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Microbubbles can temporarily open many biological barriers for polar molecules, macromolecules, and particles. Scientists have brought well-known contrast agents back to the laboratory and redesigned them as drug carriers. This article is part of a special Drug Delivery issue.
This article is part of a special issue on Drug Delivery.
In the course of three generations, ultrasound contrast agents have developed from simple air bubbles without shells to precise imaging devices. Their half-life in the bloodstream has grown from several seconds to longer than 15 minutes. Along with this great improvement, microbubbles have become able to reach not only the heart, but also organs more distant from the injection site such as the brain, the kidneys, and the liver. These advances have inspired the industry to consider using ultrasound contrast agents as targeted drug carriers (1–2).
Ultrasound drug targeting entails an "aim-and-shoot" conception. That is, the target site can be visualized by diagnostic ultrasound imaging before an operator triggers drug administration by increasing the intensity of ultrasound waves, thus destroying the microbubbles and releasing the drug. The cavitation energy released by bursting the microbubbles can render biological barriers such as cell membranes, the capillary endothelium, and the blood-brain barrier temporarily permeable for large and polar drug molecules (3–4). Some of the most promising therapeutic research on microbubbles today involves targeting chemotherapeutics to solid tumors and nucleic acids to cells for gene therapy.
One of the major challenges for the development of drug-loaded microbubbles has been to attain high drug-loading amounts. In response to this challenge, scientists have developed new structural designs of ultrasound contrast agents that offer enhanced loading potential.
Structural design of ultrasound-triggered drug carriers
Ultrasound-triggered drug-carrier particles of several distinct structures can achieve targeted drug delivery. The particles' common feature is a great difference between the propagation velocity of ultrasound within the particles and within the surrounding medium.
In general, such great structural diversity of ultrasound-active drug carriers (UAC) exists that the most popular terms in the literature (i.e., "microbubbles" and "ultrasound contrast agents") do not adequately describe them anymore. By way of example, many acoustically active constructs such as phase-shift nanoemulsions do not share the structure of a microbubble. Yet only a few of these constructs can scatter ultrasound, a characteristic that is required of contrast agents.
So far, no clear nomenclature has emerged that distinguishes ultrasound active particles' application as drug carriers versus contrast agents and takes their structure into account as well. UACs may be divided into six categories, based on the the particles' formulation design (see Table I).
Table I: Classification of ultrasound-active drug carriers according to their formulation design.
In the recent past, UACs' structural designs have evolved to incorporate therapeutic molecules with a broad array of physicochemical properties (see Figure 1). Polar drug molecules, such as small interfering RNA, may be connected to the microbubble shell through electrostatic interactions or hydrogen bonds (see Figure 1a). Amphiphilic molecules may intercalate between shell phospholipids (see Figure 1b), and oil-soluble drugs may be included in an oil layer (see Figure 1c).
Figure 1: Structural design of ultrasound active carriers (UACs) and methods of drug incorporation (in red). The drug can be attached to the outer surface (e.g., by electrostatic interactions or hydrogen bonds) (a), an amphiphilic drug may intercalate between the monolayer phospholipids (b), a lipophilic drug may be incorporated into an oil layer (c), secondary-carrier associated microbubbles may carry the drug (d), a drug may be physically encapsulated into a polymeric shell (e), a protein may be surface loaded onto microcapsules (f), drug may be loaded onto the entire shell volume of protein microcapsules (g), and protein microcapsules may be loaded layer by layer (h). Adapted from Ref. 33. (IMAGE IS COURTESY OF THE AUTHOR)
Drugs and nucleotides may be complexed into nanoparticles (i.e., secondary carriers) that are assembled in their turn onto the microbubble shell (see Figure 1d). The drug also may be encapsulated into a polymeric shell and covered by a layer of biocompatible protein, as is Point Biomedical's (San Carlos, CA) CardioSphere (see Figure 1e). Microcapsules with protein shells may be loaded by surface adsorption, by incorporating the drug into the entire shell, or by the layer-by-layer approach (5).
Microbubbles are appropriate carriers for several drugs. Scientists have loaded the particles with large molecules such as oligonucleutides, plasmids, and proteins, as well as with small molecules such as doxorubicin, docetaxel, and dexamethasone (6–10). Research also has focused on delivering formulations including paclitaxel and resveratrol using acoustically active lipospheres. In addition, secondary-carrier-associated microbubbles have been introduced for the delivery of plasmid DNA and doxorubicin (11–15).
