Coupling of drug containing liposomes to microbubbles improves ultrasound triggered drug delivery in mice.

Local extravasation and triggered drug delivery by use of ultrasound and microbubbles is a promising strategy to target drugs to their sites of action. In the past we have developed drug loaded microbubbles by coupling drug containing liposomes to the surface of microbubbles. Until now the advantages of this drug loading strategy have only been demonstrated in vitro. Therefore, in this paper, microbubbles with indocyanine green (ICG) containing liposomes at their surface or a mixture of ICG-liposomes and microbubbles was injected intravenously in mice. Immediately after injection the left hind leg was exposed to 1 MHz ultrasound and the ICG deposition was monitored 1, 4 and 7 days post-treatment by in vivo fluorescence imaging. In mice that received the ICG-liposome loaded microbubbles the local ICG deposition was, at each time point, about 2-fold higher than in mice that received ICG-liposomes mixed with microbubbles. We also showed that the perforations in the blood vessels allow the passage of ICG-liposomes up to 5h after microbubble and ultrasound treatment. An increase in tissue temperature to 41°C was observed in all ultrasound treated mice. However, ultrasound tissue heating was excluded to cause the local ICG deposition. We concluded that coupling of drug containing liposomes to microbubbles may increase ultrasound mediated drug delivery in vivo.

Targeted liposome-loaded microbubbles for cell-specific ultrasound-triggered drug delivery.

One of the main problems in cancer treatment is disease relapse through metastatic colonization, which is caused by circulating tumor cells (CTCs). This work reports on liposome-loaded microbubbles targeted to N-cadherin, a cell-cell adhesion molecule expressed by CTCs. It is shown that such microbubbles can indeed bind to N-cadherin at the surface of HMB2 cells. Interestingly, in a mixture of cells with and without N-cadherin expression, binding of the liposome-loaded microbubbles mainly occurs to the N-cadherin-expressing cells. Importantly, applying ultrasound results in the intracellular delivery of a model drug (loaded in the liposomes) in the N-cadherin-expressing cells only. As described in this paper, such liposome-loaded microbubbles may find application as theranostics and in devices aimed for the specific killing of CTCs in blood.

Design and evaluation of theranostic perfluorocarbon particles for simultaneous antigen-loading and ¹9F-MRI tracking of dendritic cells.

Perfluorocarbon (PFC) particles are currently on the rise as cell labeling agents for ¹9F-MRI tracking of dendritic cell (DC)-based vaccines. In this work, we design theranostic PFC particles for single-step loading of DCs with both antigenic protein and with a liquid PFC for ¹9F-MRI detection of the antigen-loaded cells. Upon addition to DCs in vitro, the antigen-loaded PFC particles are efficiently internalized, resulting in intracellular presence of up to 40 pmol ¹9F atoms per cell. At the same time, the DCs become loaded with antigenic proteins, that can be efficiently processed, without important effects on cell viability or altering the DC's phenotype and the cell's capacity to respond to danger signals. In addition, antigen-loaded PFC particle containing DCs are capable of inducing extensive proliferation of antigen-specific CD8⁺ T cells in vitro. Importantly, the antigen-coated PFC particles allow in vitro ¹9F-MRI-based detection of the antigen-containing DCs with detection limits as low as 10³ cells µl⁻¹. The dual-modality characteristics of the designed particles could assure that only those DCs that have taken up the antigen, and hence are responsible for an immune response, are traceable via ¹9F-MRI. Taken together, these novel dual-modality particles represent an interesting strategy in the development of a traceable DC vaccine.

Focal Delivery of AAV2/1-transgenes Into the Rat Brain by Localized Ultrasound-induced BBB Opening.

Delivery of drugs and macromolecules to the central nervous system (CNS) is hindered by the blood-brain barrier (BBB). Several approaches have been used to overcome this hindrance to facilitate the treatment of various CNS diseases. We now present results showing that chimeric adeno-associated virus 2/1 (AAV2/1) particles containing the coding region for the LacZ gene are efficiently delivered into the rat brain upon intravenous (IV) administration after BBB opening by focused ultrasound in the presence of vascular acoustic resonators. We show that the transgene is correctly and efficiently expressed in cells located in the neighborhood of the insonated focus, especially in the vicinity of small vessels and capillaries. Histochemical LacZ staining allows the identification of large amounts of cells expressing the enzymatically active protein. Using double immunofluorescence (IF) with antibodies against tubulinIII and bacterial LacZ, we identified these cells to be mostly neurons. A small proportion of the transduced cells was recognized as glial cells, reacting positive in the IF with antibodies against astrocytic markers. These results demonstrate that our approach allows a very specific, localized, and efficient expression of intravenously administered transgenes in the brain of rats upon ultrasound-induced BBB opening.Molecular Therapy - Nucleic Acids (2013) 2, e73; doi:10.1038/mtna.2012.64; published online 19 February 2013.

