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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.

Understanding ultrasound induced sonoporation: Definitions and underlying mechanisms ☆

In the past two decades, research has underlined the potential of ultrasound and microbubbles to enhance drug delivery. However, there is less consensus on the biophysical and biological mechanisms leading to this enhanced delivery. Sonoporation, i.e. the formation of temporary pores in the cell membrane, as well as enhanced endocytosis is reported. Because of the variety of ultrasound settings used and corresponding microbubble behavior, a clear overview is missing. Therefore, in this review, the mechanisms contributing to sonoporation are categorized according to three ultrasound settings: i) low intensity ultrasound leading to stable cavitation of microbubbles, ii) high intensity ultrasound leading to inertial cavitation with microbubble collapse, and iii) ultrasound application in the absence of microbubbles. Using low intensity ultrasound, the endocytotic uptake of several drugs could be stimulated, while short but intense ultrasound pulses can be applied to induce pore formation and the direct cytoplasmic uptake of drugs. Ultrasound intensities may be adapted to create pore sizes correlating with drug size. Small molecules are able to diffuse passively through small pores created by low intensity ultrasound treatment. However, delivery of larger drugs such as nanoparticles and gene complexes, will require higher ultrasound intensities in order to allow direct cytoplasmic entry.

Drug loaded microbubble design for ultrasound triggered delivery

Ultrasound imaging is widely used in both diagnosis of diseases and pregnancy follow-up. As ultrasound imaging is a harmless, cheap and portable technique, ultrasound guided drug delivery is gaining more and more attention in the drug delivery field. To be effective, in most cases, ultrasonic drug delivery makes use of microbubbles. This highlight describes why it is attractive to load the microbubbles with drugs and discusses recent attempts made in the design of drug loaded microbubbles.

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.

Ultrasound assisted siRNA delivery using PEG-siPlex loaded microbubbles.

Short interfering RNA (siRNA) attracts much attention for the treatment of various diseases. However, its delivery, especially via systemic routes, remains a challenge. Indeed, naked siRNAs are rapidly degraded, while complexed siRNAs massively aggregate in the blood or are captured by macrophages. Although this can be circumvented by PEGylation, we found that PEGylation had a strong negative effect on the gene silencing efficiency of siRNA-liposome complexes (siPlexes). Recently, ultrasound combined with microbubbles has been used to deliver naked siRNA but the gene silencing efficiency is rather low and very high amounts of siRNA are required. To overcome the negative effects of PEGylation and to enhance the efficiency of ultrasound assisted siRNA delivery, we coupled PEGylated siPlexes (PEG-siPlexes) to microbubbles. Ultrasound radiation of these microbubbles resulted in massive release of unaltered PEG-siPlexes. Interestingly, PEG-siPlexes loaded on microbubbles were able to enter cells after exposure to ultrasound, in contrast to free PEG-siPlexes, which were not able to enter cells rapidly. Furthermore, these PEG-siPlex loaded microbubbles induced, in the presence of ultrasound, much higher gene silencing than free PEG-siPlexes. Additionally, the PEG-siPlex loaded microbubbles only silenced the expression of genes in the presence of ultrasound, which allows space and time controlled gene silencing.

Ultrasound and microbubble mediated drug delivery: acoustic pressure as determinant for uptake via membrane pores or endocytosis.

Although promising results are achieved in ultrasound mediated drug delivery, its underlying biophysical mechanisms remain to be elucidated. Pore formation as well as endocytosis has been reported during ultrasound application. Due to the plethora of ultrasound settings used in literature, it is extremely difficult to draw conclusions on which mechanism is actually involved. To our knowledge, we are the first to show that acoustic pressure influences which route of drug uptake is addressed, by inducing different microbubble-cell interactions. To investigate this, FITC-dextrans were used as model drugs and their uptake was analyzed by flow cytometry. In fluorescence intensity plots, two subpopulations arose in cells with FITC-dextran uptake after ultrasound application, corresponding to cells having either low or high uptake. Following separation of the subpopulations by FACS sorting, confocal images indicated that the low uptake population showed endocytic uptake. The high uptake population represented uptake via pores. Moreover, the distribution of the subpopulations shifted to the high uptake population with increasing acoustic pressure. Real-time confocal recordings during ultrasound revealed that membrane deformation by microbubbles may be the trigger for endocytosis via mechanostimulation of the cytoskeleton. Pore formation was shown to be caused by microbubbles propelled towards the cell. These results provide a better insight in the role of acoustic pressure in microbubble-cell interactions and the possible consequences for drug uptake. In addition, it pinpoints the need for a more rational, microbubble behavior based choice of acoustic parameters in ultrasound mediated drug delivery experiments.

