Molecular mechanisms of sonoporation in cancer therapy : Optimization of sonoporation parameters and investigations of intracellular signalling

Background: Sonoporation, which is treatment with ultrasound (US) and microbubbles (MB), has shown great potential for enhancing the therapeutic efficacy of chemotherapy in cancer therapy. However, there is still very little consensus regarding the mechanism or optimal experimental and therapeutic parameters. The original assumption was that pore formation in the cell membrane was responsible for the increased uptake of drugs, but it is currently understood that the mechanisms are far more complex. The field combines US physics, MB formulation and physics, (cell) biology, pharmacology, pharmacokinetics and the biodistribution of both drugs and MBs. Hence, there is an almost endless range of experimental parameters and potential bioeffects. The current literature includes a plethora of experimental setups and parameters, which complicates the clinical translation of sonoporation. Aims and methodology: In this thesis, the effects of low-intensity US and MB parameters were investigated in vitro using custom-made ultrasound chambers and correlating commonly used measures as uptakes of impermeable dye (i.e. flow cytometry) and viability to detect intracellular signalling responses to sonoporation in different cell types. Intracellular signalling responses to sonoporation are largely unknown, and their influence on key proteins in important signalling pathways have been elucidated using phosphoflow cytometry. To gain the understanding and translatability of US + MB parameters, three commercially available MB formulations were characterized, and important parameters, such as dose and formulation, were investigated in vitro and the in vivo enhancement of chemotherapy in a mural model of pancreatic ductal adenocarcinoma (PDAC). Results and conclusions: Effective sonoporation was achieved using commercial microbubbles and low-intensity US in the diagnostic range, both in vitro and in vivo. In the low-intensity US regimen, effective sonoporation required MBs, and the efficacy increased as US intensity and MB concentrations increased. The choice of optimal MBs depended on the US parameters used, and must be carefully chosen based on the therapeutic context. The findings in vivo were correlated to those in the in vitro experiments and to simulations on MB behaviour. Sonoporation induced the immediate, transient activation of intracellular signalling (MAPK-kinases; p38, ERK1/2, CREB, STAT3, Akt) as well as changes in the phosphorylation status of the proteins involved in protein translation (i.e. ribosomal protein S6, 4E-BP1 and eIF2α). The intracellular signalling response resembles cellular recovery after pore formation by electroporation and pore-forming toxins. Based on this observation, we hypothesize that sonoporation induces a cellular stress response that is related to the membrane repair and restoration of cellular homeostasis, and it may be exploited therapeutically. Varying responses in different cell types better represent the variability within a tumour, and they indicate that the effects on the tumour microenvironment may be important for sonoporation efficacy. In the present work, cellular stress was induced using low-intensity US below the intensity limit approved for diagnostic imaging, and healthy blood peripheral cells were minimally affected

Intracellular signaling in key pathways is induced by treatment with ultrasound and microbubbles in a leukemia cell line, but not in healthy peripheral blood mononuclear cells

Treatment with ultrasound and microbubbles (sonoporation) to enhance therapeutic efficacy in cancer therapy is rapidly expanding, but there is still very little consensus as to why it works. Despite the original assumption that pore formation in the cell membrane is responsible for increased uptake of drugs, the molecular mechanisms behind this phenomenon are largely unknown. We treated cancer cells (MOLM-13) and healthy peripheral blood mononuclear cells (PBMCs) with ultrasound at three acoustic intensities (74, 501, 2079 mW/cm2) ± microbubbles. We subsequently monitored the intracellular response of a number of key signaling pathways using flow cytometry or western blotting 5 min, 30 min and 2 h post-treatment. This was complemented by studies on uptake of a cell impermeable dye (calcein) and investigations of cell viability (cell count, Hoechst staining and colony forming assay). Ultrasound + microbubbles resulted in both early changes (p38 (Arcsinh ratio at high ultrasound + microbubbles: +0.5), ERK1/2 (+0.7), CREB (+1.3), STAT3 (+0.7) and AKT (+0.5)) and late changes (ribosomal protein S6 (Arcsinh ratio at low ultrasound: +0.6) and eIF2α in protein phosphorylation). Observed changes in protein phosphorylation corresponded to changes in sonoporation efficiency and in viability, predominantly in cancer cells. Sonoporation induced protein phosphorylation in healthy cells was pronounced (p38 (+0.03), ERK1/2 (−0.03), CREB (+0.0), STAT3 (−0.1) and AKT (+0.04) and S6 (+0.2)). This supports the hypothesis that sonoporation may enhance therapeutic efficacy of cancer treatment, without causing damage to healthy cells.publishedVersio