Doxorubicin liposome-loaded microbubbles for contrast imaging and ultrasound-triggered drug delivery.

Targeted drug delivery under image guidance is gaining more interest in the drug-delivery field. The use of microbubbles as contrast agents in diagnostic ultrasound provides new opportunities in noninvasive image-guided drug delivery. In the present study, the imaging and therapeutic properties of novel doxorubicin liposome-loaded microbubbles are evaluated. The results showed that at scanning settings (1.7 MHz and mechanical index 0.2), these microbubbles scatter sufficient signal for nonlinear ultrasound imaging and can thus be imaged in real time and be tracked in vivo. In vitro therapeutic evaluation showed that ultrasound at 1 MHz and pressures up to 600 kPa in combination with the doxorubicin liposomeloaded microbubbles induced 4-fold decrease of cell viability compared with treatment with free doxorubicin or doxorubicin liposome-loaded microbubbles alone. The therapeutic effectiveness is correlated to an ultrasound-triggered release of doxorubicin from the liposomes and an enhanced uptake of the free doxorubicin by glioblastoma cells. The results obtained demonstrate that the combination of ultrasound and the doxorubicin liposome-loaded microbubbles can provide a new method of noninvasive image-guided drug delivery.

Enhancing nucleic acid delivery with ultrasound and microbubbles.

For gene therapy to work in vivo, nucleic acids need to reach the target cells without causing major side effects to the patient. In many cases the gene only has to reach a subset of cells in the body. Therefore, targeted delivery of genes to the desired tissue is a major issue in gene delivery. Many different possibilities of targeted gene delivery have been studied. A relatively novel approach to target nucleic acids and other drugs to specific regions in the body is the use of ultrasound and microbubbles. Microbubbles are gas-filled spheres with a stabilizing lipid, protein, or polymer shell. When these microbubbles enter an ultrasonic field, they start to oscillate. The bubble expansion and compression are inversely related to the pressure phases in the ultrasonic field. When microbubbles are exposed to high-intensity ultrasound they will eventually implode and fragment. This generates shockwaves and microjets which can temporarily permeate cell membranes and blood vessels. Nucleic acids or (non)-viral vectors can extravasate through these pores to gain access to the cell's cytoplasm or the surrounding tissue. The nucleic acids can either be mixed with the microbubbles or loaded on the microbubbles. Nucleic acid-loaded microbubbles can be obtained by coupling nucleic acid-containing particles (i.e., lipoplexes) to the microbubbles. Upon ultrasound-mediated implosion of the microbubbles, the nucleic acid-containing particles will be released and will deliver their nucleic acids in the ultrasound-targeted region.

Acoustical properties of individual liposome-loaded microbubbles.

A comparison between phospholipid-coated microbubbles with and without liposomes attached to the microbubble surface was performed using the ultra-high-speed imaging camera (Brandaris 128). We investigated 73 liposome-loaded microbubbles (loaded microbubbles) and 41 microbubbles without liposome loading (unloaded microbubbles) with a diameter ranging from 3-10 µm at frequencies ranging from 0.6-3.8 MHz and acoustic pressures ranging from 5-100 kPa. The experimental data showed nearly the same shell elasticity for the loaded and unloaded bubbles, but the shell viscosity was higher for loaded bubbles compared with unloaded bubbles. For loaded bubbles, a higher pressure threshold for the bubble vibrations was noticed. In addition, an "expansion-only" behavior was observed for up to 69% of the investigated loaded bubbles, which mostly occurred at low acoustic pressures (≤30 kPa). Finally, fluorescence imaging showed heterogeneity of liposome distributions of the loaded bubbles.

Crucial factors and emerging concepts in ultrasound-triggered drug delivery.

