1. Introduction - Ulster University



Magnetic Microbubble Mediated Chemo-Sonodynamic Therapy using a Combined Magnetic-Acoustic DeviceEstelle Beguin1#, Michael D. Gray1#, Keiran A. Logan2#, Heather Nesbitt2, Yingjie Sheng2, Sukanta Kamila2, Lester C. Barnsley3, Luca Bau1, Anthony P. McHale2*, John F. Callan2* and Eleanor Stride1*1. Institute of Biomedical Engineering, University of Oxford, Oxford, UK, OX3 7DQ. 2. Biomedical Sciences Research Institute, University of Ulster, Coleraine, Northern Ireland, U.K. BT52 1SA. 3. Jülich Centre for Neutron Science at Heinz Maier-Leibnitz Zentrum, Forschungszentrum Jülich GmbH, 85748 Garching, Germany# Joint first authors. * To whom correspondence should be addressed.Abstract: Recent pre-clinical studies have demonstrated the potential of combining chemotherapy and sonodynamic therapy for the treatment of pancreatic cancer. Oxygen-loaded magnetic microbubbles have been explored as a targeted delivery vehicle for this application. Despite preliminary positive results, a previous study identified a significant practical challenge regarding the co-alignment of the magnetic and ultrasound fields. The aim of this study was to determine whether this challenge could eb addressed through the use of a magnetic-acoustic device (MAD) combining a magnetic array and ultrasound transducer in a single unit, to simultaneously concentrate and activate the microbubbles at the target site. in vitro experiments were performed in tissue phantoms and followed by in vivo treatment of xenograft pancreatic cancer (BxPC-3) tumours in a murine model. In vitro, a 1.4-fold (p<0.01) increase in the deposition of a model therapeutic payload within the phantom was achieved using the MAD compared to separate magnetic and ultrasound devices. In vivo, tumours treated with the MAD had a 9% smaller mean volume 8 days after treatment, while tumours treated with separate devices or microbubbles alone were respectively 45% and 112% larger. This substantial and sustained decrease in tumour volume suggests that the proposed drug delivery approach has the potential to be an effective neo-adjuvant therapy for pancreatic cancer patients.Keywords: Microbubbles; Drug Delivery; Magnetic Targeting; Sonodynamic Therapy; Ultrasound; Medical Devices.1. IntroductionMicrobubbles (MB) and ultrasound (US) are in routine clinical use for diagnostic imaging, and are being actively investigated for a range of therapeutic applications including in recent clinical trials for cancer treatment ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.jconrel.2016.10.007","ISSN":"01683659","abstract":"BACKGROUND\nThe primary aim of our study was to evaluate the safety and potential toxicity of gemcitabine combined with microbubbles under sonication in inoperable pancreatic cancer patients. The secondary aim was to evaluate a novel image-guided microbubble-based therapy, based on commercially available technology, towards improving chemotherapeutic efficacy, preserving patient performance status, and prolonging survival. \n\nMETHODS\nTen patients were enrolled and treated in this Phase I clinical trial. Gemcitabine was infused intravenously over 30min. Subsequently, patients were treated using a commercial clinical ultrasound scanner for 31.5min. SonoVue? was injected intravenously (0.5ml followed by 5ml saline every 3.5min) during the ultrasound treatment with the aim of inducing sonoporation, thus enhancing therapeutic efficacy. \n\nRESULTS\nThe combined therapeutic regimen did not induce any additional toxicity or increased frequency of side effects when compared to gemcitabine chemotherapy alone (historical controls). Combination treated patients (n=10) tolerated an increased number of gemcitabine cycles compared with historical controls (n=63 patients; average of 8.3±6.0cycles, versus 13.8±5.6cycles, p=0.008, unpaired t-test). In five patients, the maximum tumour diameter was decreased from the first to last treatment. The median survival in our patients (n=10) was also increased from 8.9months to 17.6months (p=0.011). \n\nCONCLUSIONS\nIt is possible to combine ultrasound, microbubbles, and chemotherapy in a clinical setting using commercially available equipment with no additional toxicities. This combined treatment may improve the clinical efficacy of gemcitabine, prolong the quality of life, and extend survival in patients with pancreatic ductal adenocarcinoma.","author":[{"dropping-particle":"","family":"Dimcevski","given":"Georg","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kotopoulis","given":"Spiros","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bj?nes","given":"Tormod","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hoem","given":"Dag","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Schj?t","given":"Jan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gjertsen","given":"Bj?rn Tore","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Biermann","given":"Martin","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Molven","given":"Anders","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sorbye","given":"Halfdan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"McCormack","given":"Emmet","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Postema","given":"Michiel","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gilja","given":"Odd Helge","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Controlled Release","id":"ITEM-1","issued":{"date-parts":[["2016"]]},"title":"A human clinical trial using ultrasound and microbubbles to enhance gemcitabine treatment of inoperable pancreatic cancer","type":"article-journal"},"uris":["",""]},{"id":"ITEM-2","itemData":{"DOI":"","ISSN":"1946-6234","abstract":"The blood-brain barrier (BBB) limits the delivery of systemically administered drugs to the brain. Methods to circumvent the BBB have been developed, but none are used in standard clinical practice. The lack of adoption of existing methods is due to procedural invasiveness, serious adverse effects, and the complications associated with performing such techniques coincident with repeated drug administration, which is customary in chemotherapeutic protocols. Pulsed ultrasound, a method for disrupting the BBB, was shown to effectively increase drug concentrations and to slow tumor growth in preclinical studies. We now report the interim results of an ultrasound dose-escalating phase 1/2a clinical trial using an implantable ultrasound device system, SonoCloud, before treatment with carboplatin in patients with recurrent glioblastoma (GBM). The BBB of each patient was disrupted monthly using pulsed ultrasound in combination with systemically injected microbubbles. Contrast-enhanced magnetic resonance imaging (MRI) indicated that the BBB was disrupted at acoustic pressure levels up to 1.1 MPa without detectable adverse effects on radiologic (MRI) or clinical examination. Our preliminary findings indicate that repeated opening of the BBB using our pulsed ultrasound system, in combination with systemic microbubble injection, is safe and well tolerated in patients with recurrent GBM and has the potential to optimize chemotherapy delivery in the brain., Copyright ? 