How microbubbles work
In many experimental applications, microbubbles have proven to be suitable for drug targeting. The recent generation of microbubbles can be administered into the vein by infusion or by a bolus injection, and they possess a considerable half life of more than 15 min. (see Figure 2). During administration, diagnostic ultrasound is externally applied to the site of disease as during a common imaging procedure. Microbubbles scatter the ultrasound back and serve as contrast agents, thus allowing the clinician to find the target precisely and focus the ultrasound beam. Furthermore, the intensity of the scattered signal provides information about the amount of microbubbles that reside at the target site.
Figure 2: The mechanism of action of ultrasound-active carriers (UACs). After being applied to the patient, UACs can be excited on the target site using low-intensity ultrasound (black solid lines) and visualized through the backscattered signal (gray dashed lines). Clinicians burst the UACs by increasing the ultrasound intensity (red solid lines). The cavitation energy perforates the capillaries and increases cell permeability for the released drug (red dots). Adapted from Ref. 33. (IMAGE IS COURTESY OF THE AUTHOR)
The clinician then applies a pulse sequence of ultrasound with a higher intensity to excite the microbubbles beyond their cavitation threshold. The drug carriers burst into fragments of drug-bearing shell debris and small gas bubbles, which dissolve and are exhaled through the lungs.
The applied acoustic energy of ultrasound waves is transformed into cavitation energy and causes hydrodynamic phenomena such as microjetting, microswirling, and shock waves. These cavitation forces can cause the sonoporation of biological barriers by opening transient wall gaps and making cells and tissues permeable to the drug. After treatment is complete, the cells and vessels recover within seconds or minutes, and therapeutic effects begin.
The size range of drug molecules or drug-loaded particles that can be administered by sonoporation is limited. Capillaries and the blood-brain barrier can be opened for particles as large as several hundred nanometers (e.g., liposomes and polyplexes) (16).
The permeability of cell membranes, however, is restricted to particles of 2–3 MDa in mass (e.g., micelles, proteins, and plasmids) (17). Secondary-carrier-associated microbubbles provide one probable way to circumvent this restriction (see Figure 1d). These hybrid constructs combine site targeting, the ability of UACs to open capillaries, and the capacity of nanoparticles to enter cells by phagocytosis. The microbubbles carry the nanoparticles to the target site and open the capillary wall. The released nanoparticles penetrate the tissue and are taken up by the cells through phagocytosis, thus acting as secondary carriers for the drug (see Figure 3). Within the cell, the nanoparticles break the endosome defense mechanisms (e.g., by osmotic rupture or the proton-sponge effect) and release the plasmid into the nucleus.
Figure 3: Plasmid DNA uptake and release after capillary break and extravasation into the tissue. The secondary carriers (e.g., polyplexes) are attached to the microbubble wall (bottom left corner). After ultrasound-mediated microbubble destruction the polyplexes leave the vessel and penetrate the tissue. They are taken up by cells and escape enzymatic digestion. The therapeutic plasmid is then released to the nucleus and expressed. Adapted from Ref. 33. (IMAGE IS COURTESY OF THE AUTHOR)
Sonoporation allows the efficient targeting of delicate organs such as the brain and the heart. Unlike liposomes, ultrasound-mediated targeting is independent from the availability of capillary fenestration or specific cell ligands, which are necessary for targeting by enhanced permeability and retention or for affinity targeting.
Current applications of ultrasound drug targeting
Ultrasound-active drug carriers have been applied in numerous fields of medical research. To date, the areas of greatest interest appear to be gene delivery and tumor therapy.
Microbubbles provide a method to focus the action of chemotherapeutic drugs onto the tumor without requiring an invasive intervention. Anthracyclines are perhaps the mostly widely endorsed candidates for targeted chemotherapy. They have been included in formulations such as poly(lactic acid) microcapsules, soft-shelled phospholipid microbubbles, secondary-carrier-associated microbubbles, and as phase-shift nanoemulsions in microbubbles (8, 9, 14–15, 18–19). Delivery of taxanes such as paclitaxel and docetaxel has also been investigated (9, 11). Microbubbles also have been used to overcome the blood-brain barrier with promising results (16, 20–21). Before studies on large animals can begin, however, appropriate medical ultrasound devices must be developed.
Some gene therapy applications use mild diagnostic ultrasound to treat sensitive tissues such as the myocardium. Ultrasound-targeted gene therapy mostly focuses on promoting myocardium revascularization after ischemic stroke, or on antisense therapies of vascular hyperplasia (22, 23–24). Studies indicate that microbubbles are likely to become an effective tool for preventing or treating ischemic stroke. Furthermore, microbubbles have successfully targeted and destroyed blood thrombi, thus helping to restore the perfusion of the affected myocardium (25).