Time and space controlled drug delivery still remains a huge challenge in medicine. A novel approach that could offer a solution is ultrasound guided drug delivery. "Ultrasonic drug delivery" is often based on the use of small gas bubbles (so-called microbubbles) that oscillate and cavitate upon exposure to ultrasound waves. Some microbubbles are FDA approved contrast agents for ultrasound imaging and are nowadays widely investigated as promising drug carriers. Indeed, it has been observed that upon exposure to ultrasound waves, microbubbles may (a) release the encapsulated drugs and (b) simultaneously change the structure of the cell membranes in contact with the microbubbles which may facilitate drug entrance into cells. This review aims to highlight (a) major factors known so far which affect ultrasonic drug delivery (like the structure of the microbubbles, acoustic settings, etc.) and (b) summarizes the recent preclinical progress in this field together with a number of promising new concepts and applications.

Elucidating the mechanisms behind sonoporation with adeno-associated virus-loaded microbubbles.

Microbubbles are Food and Drug Administration (FDA) approved contrast agents for ultrasound imaging. It has been reported that applying ultrasound on drug-loaded microbubbles facilitates drug uptake by cells, due to so-named sonoporation. However, the biophysics behind sonoporation are not fully understood. It is believed that sonoporation results in a "direct" delivery of drugs in the cytoplasm of cells, though it has been suggested as well that sonoporation facilitates endocytosis which would improve the internalization of drugs by cells. To get a better understanding of sonoporation, this study reports on the ultrasound assisted delivery of adeno-associated virus (AAV) loaded on the surface of microbubbles. AAVs rely on endocytosis for efficient transduction of cells and are, consequently, an elegant tool to evaluate whether endocytosis is involved in ultrasound-induced sonoporation. Applying ultrasound on AAV-loaded microbubbles clearly improved the internalization of AAVs by cells, though transduction of the cells did not occur, indicating that by sonoporation substances become directly delivered in the cytosol of cells.

Self-assembled liposome-loaded microbubbles: The missing link for safe and efficient ultrasound triggered drug-delivery.

Liposome-loaded microbubbles have been recently introduced as a promising drug delivery platform for ultrasound guided drug delivery. In this paper we design liposome-loaded (lipid-shelled) microbubbles through the simple self-assembly of the involved compounds in a single step process. We thoroughly characterized the liposome-loading of the microbubbles and evaluated the cell killing efficiency of this material using doxorubicin (DOX) as a model drug. Importantly, we observed that the DOX liposome-loaded microbubbles allowed killing of melanoma cells even at very low doses of DOX. These findings clearly prove the potential of liposome-loaded microbubbles for ultrasound targeted drug delivery to cancer tissues.

Design and evaluation of doxorubicin-containing microbubbles for ultrasound-triggered doxorubicin delivery: cytotoxicity and mechanisms involved.

Drug delivery with microbubbles and ultrasound is gaining more and more attention in the drug delivery field due to its noninvasiveness, local applicability, and proven safety in ultrasonic imaging techniques. In this article, we tried to improve the cytotoxicity of doxorubicin (DOX)-containing liposomes by preparing DOX-liposome-containing microbubbles for drug delivery with therapeutic ultrasound. In this way, the DOX release and uptake can be restricted to ultrasound-treated areas. Compared to DOX-liposomes, DOX-loaded microbubbles killed at least two times more melanoma cells after exposure to ultrasound. After treatment of the melanoma cells with DOX-liposome-loaded microbubbles and ultrasound, DOX was mainly present in the nuclei of the cancer cells, whereas it was mainly detected in the cytoplasm of cells treated with DOX-liposomes. Exposure of cells to DOX-liposome-loaded microbubbles and ultrasound caused an almost instantaneous cellular entry of the DOX. At least two mechanisms were identified that explain the fast uptake of DOX and the superior cell killing of DOX-liposome-loaded microbubbles and ultrasound. First, exposure of DOX-liposome-loaded microbubbles to ultrasound results in the release of free DOX that is more cytotoxic than DOX-liposomes. Second, the cellular entry of the released DOX is facilitated due to sonoporation of the cell membranes. The in vitro results shown in this article indicate that DOX-liposome-loaded microbubbles could be a very interesting tool to obtain an efficient ultrasound-controlled DOX delivery in vivo.