2016, American Association for the Advancement of Science. All rights reserved.","author":[{"dropping-particle":"","family":"A.","given":"Carpentier","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"M.","given":"Canney","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"A.","given":"Vignot","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"V.","given":"Reina","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"K.","given":"Beccaria","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"C.","given":"Horodyckid","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"C.","given":"Karachi","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"D.","given":"Leclercq","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"C.","given":"Lafon","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"J.-Y.","given":"Chapelon","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"L.","given":"Capelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"P.","given":"Cornu","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"M.","given":"Sanson","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"K.","given":"Hoang-Xuan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"J.-Y.","given":"Delattre","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Science Translational Medicine","id":"ITEM-2","issue":"343","issued":{"date-parts":[["2016"]]},"page":"343re2","title":"Clinical trial of blood-brain barrier disruption by pulsed ultrasound","type":"article-journal","volume":"8"},"uris":["",""]}],"mendeley":{"formattedCitation":"[1,2]","plainTextFormattedCitation":"[1,2]","previouslyFormattedCitation":"[1,2]"},"properties":{"noteIndex":0},"schema":""}[1,2]. MBs consist of gas cavities 1-10 μm in diameter stabilised by a surfactant, lipid and/or polymer coating ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1179/175889709X446507.Microbubble","author":[{"dropping-particle":"","family":"Sirsi","given":"Shashank R.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Borden","given":"Mark A.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Bubble Sci Eng Technol.","id":"ITEM-1","issued":{"date-parts":[["2009"]]},"page":"3-17","title":"Microbubble compositions, properties and biomedical applications","type":"article-journal","volume":"1"},"uris":["",""]}],"mendeley":{"formattedCitation":"[3]","plainTextFormattedCitation":"[3]","previouslyFormattedCitation":"[3]"},"properties":{"noteIndex":0},"schema":""}[3]. Due to the compressibility of the gas core, MBs undergo volumetric oscillations when exposed to a US field, and the resulting acoustic scattering may be used to enhance the contrast between blood vessels and the surrounding tissue in US images ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1259/bjr/17790547","ISBN":"0007-1285 (Print)\\r0007-1285 (Linking)","ISSN":"00071285","PMID":"16498029","abstract":"Ultrasound is the most frequently performed ‘‘tomo- graphic’’ imaging technique which befits the perception of ultrasound as being a low cost, safe and accessible imaging modality. Widely used as a ‘‘screening’’ tool, particularly in liver imaging, ultrasound is often per- ceived as inferior to other imaging modalities such as CT and MRI in diagnostically challenging clinical situations. Both CT and MR imaging techniques make use of well established contrast agents to improve the image and hence the diagnostic potential, but with the burden of increased radiation with CT and cost with both CT and MRI. With such widespread utilization of ultrasound, it remains an enigma that until recently ultrasound had no effective contrast agent to improve imaging. The advent of microbubble contrast has brought new possibilities and, not surprisingly, recent advancement of ultrasound has been driven by research into the properties and clinical application of microbubble contrast agents.","author":[{"dropping-particle":"","family":"Stewart","given":"V. R.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sidhu","given":"Paul S.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"British Journal of Radiology","id":"ITEM-1","issue":"939","issued":{"date-parts":[["2006"]]},"page":"188-194","title":"New directions in ultrasound: Microbubble contrast","type":"article-journal","volume":"79"},"uris":["",""]}],"mendeley":{"formattedCitation":"[4]","plainTextFormattedCitation":"[4]","previouslyFormattedCitation":"[4]"},"properties":{"noteIndex":0},"schema":""}[4]. The use of sufficiently high acoustic pressures can lead to MB fragmentation and the dispersion of their coating material ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1146/annurev.bioeng.8.061505.095852","ISBN":"1523-9829","ISSN":"1523-9829","PMID":"17651012","abstract":"This review offers a critical analysis of the state of the art of med-ical microbubbles and their application in therapeutic delivery and monitoring. When driven by an ultrasonic pulse, these small gas bubbles oscillate with a wall velocity on the order of tens to hun-dreds of meters per second and can be deflected to a vessel wall or fragmented into particles on the order of nanometers. While single-session molecular imaging of multiple targets is difficult with affinity-based strategies employed in some other imaging modali-ties, microbubble fragmentation facilitates such studies. Similarly, a focused ultrasound beam can be used to disrupt delivery vehicles and blood vessel walls, offering the opportunity to locally deliver a drug or gene. Clinical translation of these vehicles will require that cur-rent challenges be overcome, where these challenges include rapid clearance and low payload. The technology, early successes with drug and gene delivery, and potential clinical applications are reviewed.","author":[{"dropping-particle":"","family":"Ferrara","given":"Katherine","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Pollard","given":"Rachel","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Borden","given":"Mark","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Annual Review of Biomedical Engineering","id":"ITEM-1","issued":{"date-parts":[["2007"]]},"page":"415-447","title":"Ultrasound Microbubble Contrast Agents: Fundamentals and Application to Gene and Drug Delivery","type":"article-journal","volume":"9"},"uris":[""]}],"mendeley":{"formattedCitation":"[5]","plainTextFormattedCitation":"[5]","previouslyFormattedCitation":"[5]"},"properties":{"noteIndex":0},"schema":""}[5]. As the MB coating can be loaded with drugs through the addition of single therapeutic molecules, drug-loaded liposomes or other nanoparticles, the ability to non-invasively trigger the release of therapeutics using US makes MBs an attractive drug delivery platform ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.addr.2008.03.005.Microbubbles","ISBN":"1873-4995 (Electronic)\\r0168-3659 (Linking)","ISSN":"01683659","PMID":"15738980","abstract":"Ultrasound contrast agents, in the form of gal-filled microbubbles, are becoming popular in perfusion monitoring; they are emplyed as molecular imaging agents. Microbubbles are manufactured from biocompatible materials, they can be injected intraveneously, and some are approved for clinical use.","author":[{"dropping-particle":"","family":"Hernot","given":"Sophie","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kilbanov","given":"Alexander L","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Advanced Drug Delivery Reviews","id":"ITEM-1","issue":"10","issued":{"date-parts":[["2008"]]},"page":"1153-1166","title":"Microbubbles in Ultrasound-Triggered Drug and Gene Delivery","type":"article-journal","volume":"60"},"uris":["",""]},{"id":"ITEM-2","itemData":{"DOI":"10.1016/j.jconrel.2006.12.015","ISBN":"1873-4995 (Electronic)","ISSN":"01683659","PMID":"17300849","abstract":"A