Ultrasound Exposure of Lipoplex Loaded Microbubbles Facilitates Direct Cytoplasmic Entry of the Lipoplexes

Recently we reported that the transfection of cells by PEGylated lipoplexes becomes significantly better by binding the PEGylated lipoplexes to the surface of microbubbles and applying ultrasound. To further optimize this gene delivery system it is important to understand the working mechanism. This paper elucidates the cellular entry path of these lipoplexes. The results clearly show that the PEGylated lipoplexes, released from the microbubbles upon applying ultrasound, are not taken up by endocytosis, the most common route for nanoparticles to enter cells. Our data demonstrate that, upon implosion of the microbubbles, the PEGylated lipoplexes are released and are most probably able to passively diffuse through the cell membrane pores or become injected in the cytoplasm of the target cells. This is attractive as the in vivo use of PEGylated nanoparticles remains currently limited due to a decreased cellular uptake and inefficient escape of the PEGylated nanoparticles from the endosomes.

Comparison of Gold Nanoparticle Mediated Photoporation: Vapor Nanobubbles Outperform Direct Heating for Delivering Macromolecules in Live Cells

There is a great interest in delivering macromolecular agents into living cells for therapeutic purposes, such as siRNA for gene silencing. Although substantial effort has gone into designing nonviral nanocarriers for delivering macromolecules into cells, translocation of the therapeutic molecules from the endosomes after endocytosis into the cytoplasm remains a major bottleneck. Laser-induced photoporation, especially in combination with gold nanoparticles, is an alternative physical method that is receiving increasing attention for delivering macromolecules in cells. By allowing gold nanoparticles to bind to the cell membrane, nanosized membrane pores can be created upon pulsed laser illumination. Depending on the laser energy, pores are created through either direct heating of the AuNPs or by vapor nanobubbles (VNBs) that can emerge around the AuNPs. Macromolecules in the surrounding cell medium can then diffuse through the pores directly into the cytoplasm. Here we present a systematic evaluation of both photoporation mechanisms in terms of cytotoxicity, cell loading, and siRNA transfection efficiency. We find that the delivery of macromolecules under conditions of VNBs is much more efficient than direct photothermal disturbance of the plasma membrane without any noticeable cytotoxic effect. Interestingly, by tuning the laser energy, the pore size could be changed, allowing control of the amount and size of molecules that are delivered in the cytoplasm. As only a single nanosecond laser pulse is required, we conclude that VNBs are an interesting photoporation mechanism that may prove very useful for efficient high-throughput macromolecular delivery in live cells.

mRNA-Lipoplex loaded microbubble contrast agents for ultrasound-assisted transfection of dendritic cells.

In cancer immunotherapy the immune system should be triggered to specifically recognize and eliminate tumor cells in the patients body. This could be achieved by loading dendritic cells (DCs) with tumor-associated antigens (TAAs). This can be achieved by transfecting DCs with messenger RNA encoding a tumor-associated antigen. Here we demonstrate transient transfection of dendritic cells by means of mRNA-lipoplexes bound to microbubbles. Microbubble-attached lipoplexes were introduced into the cells by applying ultrasound. Our data demonstrate that ultrasound-mediated delivery of mRNA-complexes led to efficient transfection of DCs. When mRNA encoding luciferase was used, maximal levels of the enzyme activity were detected 8 h after ultrasound application. Upon longer incubation protein expression gradually declined. This treatment did not affect viability of the cells. Intracellular localisation of mRNA-lipoplexes in DCs was determined by flow cytometry using fluorescently labeled lipoplexes. Over 50% of DCs contained fluorescently labeled mRNA-complexes. In the absence of additional maturation signals, transfection of immature DCs with mRNA-lipoplex loaded microbubbles and ultrasound application induced only a minor shift in the expression level of maturation markers (CD40 and CD86). However, in the presence of the activation stimulus (LPS), cells were able to further mature as shown by a significant up-regulation of CD40 expression. Thus, our results demonstrate that mRNA-lipoplex loaded microbubbles can serve as an applicable and safe tool for efficient mRNA-transfection of cultured DCs.

New strategies for nucleic acid delivery to conquer cellular and nuclear membranes

After administration to the body, nucleic acid containing nanoparticles (NANs) need to cross several extra- and intracellular barriers to reach the cytoplasm or nucleus of the target cells. In the last decade several groups tried to overcome these barriers by arming non-viral delivery systems with targeting moieties, polyethylene glycol chains, fusogenic peptides and so forth. However, the drawback of this upgrading strategy is that each of the encountered barriers requires a new functionality, leading to very complex multi-component NANs. Moreover, there are currently no components available that can efficiently transport genes or NANs inside the nucleus of non-dividing cells. In this article a new, ultrasound based delivery system that possesses the capacity to simultaneously overcome several key barriers in non-viral nucleic acid delivery is presented. Additionally, a small amphiphilic compound that induces nuclear uptake of plasmid DNA and enhances non-viral gene transfer is presented.