bacteria with some antibiotics.

Disruption of the bacterial membrane could also be directly caused by cavitation and high shearing stress generated by ultrasound [34], [35]. In these cases, the resulting small pores could be due to highly transient biochemical instability within the cell membrane and thus they will likely close quickly [20], [23], [36]. This may happen in instances wherein ultrasound alone is not lethal to the bacteria. Because antibiotic treatments alone have limited effects against intracellular bacteria, e.g., Chlamydia, Salmonella, Yersinia, and Shigella spp., ultrasound with microbubbles, echo-contrast agents, or any agent that facilitates formation of acoustic cavitation could be of benefit in the treatment of intracellular bacteria. A more recent study has shown such promise against Chlamydia [11]. It is therefore expected that continued investigations of the effects of ultrasound and antibiotic therapy against resistant pathogens will continue to provide interesting results.

In vivo experiments with thrombolytic agents

There are several therapeutic uses of ultrasound that are currently under clinical investigation. One of these is the successful in vivo [37] testing of ultrasound and thrombolytic drugs which have also lead to actual clinical use. It is the nature of this type of therapy that made in vitro [38] experimental results more reproducible in vivo and eventually clinically. An artificially made clot in a test tube in vitro may not differ physically from the one artificially induced in vivo. Apparently, the ultrasound enhanced effect of thrombolysis in either in vitro or in vivo settings proved to be similar to the actual clinical settings [39].

In vivo-simulated in vitro systems

The design of in vivo-simulated in vitro set-ups for therapeutic ultrasound experiments has proven to have value in predicting in vivo outcome, before conducting the actual animal experiments are carried out, since the latter are more cumbersome and more costly. This is also in line with the ethical considerations to use fewer animals. We recently published our work that used an in vivo-simulated in vitro set up (Figure 2) in gene delivery. The results of the in vivo experiments showed high reproducibility, such as showing successful ultrasound-mediated gene transfection (sonotransfection) in tumor cells, apoptosis induction, and tumor growth inhibition of the same malignant melanoma cell lines used in vitro [40]. Although, in this work, direct comparison of the actual cellular gene uptake in vitro and in vivo was not determined, the biological or therapeutic outcomes such as gene transfection, apoptosis induction and overall growth inhibition were comparable in both in vitro and in vivo.

Figure 2. In vivo-simulated set up. This is a novel in vitro set-up that simulates in vivo conditions. Close chamber containing cancer cells is sandwiched in between transmitting oil simulating soft tissue in the body and ultrasound absorber on the opposite side to minimize reflection of ultrasound waves.

Ex vivo systems

In some cases, in vivo-simulated set ups might be difficult to design, such that using an actual in vivo target for exposure outside of the body (e.g. removing the liver before ultrasound irradiation) might be a practical option. This is called an ex vivo set up. Unlike in vivo where active blood circulation is present; ex vivo tissues may still have a good predictability for in vivo outcome especially when freshly collected tissue is used in the experiments. In some cases, for drug delivery with ultrasound, a circulating and temperature regulating device might be added into the system to simulate the real in vivo environment [41].

In vivo systems

In vivo experimentation is a step closer to clinical application. When designing in vivo experimental set ups, eventual actual clinical application should be considered. Several in vivo set ups have been adopted for drug delivery with ultrasound. For either localized targeting or generalized ultrasound exposure, animal survival should be an important gauge for the overall success in any in vivo therapeutic experiment. The most commonly applied in vivo system for therapeutic ultrasound is direct targeting of a tumor after intratumoral injection of the drug or other agents, for example therapeutic DNA. For highly vascular tumors, intravenous administration of the drug is performed prior to direct tumor sonication. To be able to properly target and monitor the tumor during this type of treatment, real time imaging by ultrasound or MRI is commonly used. While few studies have attempted whole body exposure of animal models for sonication, a successful therapeutic outcome has been limited. Although such a model is ideal for the treatment of metastatic cancer, lethal damage by ultrasound itself can become an important issue and overall effectiveness still remains as a big challenge for investigation.

As to chemotherapeutic agents against tumors, even if the agent is effective against this particular cancer in vivo model, optimal quantities of the agent must also reach all cells within the targeted tumor [42], [43]. It is here where strategies are developed to increase drug uptake by tumor cells using ultrasound [44]. This is in addition to other strategies by which ultrasound is being applied for therapy such as thermal ablation, hyperthermia, and interaction with drug carriers and vasculature [45].

Mechanisms involving physics and chemistry

The mechanisms by which ultrasound augments the activity of drugs and other agents remain poorly understood, although several explanations have been proposed. In vitro, (a) increased cellular membrane permeabilization of drugs, also called sonoporation, (b) increased sensitivity of the cells to the agent, (c) potentiation of the agent, (d) partial damage caused by one of the modalities alone was rendered irreversible when the two are combined, and (e) the thermal effect, were cited as possible mechanisms. All these in vitro effects could probably be present to varying extents in vivo, as well. Furthermore, in vivo, vascular effects and immune response [46] of the animal model may also uniquely produce interactions between ultrasound and anticancer agent, resulting in enhanced effects.