new acoustically-active delivery vehicle was developed by conjugating liposomes and microbubbles, using the high affinity interaction between avidin and biotin. Binding between microbubbles and liposomes, each containing 5% DSPE-PEG2kBiotin, was highly dependent on avidin concentration and observed above an avidin concentration of 10 nM. With an optimized avidin and liposome concentration, we measured and calculated as high as 1000 to 10,000 liposomes with average diameters of 200 and 100 nm, respectively, attached to each microbubble. Replacing avidin with neutravidin resulted in 3-fold higher binding, approaching the calculated saturation level. High-speed photography of this new drug delivery vehicle demonstrated that the liposome-bearing microbubbles oscillate in response to an acoustic pulse in a manner similar to microbubble contrast agents. Additionally, microbubbles carrying liposomes could be spatially concentrated on a monolayer of PC-3 cells at the focal point of ultrasound beam. As a result of cell-vehicle contact, the liposomes fused with the cells and internalization of NBD-cholesterol occurred shortly after incubation at 37 °C, with internalization of NBD-cholesterol substantially enhanced in the acoustic focus. ? 2007 Elsevier B.V. All rights reserved.","author":[{"dropping-particle":"","family":"Kheirolomoom","given":"Azadeh","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Dayton","given":"Paul A.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Lum","given":"Aaron F H","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Little","given":"Erika","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Paoli","given":"Eric E.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zheng","given":"Hairong","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ferrara","given":"Katherine W.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Controlled Release","id":"ITEM-2","issue":"3","issued":{"date-parts":[["2007"]]},"page":"275-284","title":"Acoustically-active microbubbles conjugated to liposomes: Characterization of a proposed drug delivery vehicle","type":"article-journal","volume":"118"},"uris":[""]},{"id":"ITEM-3","itemData":{"DOI":"10.1002/adfm.200700106","ISBN":"1043-0342","ISSN":"1616301X","abstract":"Cationic poly(ethylene glycol)ylated (PEGylated) liposomes are one of the most important gene transfer reagents in non-viral gene therapy. However, the low transfection efficiencies of highly PEGylated lipoplexes currently hamper their clinical use. Recently, ultrasound has been used in combination with microbubbles to enhance the uptake of genes in different cell types. However, the gene transfer efficiency still remains low in these experiments. To overcome the limitations of both techniques, we present the attachment of PEGylated lipoplexes to microbubbles via biotin-avidin-biotin linkages. Exposure of these lipoplex-loaded microbubbles to ultrasound results in the release of unaltered lipoplexes. Furthermore, these lipoplex-loaded microbubbles exhibit much higher transfection efficiencies than ldquofreerdquo PEGylated lipoplexes or naked plasmid DNA (pDNA) when combined with microbubbles and ultrasound. Interestingly, the lipoplex-loaded microbubbles only transfect cells when exposed to ultrasound, which is promising for space- and time-controlled gene transfer. Finally, this novel Trojan-horse-like concept can also be exploited to achieve the ultrasound-triggered release of nanoparticles containing other therapeutic agents such as anticancer drugs.","author":[{"dropping-particle":"","family":"Lentacker","given":"Ine","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Smedt","given":"Stefaan C.","non-dropping-particle":"De","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Demeester","given":"Joseph","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Marck","given":"Veerle","non-dropping-particle":"Van","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bracke","given":"Marc","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sanders","given":"Niek N.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Advanced Functional Materials","id":"ITEM-3","issue":"12","issued":{"date-parts":[["2007"]]},"page":"1910-1916","title":"Lipoplex-loaded microbubbles for gene delivery: A trojan horse controlled by ultrasound","type":"article-journal","volume":"17"},"uris":["",""]}],"mendeley":{"formattedCitation":"[6–8]","plainTextFormattedCitation":"[6–8]","previouslyFormattedCitation":"[6–8]"},"properties":{"noteIndex":0},"schema":""}[6–8]. Additionally, US targeted MB destruction (UTMD) has been associated with increased payload distribution ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1093/jnci/djt305","ISBN":"1460-2105 (Electronic)\\r0027-8874 (Linking)","ISSN":"14602105","PMID":"24168971","abstract":"BACKGROUND: Oncolytic viruses are among the most powerful and selective cancer therapeutics under development and are showing robust activity in clinical trials, particularly when administered directly into tumor nodules. However, their intravenous administration to treat metastatic disease has been stymied by unfavorable pharmacokinetics and inefficient accumulation in and penetration through tumors.\\n\\nMETHODS: Adenovirus (Ad) was \"stealthed\" with a new N-(2-hydroxypropyl)methacrylamide polymer, and circulation kinetics were characterized in Balb/C SCID mice (n = 8 per group) bearing human ZR-75-1 xenograft tumors. Then, to noninvasively increase extravasation of the circulating polymer-coated Ad into the tumor, it was coinjected with gas microbubbles and the tumor was exposed to 0.5 MHz focused ultrasound at peak rarefactional pressure of 1.2 MPa. These ultrasound exposure conditions were designed to trigger inertial cavitation, an acoustic phenomenon that produces shock waves and can be remotely monitored in real-time. Groups were compared with Student t test or one-way analysis of variance with Tukey correction where groups were greater than two. All statistical tests were two-sided.\\n\\nRESULTS: Polymer-coating of Ad reduced hepatic sequestration, infection (>8000-fold; P < .001), and toxicity and improved circulation half-life (>50-fold; P = .001). Combination of polymer-coated Ad, gas bubbles, and focused ultrasound enhanced tumor infection >30-fold; (4 × 10(6) photons/sec/cm(2); standard deviation = 3 × 10(6) with ultrasound vs 1.3 × 10(5); standard deviation = 1 × 10(5) without ultrasound; P = .03) and penetration, enabling kill of cells more than 100 microns from the nearest blood vessel. This led to substantial and statistically significant retardation of tumor growth and increased survival.\\n\\nCONCLUSIONS: Combining drug stealthing and ultrasound-induced cavitation may ultimately enhance the efficacy of a range of powerful therapeutics, thereby improving the treatment of metastatic cancer.","author":[{"dropping-particle":"","family":"Carlisle","given":"Robert","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Choi","given":"James","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bazan-Peregrino","given":"Miriam","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Laga","given":"Richard","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Subr","given":"Vladimir","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kostka","given":"Libor","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ulbrich","given":"Karel","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Coussios","given":"Constantin C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Seymour","given":"Leonard W.