Formulation and characterisation of drug-loaded antibubbles for image-guided and ultrasound-triggered drug delivery

The aim of this study was to develop high load-capacity antibubbles that can be visualized using diagnostic ultrasound and the encapsulated drug can be released and delivered using clinically translatable ultrasound. The antibubbles were developed by optimising a silica nanoparticle stabilised double emulsion template. We produced an emulsion with a mean size diameter of 4.23 ± 1.63 µm where 38.9 ± 3.1% of the droplets contained a one or more cores. Following conversion to antibubbles, the mean size decreased to 2.96 ± 1.94 µm where 99% of antibubbles were <10 µm. The antibubbles had a peak attenuation of 4.8 dB/cm at 3.0 MHz at a concentration of 200 × 103 particles/mL and showed distinct attenuation spikes at frequencies between 5.5 and 13.5 MHz. No increase in subharmonic response was observed for the antibubbles in contrast to SonoVue®. High-speed imaging revealed that antibubbles can release their cores at MIs of 0.6. In vivo imaging indicated that the antibubbles have a long half-life of 68.49 s vs. 40.02 s for SonoVue®. The antibubbles could be visualised using diagnostic ultrasound and could be disrupted at MIs of ≥0.6. The in vitro drug delivery results showed that antibubbles can significantly improve drug delivery (p < 0.0001) and deliver the drug within the antibubbles. In conclusion antibubbles are a viable concept for ultrasound guided drug delivery.publishedVersio

Formulation and characterisation of drug-loaded antibubbles for image-guided and ultrasound-triggered drug delivery

The aim of this study was to develop high load-capacity antibubbles that can be visualized using diagnostic ultrasound and the encapsulated drug can be released and delivered using clinically translatable ultrasound. The antibubbles were developed by optimising a silica nanoparticle stabilised double emulsion template. We produced an emulsion with a mean size diameter of 4.23 ± 1.63 µm where 38.9 ± 3.1% of the droplets contained a one or more cores. Following conversion to antibubbles, the mean size decreased to 2.96 ± 1.94 µm where 99% of antibubbles were <10 µm. The antibubbles had a peak attenuation of 4.8 dB/cm at 3.0 MHz at a concentration of 200 × 103 particles/mL and showed distinct attenuation spikes at frequencies between 5.5 and 13.5 MHz. No increase in subharmonic response was observed for the antibubbles in contrast to SonoVue®. High-speed imaging revealed that antibubbles can release their cores at MIs of 0.6. In vivo imaging indicated that the antibubbles have a long half-life of 68.49 s vs. 40.02 s for SonoVue®. The antibubbles could be visualised using diagnostic ultrasound and could be disrupted at MIs of ≥0.6. The in vitro drug delivery results showed that antibubbles can significantly improve drug delivery (p < 0.0001) and deliver the drug within the antibubbles. In conclusion antibubbles are a viable concept for ultrasound guided drug delivery.publishedVersio

SonoVue® vs. Sonazoid™ vs. Optison™: Which Bubble Is Best for Low-Intensity Sonoporation of Pancreatic Ductal Adenocarcinoma?

The use of ultrasound and microbubbles to enhance therapeutic efficacy (sonoporation) has shown great promise in cancer therapy from in vitro to ongoing clinical studies. The fastest bench-to-bedside translation involves the use of ultrasound contrast agents (microbubbles) and clinical diagnostic scanners. Despite substantial research in this field, it is currently not known which of these microbubbles result in the greatest enhancement of therapy within the applied conditions. Three microbubble formulations—SonoVue®, Sonazoid™, and Optison™—were physiochemically and acoustically characterized. The microbubble response to the ultrasound pulses used in vivo was simulated via a Rayleigh–Plesset type equation. The three formulations were compared in vitro for permeabilization efficacy in three different pancreatic cancer cell lines, and in vivo, using an orthotopic pancreatic cancer (PDAC) murine model. The mice were treated using one of the three formulations exposed to ultrasound from a GE Logiq E9 and C1-5 ultrasound transducer. Characterisation of the microbubbles showed a rapid degradation in concentration, shape, and/or size for both SonoVue® and Optison™ within 30 min of reconstitution/opening. Sonazoid™ showed no degradation after 1 h. Attenuation measurements indicated that SonoVue® was the softest bubble followed by Sonazoid™ then Optison™. Sonazoid™ emitted nonlinear ultrasound at the lowest MIs followed by Optison™, then SonoVue®. Simulations indicated that SonoVue® would be the most effective bubble using the evaluated ultrasound conditions. This was verified in the pre-clinical PDAC model demonstrated by improved survival and largest tumor growth inhibition. In vitro results indicated that the best microbubble formulation depends on the ultrasound parameters and concentration used, with SonoVue® being best at lower intensities and Sonazoid™ at higher intensities.publishedVersio