Future directions for ultrasound drug targeting
The wide and established application of ultrasound contrast agents has provided a solid foundation for research into ultrasound drug targeting. Since the US Food and Drug Administration published safety recommendations for the use of diagnostic ultrasound in 1976, a considerable amount of data has been collected and evaluated. Researchers now must bridge the gap between diagnostic and therapeutic applications.
More than 20 years of clinical experience with contrast imaging has helped scientists map the body organs most accessible to ultrasound. These organs represent suitable targets for microbubble targeting.
Although FDA has approved ultrasound contrast agents only for a few limited cardiac-imaging applications to date, the agents are used much more broadly in Europe and Asia (e.g., to image the liver). A growing regulatory acceptance of contrast applications is necessary to prepare the ground for clinical studies on therapeutic drug targeting.
Several safety concerns with regard to microbubbles still pose open questions to the authorities. In 2008, FDA issued an alert about Optison (GE Healthcare, Waukesha, WI) and Definity (Bristol-Myers Squibb, New York), the two microbubble contrast agents approved in the US. The authorities revised the contraindications and warnings for these products after several cases of severe cardiopulmonary reactions occurred in patients with pulmonary hypertension shortly after the contrast agents were applied. Recent postmarketing observations, however, provided reassurance about the safety of ultrasound contrast agents (26).
Those safety incidents relate to microbubbles' ability to pass through narrow lung capillaries. Third-generation microbubbles are smaller than red blood cells and pass through the lung capillaries without impeding circulation. Severe adverse effects after microbubble administration have been observed in patients with multiple morbidities and large cardiopulmonary disturbances. Nevertheless, these concerns may potentially affect microbubble applications such as drug administration, where fairly enhanced dosages may be needed to reach therapeutic concentrations at the target site.
One question relevant to all drug-targeting approaches in general is how to estimate a drug's pharmacokinetic therapeutic window. In a targeting approach, the active drug must be enriched at the disease site, and the therapeutic window must be reached there rather than in the systemic circulation.
An important milestone on the way toward creating a successful product is the establishment of a lean and scalable manufacturing process that conforms to good manufacturing practice. The formulator should bear regulatory requirements in mind as early as in the prepatent stage of product development. If a production process is not designed to allow terminal sterile filtration, for example, during a drug's preformulation stage, it is problematic to redesign the process during the production of clinical batches.
With their dimensions of between several hundred nanometers and 5–6 µm, UCA cannot be subjected to sterile filtration. Their sensitivity to pressure excursions is also a hurdle to terminal steam sterilization. Sterilization through gamma irradiation is, in most cases, not an option because of the sensitivity of glass and most active ingredients to gamma rays. A sterile filtration of the contrast agent Definity is possible before the fill–finish step because the microbubbles are formed at the patient's bed side by mechanical agitation in the final container. If the product is supposed to include preformed microbubbles, however, some aseptic processing steps seem to be inevitable.
The development of techniques for safe and precise ultrasound focusing in sensitive areas such as behind the cranial wall remains a vital need. The ultrasound cycle should correspond to the maximal tissue replenishment achieved with microbubbles (e.g., by using electrocardiographic triggering to target the myocardium and the coronary vessels).
Finally, microbubbles pose a serious challenge for establishing methods for quality control and particle characterization. The final product often does not contain a single particle species; it includes at least two. Phospholipid microbubbles, for example, are always accompanied by liposomes. Furthermore, depending on their diameter, microbubbles rise upwards at various speeds. This feature requires scientists to consider the analytical approach for particle sizing and determining Zeta potential.
Undoubtedly, many aspects of UACs still must be sufficiently explored before an advanced drug-targeting strategy can be developed. Nevertheless, the industry has witnessed the emergence of the first fully established and marketed approach for active drug targeting: antibody drug conjugates. Inevitably, this approach will be followed by other, even more sophisticated means to guide the drug through the body to the desired site of action. The field of ultrasound drug targeting will remain active and exciting.
This article was funded by the BioFuture grant of Germany's Federal Ministry of Education and Research (Raffi Bekeredjian, Heidelberg, Germany).
Steliyan Tinkov* is an alumnus, Conrad Coester is an associate professor of pharmaceutical technology and biopharmaceutics, and Gerhard Winter is a professor of pharmaceutical technology and biopharmaceutics, all at Ludwig-Maximilians University Munich, Butenandtstr. 5-13, D-81377 Munich, Germany, firstname.lastname@example.org. Raffi Bekeredjian is an associate professor of cardiology at Ruprecht-Karls University in Heidelberg, Germany.
*To whom all correspondence should be addressed.
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