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of the National Cancer Institute","id":"ITEM-1","issue":"22","issued":{"date-parts":[["2013"]]},"page":"1701-1710","title":"Enhanced tumor uptake and penetration of virotherapy using polymer stealthing and focused ultrasound.","type":"article-journal","volume":"105"},"uris":["",""]}],"mendeley":{"formattedCitation":"[9]","plainTextFormattedCitation":"[9]","previouslyFormattedCitation":"[9]"},"properties":{"noteIndex":0},"schema":""}[9] and sonoporation ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.addr.2013.11.008","ISBN":"1872-8294 (Electronic)\r0169-409X (Linking)","ISSN":"18728294","PMID":"24270006","abstract":"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. ? 2013 Elsevier B.V.","author":[{"dropping-particle":"","family":"Lentacker","given":"I.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Cock","given":"I.","non-dropping-particle":"De","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Deckers","given":"R.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Smedt","given":"S. C.","non-dropping-particle":"De","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Moonen","given":"C. T W","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Advanced Drug Delivery Reviews","id":"ITEM-1","issued":{"date-parts":[["2014"]]},"page":"49-64","publisher":"Elsevier B.V.","title":"Understanding ultrasound induced sonoporation: Definitions and underlying mechanisms","type":"article-journal","volume":"72"},"uris":[""]}],"mendeley":{"formattedCitation":"[10]","plainTextFormattedCitation":"[10]","previouslyFormattedCitation":"[10]"},"properties":{"noteIndex":0},"schema":""}[10] in tissue. Recent reviews of UTMD have summarised the use of this method for the delivery of chemotherapies ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.14366/usg.17021","ISSN":"2288-5919","PMID":"28607323","abstract":"Recent advancements in ultrasound and microbubble (USMB) mediated drug delivery technology has shown that this approach can improve spatially confined delivery of drugs and genes to target tissues while reducing systemic dose and toxicity. The mechanism behind enhanced delivery of therapeutics is sonoporation, the formation of openings in the vasculature, induced by ultrasound-triggered oscillations and destruction of microbubbles. In this review, progress and challenges of USMB mediated drug delivery are summarized, with special focus on cancer therapy.","author":[{"dropping-particle":"","family":"Mullick Chowdhury","given":"Sayan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Lee","given":"Taehwa","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Willmann","given":"Jürgen K","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Ultrasonography","id":"ITEM-1","issue":"3","issued":{"date-parts":[["2017"]]},"page":"171-184","title":"Ultrasound-guided drug delivery in cancer","type":"article-journal","volume":"36"},"uris":["",""]},{"id":"ITEM-2","itemData":{"ISBN":"1334601704","author":[{"dropping-particle":"","family":"Jain","given":"Ankit","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Tiwari","given":"Ankita","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Verma","given":"Amit","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Jain","given":"Sanjay K","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Drug Deliv. and Transl. Res.","id":"ITEM-2","issued":{"date-parts":[["2017"]]},"publisher":"Drug Delivery and Translational Research","title":"Ultrasound-based triggered drug delivery to tumors","type":"article-journal"},"uris":["",""]}],"mendeley":{"formattedCitation":"[11,12]","plainTextFormattedCitation":"[11,12]","previouslyFormattedCitation":"[11,12]"},"properties":{"noteIndex":0},"schema":""}[11,12], genes and thrombolytic drugs ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1111/j.1747-0285.2012.01428.x.Identification","ISBN":"6176321972","ISSN":"1878-5832","PMID":"21959306","author":[{"dropping-particle":"","family":"Heath Martin","given":"K.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Dayton","given":"Paul A.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Wiley Interdiscip Rev Nanomed Nanobiotechnology","id":"ITEM-1","issue":"4","issued":{"date-parts":[["2013"]]},"page":"631-637","title":"Current Status and Prospects for Microbubbles in Ultrasound Theranostics","type":"article-journal","volume":"5"},"uris":["",""]}],"mendeley":{"formattedCitation":"[13]","plainTextFormattedCitation":"[13]","previouslyFormattedCitation":"[13]"},"properties":{"noteIndex":0},"schema":""}[13]. Several complementary targeting techniques have been developed to increase treatment localisation and so reduce potential side effects resulting from systemic administration. For example, MBs have been functionalised by attachment of antibodies enabling their binding to target tissues ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.2310/7290.2006.00016","ISBN":"1535-3508 (Print)\\n1535-3508 (Linking)","ISSN":"15353508","PMID":"16954028","abstract":"Radiation force produced by low-amplitude ultrasound at clinically relevant frequencies remotely translates freely flowing microbubble ultrasound contrast agents over distances up to centimeters from the luminal space to the vessel wall in order to enhance ligand-receptor contact in targeting applications. The question arises as to how the microbubble shell might be designed at the molecular level to fully take advantage of such physical forces in targeted adhesion for molecular imaging and controlled therapeutic release. Herein, we report on a novel surface architecture in which the tethered ligand is buried in a polymeric overbrush. Our results, with biotin-avidin as the model ligand-receptor pair, show that the overbrush conceals the ligand, thereby reducing immune cell binding and increasing circulation persistence. Targeted adhesion is achieved through application of ultrasound radiation force to instantly reveal the ligand within a well-defined focal zone and simultaneously bind the ligand and receptor. Our data illustrate how the adhesive properties of the contrast agent surface can be reversibly changed, from stealth to sticky, through the physical effects of ultrasound. This technique can be combined with any ligand-receptor pair to optimize targeted adhesion for ultrasonic molecular imaging.","author":[{"dropping-particle":"","family":"Borden","given":"Mark A.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sarantos","given":"Melissa R.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stieger","given":"Susanne M.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Simon","given":"Scott I.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ferrara","given":"Katherine W.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Dayton","given":"Paul A.