While sonoporation is a widely accepted mechanism in the enhanced effect of drug against the target cells, there is also evidences showing that other mechanisms may contribute to the overall enhancement such as thermal, sonochemical and biomolecular effects [20], [22], [47].

Mechanical: cavitational and non-cavitational

The mechanism by which drugs or therapeutic genes [12] are taken up by cells during sonication generally still remains clear. However, several findings point to the mechanical effects of cavitation as the likely mechanism involved. Acoustic cavitation, or formation, oscillation and collapse of bubbles due to acoustic waves, create mechanical turbulence, and in particular the implosion of a bubble during the so called “inertial cavitation” will produce microjets of fluid that can potentially carry drug with it, directly into cells [44] (Figure 3). Non-collapsing but oscillating bubbles close to a cell may create disturbance in the cell membrane, allowing increased inflow of extracellular agents. This concept has been cited in previous publications [41], [47], while more recently the direct visualization of oscillating (but not collapsing) bubble and the resulting uptake of propidium iodide (PI) has been attempted [48]. Even the use of ultrasound at diagnostic power levels has been found to promote trans-membrane lipid delivery to cancer cells [49].

Figure 3. Targeted drug delivery by ultrasound with microbubbles. Microbubbles can be engineered to carry therapeutic agents and at the same time carry with them ligands that specifically latches on to particular cells (e.g. cancer cells belonging to a cancer cell line). Conceptually, these engineered microbubbles will be injected intravenously into a cancer patient, allowing time for the microbubble to localize on the cancer cells before sonication. Sonication will release and deliver the therapeutic agent into the target cells. Ultrasound in this case should reach an acoustic pressure sufficient to destroy the bubbles, but may generate mechanical and thermal effects in the surrounding tissue without or with fewer microbubbles and less drug. The expected effect would be greater on the target cells and tissues, with minimal side effects.

Thermal

A rise in temperature is known to increase transdermal absorption [50], cellular uptake [51], drug release from a carrier [52], [53], or activation of certain drugs [54]. In this regard ultrasound is being utilized to increase tissue temperature in facilitating the release of drugs locally [55], [56], thus enhancing the therapeutic efficacy of the drug. For example, a study showed that a greater than 7 fold increase in concentration of anti-cancer doxorubicin was observed in a tumor heated (about 40^oC) by high-intensity focused ultrasound as compared to unsonicated tumors, when rabbits were administered with clinical-grade doxorubicin encapsulated low temperature sensitive liposomes prior to sonication [57].

While delivering energy, ultrasound irradiation can be administered extracorporeally. This energy can then be transformed into heat in a deep target. This method is proven to be easier and potentially safer to apply in actual therapy.

Sonochemical and biomolecular

The concept on the sonochemical effects of ultrasound [58] was first introduced when production of active oxygen species [59] was observed during sonication in a fluid that allows inertial cavitation to take place. Several free radicals have been implicated in chemical reactions following sonication. Electro-paramagnetic resonance (EPR) analysis has been done to identify and quantify the amount of radicals being produced by a sonication system [13]. However, it could not be clearly established whether radical production has direct bearing in the delivery of drugs into cells.

Recent studies have demonstrated potent toxicity of nanoparticle titanium dioxide (TiO₂) [60], [61], when combined with ultrasound irradiation against melanoma C32 cells in vitro, and also growth inhibition of tumors in a mouse xenograft model [7]. These findings suggested that TiO₂ was directly activated under the irradiation of ultrasound. Activation of TiO₂ is known to be associated with reduction and oxidative activities resulting in the formation of reactive oxygen species [62].

Several findings have shown that free radicals may participate in the chemical changes either of the drug or the cell membrane resulting into an enhanced drug uptake. On the other hand, a similar reaction can be counterproductive as the chemical reaction may inhibit drug uptake instead. Generally, despite the cited role of radicals in enhanced drug uptake, this can be considered a minor mechanism and certainly not a consistent contributing factor.

Future directions

Experimental set ups are likely to become more and more sophisticated in the future, particularly the design of an in vivo-simulated set ups. Together with technological advances of applying, guiding and monitoring ultrasound, a variety of ultrasound therapeutic applications will continue to be unraveled. Of particular interest is the use of thermally responsive polymers [53] to deliver drug to a particular target in the body (e.g. a tumor) and the use of ultrasound to induce hyperthermia that would locally release the drug from the polymers. These complimentary methods of targeted drug delivery and localized release by ultrasound-induced hyperthermia would certainly revolutionize drug delivery in the future.

In general, the increased level of understanding of ultrasound and of how it interacts with biomolecular materials will likely lead to more exciting discoveries in ultrasound-mediated delivery of drugs for the treatment of cancer and other diseases.

Declaration of interest: The authors report no declarations of interest.