Molecular mechanisms of sonoporation in cancer therapy : Optimization of sonoporation parameters and investigations of intracellular signalling

Background: Sonoporation, which is treatment with ultrasound (US) and microbubbles (MB), has shown great potential for enhancing the therapeutic efficacy of chemotherapy in cancer therapy. However, there is still very little consensus regarding the mechanism or optimal experimental and therapeutic parameters. The original assumption was that pore formation in the cell membrane was responsible for the increased uptake of drugs, but it is currently understood that the mechanisms are far more complex. The field combines US physics, MB formulation and physics, (cell) biology, pharmacology, pharmacokinetics and the biodistribution of both drugs and MBs. Hence, there is an almost endless range of experimental parameters and potential bioeffects. The current literature includes a plethora of experimental setups and parameters, which complicates the clinical translation of sonoporation. Aims and methodology: In this thesis, the effects of low-intensity US and MB parameters were investigated in vitro using custom-made ultrasound chambers and correlating commonly used measures as uptakes of impermeable dye (i.e. flow cytometry) and viability to detect intracellular signalling responses to sonoporation in different cell types. Intracellular signalling responses to sonoporation are largely unknown, and their influence on key proteins in important signalling pathways have been elucidated using phosphoflow cytometry. To gain the understanding and translatability of US + MB parameters, three commercially available MB formulations were characterized, and important parameters, such as dose and formulation, were investigated in vitro and the in vivo enhancement of chemotherapy in a mural model of pancreatic ductal adenocarcinoma (PDAC). Results and conclusions: Effective sonoporation was achieved using commercial microbubbles and low-intensity US in the diagnostic range, both in vitro and in vivo. In the low-intensity US regimen, effective sonoporation required MBs, and the efficacy increased as US intensity and MB concentrations increased. The choice of optimal MBs depended on the US parameters used, and must be carefully chosen based on the therapeutic context. The findings in vivo were correlated to those in the in vitro experiments and to simulations on MB behaviour. Sonoporation induced the immediate, transient activation of intracellular signalling (MAPK-kinases; p38, ERK1/2, CREB, STAT3, Akt) as well as changes in the phosphorylation status of the proteins involved in protein translation (i.e. ribosomal protein S6, 4E-BP1 and eIF2α). The intracellular signalling response resembles cellular recovery after pore formation by electroporation and pore-forming toxins. Based on this observation, we hypothesize that sonoporation induces a cellular stress response that is related to the membrane repair and restoration of cellular homeostasis, and it may be exploited therapeutically. Varying responses in different cell types better represent the variability within a tumour, and they indicate that the effects on the tumour microenvironment may be important for sonoporation efficacy. In the present work, cellular stress was induced using low-intensity US below the intensity limit approved for diagnostic imaging, and healthy blood peripheral cells were minimally affected

The synthesis, crystal properties, cyclodextrin complexation and photochemical stability of selected curcuminoids

ABSTRACT R1 R2 Mw RHC-1 -OCH3 -OCH3 396.42 RHC-2 -OCH3 -OH 368.37 RHC-3 -H -OH 308.32 RHC-4 -H -OCH3 336.39 RHC-5 -OH -OH 340.32 A selection of curcuminoids has been synthesized and complexed to HPβCD, MβCD and HPγCD. The influence of concentration of cyclodextrin (CD), of ionic strength, choice of buffer salt and pH on aqueous phase solubility was investigated. In addition it was investigated if the use Mg2+ together with the CDs could increase the aqueous solubility of curcuminoids. Melting point and polymorphic forms of the curcuminoids were investigated using differential scanning calorimetry (DSC), and photochemical stability was investigated in hydrogen bonding organic solvent, EtOH, a mixture of this organic solvent and water and in aqueous solution of 10% HPβCD and HPγCD. The ionic strength or addition of Mg2+ did not influence the solubility, nor did pH when kept at pH 5 or lower. The stoichiometry of the curcuminoids-CD complexes was not unequivocally determined, but some sort of higher-order complex or non-inclusion complexation seems to be present. Solubility was found to be best for curcumin in HPγCD and best for bisdemethoxycurcumin in the βCDs. Different batches of the curcuminoids have formed different crystal forms, with slightly different melting points. This is assumed to have an effect on the aqueous solubility. Photochemical stability was found to be generally better for curcumin than for the other curcuminoids, presumably due to intramolecular bondings. The stability was best in hydrogen bonding organic solvent for all the curcuminoids. An attempt was made to synthesize a curcumin galactoside, with postulated increased aqueous solubility. This was not successful