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Molecular imaging : official journal of the Society for Molecular Imaging","id":"ITEM-1","issue":"3","issued":{"date-parts":[["2006"]]},"page":"139-147","title":"Ultrasound radiation force modulates ligand availability on targeted contrast agents.","type":"article-journal","volume":"5"},"uris":["",""]}],"mendeley":{"formattedCitation":"[14]","plainTextFormattedCitation":"[14]","previouslyFormattedCitation":"[14]"},"properties":{"noteIndex":0},"schema":""}[14]. The short half-life of MBs (<5 minutes in circulation) has presented a challenge for this method, and so acoustic radiation force has been investigated as a way to concentrate MBs and increase their binding to target sites ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/S0301-5629(99)00062-9","ISBN":"0301-5629","ISSN":"03015629","PMID":"10576262","abstract":"The goal of targeted imaging is to produce an enhanced view of physiological processes or pathological tissue components. Contrast agents may improve the specificity of imaging modalities through selective targeting, and this may be particularly significant when using ultrasound (US) to image inflammatory processes or thrombi. One means of selective targeting involves the attachment of contrast agents to the desired site with the use of a specific binding mechanism. Because molecular binding mechanisms are effective over distances on the order of nanometers, targeting effectiveness would be greatly increased if the agent is initially concentrated in a particular region, and if the velocity of the agent is decreased as it passes the potential binding site. Ultrasonic transmission produces a primary radiation force that can manipulate microbubbles with each acoustic pulse. Observations demonstrate that primary radiation force can displace US contrast agents from the center of the streamline to the wall of a 200-μm cellulose vessel in vitro. Here, the effects of radiation force on contrast agents in vivo are presented for the first time. Experimental results demonstrate that radiation force can displace a contrast agent to the wall of a 50-μm blood vessel in the mouse cremaster muscle, can significantly reduce the velocity of flowing contrast agents, and can produce a reversible aggregation. Acoustic radiation force presents a means to localize and concentrate contrast agents near a vessel wall, which may assist the delivery of targeted agents. Copyright (C) 1999 World Federation for Ultrasound in Medicine and Biology.","author":[{"dropping-particle":"","family":"Dayton","given":"Paul","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Klibanov","given":"Alexander","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Brandenburger","given":"Gary","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ferrara","given":"Kathy","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Ultrasound in Medicine and Biology","id":"ITEM-1","issue":"8","issued":{"date-parts":[["1999"]]},"page":"1195-1201","title":"Acoustic radiation force in vivo: A mechanism to assist targeting of microbubbles","type":"article-journal","volume":"25"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.2310/7290.2006.00016","ISBN":"1535-3508 (Print)\\n1535-3508 (Linking)","ISSN":"15353508","PMID":"16954028","abstract":"Radiation force produced by low-amplitude ultrasound at clinically relevant frequencies remotely translates freely flowing microbubble ultrasound contrast agents over distances up to centimeters from the luminal space to the vessel wall in order to enhance ligand-receptor contact in targeting applications. The question arises as to how the microbubble shell might be designed at the molecular level to fully take advantage of such physical forces in targeted adhesion for molecular imaging and controlled therapeutic release. Herein, we report on a novel surface architecture in which the tethered ligand is buried in a polymeric overbrush. Our results, with biotin-avidin as the model ligand-receptor pair, show that the overbrush conceals the ligand, thereby reducing immune cell binding and increasing circulation persistence. Targeted adhesion is achieved through application of ultrasound radiation force to instantly reveal the ligand within a well-defined focal zone and simultaneously bind the ligand and receptor. Our data illustrate how the adhesive properties of the contrast agent surface can be reversibly changed, from stealth to sticky, through the physical effects of ultrasound. This technique can be combined with any ligand-receptor pair to optimize targeted adhesion for ultrasonic molecular imaging.","author":[{"dropping-particle":"","family":"Borden","given":"Mark A.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sarantos","given":"Melissa R.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stieger","given":"Susanne M.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Simon","given":"Scott I.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ferrara","given":"Katherine W.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Dayton","given":"Paul A.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Molecular imaging : official journal of the Society for Molecular Imaging","id":"ITEM-2","issue":"3","issued":{"date-parts":[["2006"]]},"page":"139-147","title":"Ultrasound radiation force modulates ligand availability on targeted contrast agents.","type":"article-journal","volume":"5"},"uris":["",""]}],"mendeley":{"formattedCitation":"[14,15]","plainTextFormattedCitation":"[14,15]","previouslyFormattedCitation":"[14,15]"},"properties":{"noteIndex":0},"schema":""}[14,15]. Another approach has been to incorporate magnetic material into MBs and accumulate them in a target region using an external magnetic field ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.3109/02656736.2012.668639","ISBN":"0265-6736","ISSN":"0265-6736","PMID":"22621737","abstract":"This paper reviews the uses of magnetism and ultrasound in therapeutic delivery applications. Emphasis is placed upon magnetic nanoparticles and microbubble ultrasound contrast agents. The underlying physical principles, history, key developments and limitations of these techniques for drug and gene delivery in vitro and in vivo are explored. The combination of ultrasonic and magnetic techniques is also reviewed with particular focus on magnetic microbubbles as delivery agents with the potential to combine the advantages of both methods whilst addressing many of their limitations. Finally, results are presented from a study of a new magnetic microbubble formulation which shows great applicability as a therapeutic delivery vehicle.","author":[{"dropping-particle":"","family":"Owen","given":"Joshua","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Pankhurst","given":"Quentin","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"International Journal of Hyperthermia","id":"ITEM-1","issue":"4","issued":{"date-parts":[["2012"]]},"page":"362-373","title":"Magnetic targeting and ultrasound mediated drug delivery: Benefits, limitations and combination","type":"article-journal","volume":"28"},"uris":["",""]}],"mendeley":{"formattedCitation":"[16]","plainTextFormattedCitation":"[16]","previouslyFormattedCitation":"[16]"},"properties":{"noteIndex":0},"schema":""}[16]. This method was used in a recent study by the authors in which magnetically responsive oxygen MBs (MagO2MBs) were used to deliver a combination of an antimetabolite drug (5-fluouracil) and sonodynamic therapy (SDT) to pancreatic tumours ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.jconrel.2017.07.040","ISSN":"01683659","abstract":"Magnetically responsive microbubbles (MagMBs), consisting of an oxygen gas core and a phospholipid coating functionalised with Rose Bengal (RB) and/or 5-fluorouracil (5-FU), were assessed as a delivery vehicle for the targeted treatment of pancreatic cancer using combined antimetabolite and sonodynamic therapy (SDT). MagMBs delivering the combined 5-FU/SDT treatment produced