Facilitating Intracellular Drug Delivery by Ultrasound-Activated Microbubbles

Abstract

The goal of this thesis was to investigate the combination of ultrasound and microbubbles (USMB) for intracellular delivery of (model) drugs in vitro. We have focused on clinically approved drugs, i.e. cisplatin, and microbubbles, i.e. SonoVue™, to facilitate clinical translation. In addition, model drugs, predominantly SYTOX Green, were used to increase the understanding of the mechanisms involved in USMB-induced cellular uptake, in particular during cellular recovery after membrane permeabilization. Chemotherapy is one of the major treatment modalities in cancer, next to radiotherapy and surgery. However, in certain types of cancer, chemotherapy shows low efficacy while inducing serious adverse effects. Much research has focused on increasing the efficacy or decreasing the toxicity of these chemotherapeutic drugs, with limited success. The combination of ultrasound and microbubbles is a promising new technique to increase the efficacy of chemotherapeutic drugs, by increasing drug extravasation in the tumor and enhancing cellular uptake of drugs. The variety in treatment strategies for USMB-induced drug delivery were discussed in Chapter 2, focusing on drugs and microbubbles, route of administration, ultrasound equipment and treatment schedules. Chemotherapeutic drugs can be loaded into the shell of the microbubble, resulting in a microbubble that not only acts as a cavitation-enhancing agent, but also as a drug carrier. In this case, ultrasound-induced destruction of the microbubble results not only in vascular- and membrane permeabilization, but also in concurrent local drug release. This may lead to higher efficacy and lower toxicity compared to the co-administration approach, where conventional microbubbles and free drugs are administered simultaneously. However, the co-administration approach is likely the fastest way towards the clinic, as both microbubbles and drugs are clinically approved, in contrast to the drug-loaded microbubbles. While drugs and microbubbles can be injected directly into the tumor to ensure direct contact between drugs, microbubbles and tumor cells, most in vivo studies opt for intravenous administration. As microbubbles are confined to the vascular space due to their micrometer size, they will be primarily in contact with endothelial cells, rather than tumor cells. Nevertheless, enhanced chemotherapeutic efficacy when combined with USMB has been reported for both routes of administration. Although pre-clinical success has been demonstrated in vitro and in vivo, the translation towards the clinic has been limited thus far. One clinical case study has been published, where pancreatic cancer patients were treated with gemcitabine and concurrent USMB, using the clinically approved SonoVue™ microbubbles. Patients who received gemcitabine in combination with USMB showed enhanced treatment efficacy compared to a historical control group of patients that received only gemcitabine [1]. As discussed in Chapter 1 of this thesis, chemotherapy is often combined with surgery or radiotherapy in an (neo)adjuvant setting. Nevertheless, practically all studies on chemotherapeutic drug delivery by USMB focus on chemotherapy as a single modality treatment. Cisplatin is one of the most widely used chemotherapeutics in combination with radiotherapy, where both treatments act synergistically to enhance treatment efficacy. In advanced staged (stage III and IV) head and neck cancer patients with high-risk features, treatment often includes concurrent cisplatin and radiotherapy treatment. Therefore, Chapter 3 addressed the potential of USMB to enhance the efficacy of chemoradiation by increasing the intracellular uptake of cisplatin in a head and neck cancer model in vitro. Measurements with inductively coupled plasma mass spectrometry demonstrated that intracellular cisplatin levels were 2.7-fold higher when cisplatin treatment was combined with USMB, compared to cisplatin only. These enhanced intracellular cisplatin levels were associated with increased DNA damage, which was 82% higher after cisplatin + USMB treatment compared to cisplatin only treatment, while USMB alone did not enhance DNA damage. Subsequently, USMB significantly enhanced the efficacy of the combination cisplatin and radiotherapy, without affecting radiotherapy efficacy in the absence of cisplatin. While these results demonstrate the potential of USMB to enhance the efficacy of the combination cisplatin and radiotherapy in vitro, future research should confirm if similar results can be obtained in vivo. The next chapters aimed to increase the understanding of the biological mechanisms involved in USMB-induced membrane permeabilization and drug uptake. The majority of research into the mechanism of USMB focused on the direct effects on the cell membrane, e.g. by investigating the pore formation or the upregulation of endocytosis [2], [3]. However, less attention has been paid to cellular recovery after USMB exposure and how this relates to prolonged membrane permeability and (model) drug uptake. In Chapter 4, we studied the duration of cell membrane permeability after a single USMB treatment by assessing the intracellular uptake of model drug SYTOX Green as a function of acoustic pressure and cell line. Fluorescence microscopy demonstrated that the number of cells with SYTOX Green uptake was highest when cells were sonicated in the presence of the dye. The number of SYTOX Green positive cells decreased with increasing time interval between USMB treatment and addition of the model drug, suggesting that more cells recovered from USMB-induced damage with increasing