Design and evaluation of drug loaded microbubbles for ultrasound guided cancer therapy

Cancer is a genetic disease, caused by mutations in the genome of normal cells. Chemical and physical damage to the cellular genome can induce these mutations resulting in the transformation of a healthy cell into a tumor cell. Over the past years, researchers have acquired a basic understanding of tumor onset. Several important proto-oncogenes and proteins involved in angiogenesis have been identified, leading to the development and clinical use of several new anticancer agents. We succeeded in preparing microbubbles which are able to carry genetic (pDNA, siRNA) and chemotherapeutic (doxorubicin) drugs. We proved that these microbubbles can selectively deliver their genetic or chemotherapeutic drugs to melanoma cells upon ultrasound exposure. We also showed that the activity of currently available drug carriers (pDNA or siRNA lipoplexes and doxorubicin-liposomes) was significantly improved by microbubble coupling and ultrasound exposure. This enables us to obtain a space and time controlled drug delivery guided by ultrasound. The fact that ultrasound and microbubbles have already been approved for ultrasound contrast imaging and are currently used in daily clinic, makes it very plausible that the microbubble concept reported in this thesis may find its way as an advanced drug delivery system

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

Choose your models wisely: how different murine bone marrow-derived dendritic cell protocols influence the success of nanoparticulate vaccines in vitro

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New physical and chemical approaches for the cytosolic delivery of bio- therapeutics and nanoparticles into cells

Delivery of bio-therapeutics and nanomaterials into living cells is an important step not only for cell studies but also for therapy and bio-imaging. Clear examples are the intracellular delivery of various classes of nucleic acids (siRNA, µRNA, mRNA, pDNA), peptides and proteins for therapy purposes. As another example, all types of (inorganic/organic) nanoparticles are under investigation as intracellular labels for imaging purposes. Meanwhile it generally accepted that after uptake by cells, nanomaterials typically end up in endo-lysosomal vesicles in which they remain entrapped while they should escape from such compartments and arrive in the cytosolic fluids of the cells. In recent years our team undertook major efforts to understand the biophysics which play a role in (a lack of) escape of nanomaterials from endo-lysosomal vesicles. Vere recently we also discovered new chemical strategies (so named ‘escape adjuvants’) (1) which seems promising to ‘liberate’ nucleic acids (like siRNA) from endo-lysosomal vesicles into the cytosol. Furthermore we explored physical methods (either light (2,3) or ultrasound (4) driven) which directly deliver bio-therapeutics into the cytosol, thereby bypassing the endo-lysosomal routes. This lecture will explain our recent findings in this area, as reported in a serious of recently published papers (1-4). Both pharmaceutical, biological and engineering aspects of our work will be highlighted in the lecture. References 1) Repurposing cationic amphiphilic drugs as adjuvants to induce lysosomal siRNA escape in nanogel transfected cells F. Joris, L. De Backer, T. Van de Vyver, C. Bastiancich, S.C. De Smedt, K. Raemdonck Journal of Controlled Release 2018, in Press 2) Comparison of gold nanoparticle mediated photoporation: vapour nanobubbles outperform direct heating for delivering macromolecules in live cells R.H. Xiong, K. Raemdonck, K. Peynshaert, I. Lentacker, I. De Cock, J. Demeester, S.C. De Smedt, A.G. Skirtach, K. Braeckmans ACS Nano 2014, 8(6): 6288-6296 3) Cytosolic Delivery of Nanolabels Prevents Their Asymmetric Inhentance and Enables Extended Quantitative in Vivo Cell Imaging R.H. Xiong, F. Joris, S.Y. Liang, R. De Rycke, S. Lippens, J. Demeester, A. Skirtach, K. Raemdonck, U. Himmelreich, S.C. De Smedt, K. Braeckmans Nano Letters 2016, 16(10): 5975-5986 4) Sonoprinting and the importance of microbubble loading for the ultrasound mediated cellular delivery of nanoparticles I. De Cock, G.P.R. Lajoinie, M. Versluis, S.C. De Smedt*, I. Lentacker Biomaterials 2016, 83: 294-30

The role of ultrasound-driven microbubble dynamics in drug delivery : from microbubble fundamentals to clinical translation

In the last couple of decades, ultrasound-driven microbubbles have proven excellent candidates for local drug delivery applications. Besides being useful drug carriers, microbubbles have demonstrated the ability to enhance cell and tissue permeability and, as a consequence, drug uptake herein. Notwithstanding the large amount of evidence for their therapeutic efficacy, open issues remain. Because of the vast number of ultrasound- and microbubble-related parameters that can be altered and the variability in different models, the translation from basic research to (pre)clinical studies has been hindered. This review aims at connecting the knowledge gained from fundamental microbubble studies to the therapeutic efficacy seen in in vitro and in vivo studies, with an emphasis on a better understanding of the response of a microbubble upon exposure to ultrasound and its interaction with cells and tissues. More specifically, we address the acoustic settings and microbubble-related parameters (i.e., bubble size and physicochemistry of the bubble shell) that play a key role in microbubble cell interactions and in the associated therapeutic outcome. Additionally, new techniques that may provide additional control over the treatment, such as monodisperse microbubble formulations, tunable ultrasound scanners, and cavitation detection techniques, are discussed. An in-depth understanding of the aspects presented in this work could eventually lead the way to more efficient and tailored microbubble-assisted ultrasound therapy in the future