a reduction in cell viability of over 50% when tested against a panel of four pancreatic cancer cell lines in vitro. Intravenous administration of the MagMBs to mice bearing orthotopic human xenograft BxPC-3 tumours yielded a 48.3% reduction in tumour volume relative to an untreated control group (p < 0.05) when the tumour was exposed to both external magnetic and ultra- sound fields during administration of the MagMBs. In contrast, application of an external ultrasound field alone resulted in a 27% reduction in tumour volume. In addition, activated caspase and BAX protein levels were both observed to be significantly elevated in tumours harvested from animals treated with the MagMBs in the pre- sence of magnetic and ultrasonic fields when compared to expression of those proteins in tumours from either the control or ultrasound field only groups (p < 0.05). These results suggest MagMBs have considerable po- tential as a platform to enable the targeted delivery of combined sonodynamic/antimetabolite therapy in pan- creatic cancer.","author":[{"dropping-particle":"","family":"Sheng","given":"Yingjie","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Beguin","given":"Estelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nesbitt","given":"Heather","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kamila","given":"Sukanta","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Owen","given":"Joshua","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Barnsley","given":"Lester C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"Bridgeen","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"O'Kane","given":"Christopher","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nomikou","given":"Nikolitsa","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hamoudi","given":"Rifat","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Taylor","given":"Mark A.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Love","given":"Mark","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kelly","given":"Paul","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"O'Rourke","given":"Declan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"McHale","given":"Anthony P.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"John F.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Controlled Release","id":"ITEM-1","issue":"June","issued":{"date-parts":[["2017"]]},"page":"192-200","publisher":"Elsevier","title":"Magnetically responsive microbubbles as delivery vehicles for targeted sonodynamic and antimetabolite therapy of pancreatic cancer","type":"article-journal","volume":"262"},"uris":[""]}],"mendeley":{"formattedCitation":"[17]","plainTextFormattedCitation":"[17]","previouslyFormattedCitation":"[17]"},"properties":{"noteIndex":0},"schema":""}[17]. While the combination of magnetic and US fields demonstrated an improved tumour growth delay and increased apoptotic cell signalling compared to the treatment with US only, the simultaneous application and alignment of magnetic and US fields represented a significant practical challenge in vivo. This problem is particularly acute in small animal models due to space constraints and may greatly limit the potential synergistic benefits of magnetic-acoustic targeting. In the present study, this was addressed by using a prototype probe enabling co-aligned US and magnetic fields to be applied simultaneously ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1002/admt.201800081","ISSN":"2365709X","author":[{"dropping-particle":"","family":"Barnsley","given":"Lester C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gray","given":"Michael D.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Beguin","given":"Estelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Carugo","given":"Dario","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Advanced Materials Technologies","id":"ITEM-1","issue":"7","issued":{"date-parts":[["2018"]]},"page":"1800081","title":"A Combined Magnetic-Acoustic Device for Simultaneous, Coaligned Application of Magnetic and Ultrasonic Fields","type":"article-journal","volume":"3"},"uris":["",""]}],"mendeley":{"formattedCitation":"[20]","plainTextFormattedCitation":"[20]","previouslyFormattedCitation":"[19]"},"properties":{"noteIndex":0},"schema":""}[20]. The aim of the experiments described in the next section was to determine the effect upon drug delivery first in vitro in a tissue mimicking phantom and subsequently in vivo in a murine pancreatic cancer model.2. Materials and methodsThis section details the materials and suppliers used, followed by a description of the chemo/sonodynamic therapy complex used in all of the experiments and its in vitro evaluation with cultured cancer cells. The formulation and characterisation of drug-loaded, oxygen-filled, magnetically functionalised microbubbles are then presented and the devices for non-invasively providing magnetic and ultrasonic fields introduced. The section concludes with descriptions of the in vitro and in vivo experiments undertaken to determine the delivery potential of the drug loaded bubbles under different combinations of magnetic and ultrasound fields.2.1 Reagents, equipment and softwareBiotinylated Rose Bengal (RB) (compound 9) was prepared as described in ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.jconrel.2015.02.004","ISSN":"1873-4995","PMID":"25660073","abstract":"Tumour hypoxia represents a major challenge in the effective treatment of solid cancerous tumours using conventional approaches. As oxygen is a key substrate for Photo-/Sono-dynamic Therapy (PDT/SDT), hypoxia is also problematic for the treatment of solid tumours using these techniques. The ability to deliver oxygen to the vicinity of the tumour increases its local partial pressure improving the possibility of ROS generation in PDT/SDT. In this manuscript, we investigate the use of oxygen-loaded, lipid-stabilised microbubbles (MBs), decorated with a Rose Bengal sensitiser, for SDT-based treatment of a pancreatic cancer model (BxPc-3) in vitro and in vivo. We directly compare the effectiveness of the oxygen-loaded MBs with sulphur hexafluoride (SF6)-loaded MBs and reveal a significant improvement in therapeutic efficacy. The combination of oxygen-carrying, ultrasound-responsive MBs, with an ultrasound-responsive therapeutic sensitiser, offers the possibility of delivering and activating the MB-sensitiser conjugate at the tumour site in a non-invasive manner, providing enhanced sonodynamic activation at that site.","author":[{"dropping-particle":"","family":"McEwan","given":"Conor","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Owen","given":"Joshua","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Fowley","given":"Colin","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nesbitt","given":"Heather","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Cochrane","given":"David","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Coussios","given":"Constantin C","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Borden","given":"M","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nomikou","given":"Nikolitsa","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"McHale","given":"Anthony P","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"John F","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Controlled Release","id":"ITEM-1","issued":{"date-parts":[["2015","4","10"]]},"note":"We investigate the use of oxygen-loaded, lipid-stabilised microbubbles(MBs), decorated with a Rose Bengal sensitiser, for SDT-based treatment of a pancreatic cancermodel (BxPc-3) in vitro and in vivo.