time after sonication. The duration of membrane permeability in the breast cancer cell line MDA-MB-468 was only slightly affected by the acoustic pressures used in the study, and returned to the level of nonsonicated cells within 1 – 2 hours after USMB treatment. Larger differences in duration of membrane permeability after USMB exposure were found between cell lines. As mentioned before, the membrane permeability of MDA-MB-468 cells returned to control level within 2 hours, while this lasted more than 3 hours for the breast cancer cell line 4T1, and less than 1 hour for the endothelial cell line HUVEC. This difference in time window of drug uptake following sonication may reflect the heterogeneity between cell lines in their ability to recover from the biophysical stress induced by USMB. Intracellular drug uptake by USMB has been associated with disruption of the cellular membrane. Direct microscopic observations in addition to transmembrane current measurements have demonstrated that membrane pores can be created when cells are exposed to USMB, which facilitate the intracellular uptake of (model) drugs. However, it recently has been suggested that USMB may also disrupt the tightly regulated lipid composition of the cell membrane. Therefore, the effect of USMB on the membrane lipid phosphatidylserine (PS) was studied and presented in Chapter 5. Fluorescence microscopy demonstrated that USMB induced a transient PS translocation from the inner leaflet of the plasma membrane to the outer leaflet. The duration of PS externalization was positively correlated with model drug uptake, suggesting that it is related with USMB-induced membrane permeability. While PS externalization is normally regarded as a precursor of apoptosis, USMB-induced PS externalization was found to be transient and not associated with loss of cell viability. In our experiments, we did not find evidence that USMB-induced PS externalization occurred via calcium-stimulated activation of the phospholipid scramblase. Therefore, it was hypothesized that the membrane pores could provide a new pathway for PS translocation between the inner and outer leaflet of the plasma membrane. During membrane recovery, external PS may be internalized by the transmembrane lipid transporter protein flippase or by endocytosis and exocytosis. SYTOX Green and propidium iodide (PI) are membrane impermeant dyes that exhibit 500- fold and 30-fold fluorescence intensity enhancement, respectively, upon binding to nucleic acids. They are widely used as model drugs to study USMB-induced membrane permeabilization and cellular uptake, as was shown in Chapters 4 and 5. The fluorescence intensity enhancement after USMB-mediated cellular internalization has been associated with cellular uptake kinetics [4], where others related the duration of fluorescence intensity enhancement to pore size [5]. However, SYTOX Green fluorescence intensity enhancement is induced by nucleic acid binding, which is an indirect result of cellular uptake. To increase the understanding and interpretation of the fluorescence intensity enhancement of model drugs like SYTOX Green after cellular internalization, Chapter 6 characterized the fluorescence intensity enhancement upon intracellular SYTOX Green uptake in vitro, and how this is influenced by experimental parameters. Swept-field confocal microscopy of single cells showed that a membrane pore was created upon exposure to USMB. SYTOX Green fluorescence intensity increased around the pore, suggesting that the model drug entered the cell through this pore. Next, the SYTOX Green fluorescence signal spread throughout the cell and accumulated in the nucleus during the 9-minute acquisition, whereas the cytosolic signal reached a maximum fluorescence intensity already after 15 seconds. These results underlined that SYTOX Green fluorescence intensity depends primarily on nuclear accumulation and binding DNA and not just on cellular uptake. The prolonged signal intensity enhancement of SYTOX Green after USMB exposure was confirmed in populations of cells on a fibered confocal fluorescence microscope. The cellular fluorescence signal of SYTOX Green increased at least for 10 minutes, and often more than 30 minutes, after USMB exposure, and was substantially affected by the experimental conditions. It was found that the duty cycle of the fluorescent laser largely influenced the signal enhancement of SYTOX Green upon USMB-induced cellular uptake, due to a 6.4-fold enhancement of the photobleaching rate. In addition, the rate of fluorescence enhancement upon cellular internalization was demonstrated to be dependent on the extracellular dye concentration, as we found a positive linear relation between the dye concentration and fluorescence rate constant. While it has been postulated in the literature that increased acoustic pressure results in larger pore sizes, we did not find a relation between the fluorescence rate constants and the acoustic range tested in this study (350 kPa – 850 kPa). Furthermore, we found that the rate of fluorescence enhancement differs between the glioma cell line C6 and the head and neck squamous cell carcinoma cell line FaDu in both ultrasound- and chemically permeabilized cells. This suggests that the differences in dynamics of fluorescence enhancement cannot be solely attributed to different susceptibility to ultrasound exposure. The data in this chapter demonstrated that intercalating model drugs like SYTOX Green are useful as a biomarker for membrane permeability, but the dynamics of signal enhancement upon cellular internalization should be carefully interpreted before drawing conclusions on the underlying biology, such as pore resealing.