Molecular mechanisms of nutlin-3 involve acetylation of p53, histones and heat shock proteins in acute myeloid leukemia

Background The small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). We and others have previously reported a vigorous acetylation of the p53 protein by nutlin-treatment. In this study we aimed to investigate the functional role of this p53 acetylation in nutlin-sensitivity, and further to explore if nutlin-induced protein acetylation in general could indicate novel targets for the enhancement of nutlin-based therapy. Results Nutlin-3 was found to enhance the acetylation of p53 in the human AML cell line MOLM-13 (wild type TP53) and in TP53 null cells transfected with wild type p53 cDNA. Stable isotope labeling with amino acids in cell culture (SILAC) in combination with immunoprecipitation using an anti-acetyl-lysine antibody and mass spectrometry analysis identified increased levels of acetylated Histone H2B, Hsp27 and Hsp90 in MOLM-13 cells after nutlin-treatment, accompanied by downregulation of total levels of Hsp27 and Hsp90. Intracellular levels of heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were correlated to nutlin-sensitivity for primary AML cells (n = 40), and AML patient samples with low sensitivity to nutlin-3 tended to express higher levels of heat shock proteins than more responsive samples. Combination therapy of nutlin-3 and Hsp90 inhibitor geldanamycin demonstrated synergistic induction of apoptosis in AML cell lines and primary AML cells. Finally, TP53 null cells transfected with a p53 acetylation defective mutant demonstrated decreased heat shock protein acetylation and sensitivity to nutlin-3 compared to wild type p53 expressing cells. Conclusions Altogether, our results demonstrate that nutlin-3 induces acetylation of p53, histones and heat shock proteins, and indicate that p53 acetylation status and the levels of heat shock proteins may participate in modulation of nutlin-3 sensitivity in AML

Intracellular signaling in key pathways is induced by treatment with ultrasound and microbubbles in a leukemia cell line, but not in healthy peripheral blood mononuclear cells

Treatment with ultrasound and microbubbles (sonoporation) to enhance therapeutic efficacy in cancer therapy is rapidly expanding, but there is still very little consensus as to why it works. Despite the original assumption that pore formation in the cell membrane is responsible for increased uptake of drugs, the molecular mechanisms behind this phenomenon are largely unknown. We treated cancer cells (MOLM-13) and healthy peripheral blood mononuclear cells (PBMCs) with ultrasound at three acoustic intensities (74, 501, 2079 mW/cm2) ± microbubbles. We subsequently monitored the intracellular response of a number of key signaling pathways using flow cytometry or western blotting 5 min, 30 min and 2 h post-treatment. This was complemented by studies on uptake of a cell impermeable dye (calcein) and investigations of cell viability (cell count, Hoechst staining and colony forming assay). Ultrasound + microbubbles resulted in both early changes (p38 (Arcsinh ratio at high ultrasound + microbubbles: +0.5), ERK1/2 (+0.7), CREB (+1.3), STAT3 (+0.7) and AKT (+0.5)) and late changes (ribosomal protein S6 (Arcsinh ratio at low ultrasound: +0.6) and eIF2α in protein phosphorylation). Observed changes in protein phosphorylation corresponded to changes in sonoporation efficiency and in viability, predominantly in cancer cells. Sonoporation induced protein phosphorylation in healthy cells was pronounced (p38 (+0.03), ERK1/2 (−0.03), CREB (+0.0), STAT3 (−0.1) and AKT (+0.04) and S6 (+0.2)). This supports the hypothesis that sonoporation may enhance therapeutic efficacy of cancer treatment, without causing damage to healthy cells