\n\nDuring in vivo study the MBs were directly injected into the mices' tumors.\nThe average size of the MBs was 1–2 μm with a concentration of approximately 1 × 109 MB/ml","page":"51-6","title":"Oxygen carrying microbubbles for enhanced sonodynamic therapy of hypoxic tumours.","type":"article-journal","volume":"203"},"uris":[""]}],"mendeley":{"formattedCitation":"[21]","plainTextFormattedCitation":"[21]","previouslyFormattedCitation":"[20]"},"properties":{"noteIndex":0},"schema":""}[21]. Reagents used for the preparation of the chemo-sonodynamic drug complex combining the sonosensitiser Rose Bengal (RB) and antimetabolite drug gemcitabine (Gem) (compound 8) were purchased from Sigma Aldrich (Gillingham, Dorset, UK) at the highest grade available with the exception of biotin, di(N-succinimidyl) carbonate and 2-aminoethanol which were purchased from Tokyo Chemical Industry UK Ltd. NMR spectra were obtained on a Varian 500 MHz instrument (Palo Alto, CA, USA) at 25.0 ± 1oC and processed using Bruker software (Billerica, MA, USA). Mass spectra were obtained on a Finnigan LCQMS instrument (San Jose, CA, USA).The microbubbles were produced from a lipid mixture of 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) -2000] (DSPE-PEG2000) and DSPE-PEG2000-biotin from Avanti Polar Lipids (Alabaster, Alabama, USA). All reagents and equipment used for magnetic oxygen MB production were as previously described in ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.jconrel.2017.07.040","ISSN":"01683659","abstract":"Magnetically responsive microbubbles (MagMBs), consisting of an oxygen gas core and a phospholipid coating functionalised with Rose Bengal (RB) and/or 5-fluorouracil (5-FU), were assessed as a delivery vehicle for the targeted treatment of pancreatic cancer using combined antimetabolite and sonodynamic therapy (SDT). MagMBs delivering the combined 5-FU/SDT treatment produced a reduction in cell viability of over 50% when tested against a panel of four pancreatic cancer cell lines in vitro. Intravenous administration of the MagMBs to mice bearing orthotopic human xenograft BxPC-3 tumours yielded a 48.3% reduction in tumour volume relative to an untreated control group (p < 0.05) when the tumour was exposed to both external magnetic and ultra- sound fields during administration of the MagMBs. In contrast, application of an external ultrasound field alone resulted in a 27% reduction in tumour volume. In addition, activated caspase and BAX protein levels were both observed to be significantly elevated in tumours harvested from animals treated with the MagMBs in the pre- sence of magnetic and ultrasonic fields when compared to expression of those proteins in tumours from either the control or ultrasound field only groups (p < 0.05). These results suggest MagMBs have considerable po- tential as a platform to enable the targeted delivery of combined sonodynamic/antimetabolite therapy in pan- creatic cancer.","author":[{"dropping-particle":"","family":"Sheng","given":"Yingjie","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Beguin","given":"Estelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nesbitt","given":"Heather","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kamila","given":"Sukanta","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Owen","given":"Joshua","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Barnsley","given":"Lester C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"Bridgeen","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"O'Kane","given":"Christopher","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nomikou","given":"Nikolitsa","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hamoudi","given":"Rifat","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Taylor","given":"Mark A.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Love","given":"Mark","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kelly","given":"Paul","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"O'Rourke","given":"Declan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"McHale","given":"Anthony P.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"John F.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Controlled Release","id":"ITEM-1","issue":"June","issued":{"date-parts":[["2017"]]},"page":"192-200","publisher":"Elsevier","title":"Magnetically responsive microbubbles as delivery vehicles for targeted sonodynamic and antimetabolite therapy of pancreatic cancer","type":"article-journal","volume":"262"},"uris":[""]}],"mendeley":{"formattedCitation":"[17]","plainTextFormattedCitation":"[17]","previouslyFormattedCitation":"[17]"},"properties":{"noteIndex":0},"schema":""}[17] with the exception of the superparamagnetic iron oxide nanoparticles (IONPs) and therapeutic agent (i.e. 8). The IONPs (50 nm hydrodynamic diameter) were custom-conjugated by Ocean NanoTech (San Diego, CA, USA). The design and calibration of the magnetic-acoustic device (MAD) having co-aligned acoustic and magnetic fields is described in ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1002/admt.201800081","ISSN":"2365709X","author":[{"dropping-particle":"","family":"Barnsley","given":"Lester C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gray","given":"Michael D.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Beguin","given":"Estelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Carugo","given":"Dario","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Advanced Materials Technologies","id":"ITEM-1","issue":"7","issued":{"date-parts":[["2018"]]},"page":"1800081","title":"A Combined Magnetic-Acoustic Device for Simultaneous, Coaligned Application of Magnetic and Ultrasonic Fields","type":"article-journal","volume":"3"},"uris":["",""]}],"mendeley":{"formattedCitation":"[20]","plainTextFormattedCitation":"[20]","previouslyFormattedCitation":"[19]"},"properties":{"noteIndex":0},"schema":""}[20]. The paraffin wax and US gel used during MAD testing were obtained from FullMoons Cauldron (Berkshire, UK) and Ana Wiz Ltd. (Anagel, Surrey, UK), respectively. UltraPure low melting point agarose was purchased from Thermo Fischer Scientific, (Paisley, UK) and the syringe pump was an AL-1000 from World Precision Instruments (Sarasota, FL, USA). Ultrasound drive signals were provided by a waveform generator (33500B, Keysight, Santa Rosa, CA, USA) and passed to a power amplifier (1040L, E&I Ltd., Rochester, NY, USA). All ultrasound data sets were processed using MATLAB (Mathworks, Natick, MA, USA). Passive cavitation detection (PCD) was enabled by a single element focused transducer (7.5?MHz center frequency, 12.7?mm diameter, 75?mm focal distance, Olympus NDT, Essex, UK). Signals were passed to a single stage preamplifier (SR455A, Stanford Research Systems, Sunnyvale, CA, USA) before streaming to computer disk using a two-channel digitizer (HS-3, TiePie Engineering, Sneek, Netherlands). Additional description of the methods used to acquire and analyse the PCD data are provided in the subsequent sections and summarized in the supporting information (section 5).MBs were ruptured using an US bath (Eumax, UD150SH-6L, 40 kHz, 150 W) prior to determining drug loading using a FLUOstar Omega multi-purpose plate reader from BMG Labtech (Aylesbury, Bucks, UK). The iron loading on MBs was determined by inductively coupled plasma - optical emission spectroscopy (ICP-OES) using an Optima 8000 instrument from Perkin Elmer (Seer Green, UK). Singlet oxygen sensor green (SOSG) was purchased from Thermo Fisher Scientific (Paisley, UK).2.2 Preparation of chemo/sonodynamic therapy complex (biotin-RB-Gem) In our previous work, we investigated the combination of 5-fluouracil (chemotherapy) and Rose-Bengal (sonodynamic therapy). As gemcitabine has been reported as the antimetabolite therapy of choice for pancreatic cancer, superseding treatments with 5-fluouracil ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1126/science.1198443","ISBN":"0732-183X (Print)\\n0732-183X (Linking)","ISSN":"0732-183X","PMID":"9196156","abstract":"PURPOSE: Most patients with advanced pancreas cancer experience pain and must limit their daily activities because of tumor-related symptoms. To date, no treatment has had a significant impact on the disease. In early studies with gemcitabine, patients with pancreas cancer experienced an improvement in disease-related symptoms. Based on those findings, a definitive trial was performed to assess the effectiveness of gemcitabine in patients with newly diagnosed advanced pancreas cancer. PATIENTS AND METHODS: One hundred twenty-six patients with advanced symptomatic pancreas cancer completed a lead-in period to characterize and stabilize pain and were randomized to receive either gemcitabine 1,000 mg/m2 weekly x 7 followed by 1 week of rest, then weekly x 3 every 4 weeks thereafter (63 patients), or to fluorouracil (5-FU) 600 mg/m2 once weekly (63 patients). The primary efficacy measure was clinical benefit response, which was a composite of measurements of pain (analgesic consumption and pain intensity), Karnofsky performance status, and weight. Clinical benefit required a sustained (> or = 4 weeks) improvement in at least one parameter without worsening in any others. Other measures of efficacy included response rate, time to progressive disease, and survival. RESULTS: Clinical benefit response was experienced by 23.8% of gemcitabine-treated patients compared with 4.8% of 5-FU-treated patients (P = .0022). The median survival durations were 5.65 and 4.41 months for gemcitabine-treated and 5-FU-treated patients, respectively (P = .0025). The survival rate at 12 months was 18% for gemcitabine patients and 2% for 5-FU patients. Treatment was well tolerated. CONCLUSION: This study demonstrates that gemcitabine is more effective than 5-FU in alleviation of some disease-related symptoms in patients with advanced, symptomatic pancreas cancer. Gemcitabine also confers a modest survival advantage over treatment with 5-FU.","author":[{"dropping-particle":"","family":"Burris","given":"HA A","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Moore","given":"MJ J","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Andersen","given":"J","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Green","given":"M R","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Rothenberg","given":"M L","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Modiano","given":"M R","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Cripps","given":"M C","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Portenoy","given":"R K","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Storniolo","given":"A M","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Tarassoff","given":"P","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nelson","given":"R","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Dorr","given":"F A","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stephens","given":"C D","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hoff","given":"D D","non-dropping-particle":"Von","parse-names":false,"suffix":""}],"container-title":"Journal of Clinical Oncology","id":"ITEM-1","issue":"6","issued":{"date-parts":[["1997"]]},"page":"2403-2413","title":"Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial.","type":"article-journal","volume":"15"},"uris":[""]}],"mendeley":{"formattedCitation":"[18]","plainTextFormattedCitation":"[18]"},"properties":{"noteIndex":0},"schema":""}[18], recent work by the authors has also presented microbubbles loaded separately with gemcitabine and Rose Bengal ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.jconrel.2018.04.018","ISSN":"18734995","abstract":"Pancreatic cancer remains one of the most lethal forms of cancer with a 10-year survival of <1%. With little improvement in survival rates observed in the past 40 years, there is a significant need for new treatments or more effective strategies to deliver existing treatments. The antimetabolite gemcitabine (Gem) is the most widely used form of chemotherapy for pancreatic cancer treatment, but is known to produce significant side effects when administered systemically. We have previously demonstrated the benefit of combined chemo-sonodynamic therapy (SDT), delivered using oxygen carrying microbubbles (O2MB), as a targeted treatment for pancreatic cancer in a murine model of the disease. In this manuscript, we report the preparation of a biotin functionalised Gem ligand for attachment to O2MBs (O2MB-Gem). We demonstrate the effectiveness of chemo-sonodynamic therapy following ultrasound-targeted-microbubble-destruction (UTMD) of the O2MB-Gem and a Rose Bengal loaded O2MB (O2MB-RB) as a targeted treatment for pancreatic cancer. Specifically, UTMD using the O2MB-Gem and O2MB-RB conjugates reduced the viability of MIA PaCa-2, PANC-1, BxPC3 and T110299 pancreatic cancer cells by >60% (p < 0.001) and provided significant tumour growth delay (>80%, p < 0.001) compared to untreated animals when human xenograft MIA PaCa-2 tumours were treated in SCID mice. The toxicity of the O2MB-Gem conjugate was also determined in healthy non-tumour bearing MF1 mice and revealed no evidence of renal or hepatic damage. Therefore, the results presented in this manuscript suggest that chemo-sonodynamic therapy using the O2MB-Gem and O2MB-RB conjugates, is potentially an effective targeted and safe treatment modality for pancreatic cancer.","author":[{"dropping-particle":"","family":"Nesbitt","given":"Heather","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sheng","given":"Yingjie","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kamila","given":"Sukanta","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Logan","given":"Keiran","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Thomas","given":"Keith","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"Bridgeen","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Taylor","given":"Mark A.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Love","given":"Mark","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"O'Rourke","given":"Declan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kelly","given":"Paul","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Beguin","given":"Estelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"McHale","given":"Anthony P.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"John F.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Controlled Release","id":"ITEM-1","issue":"December 2017","issued":{"date-parts":[["2018"]]},"page":"8-16","publisher":"Elsevier","title":"Gemcitabine loaded microbubbles for targeted chemo-sonodynamic therapy of pancreatic cancer","type":"article-journal","volume":"279"},"uris":[""]}],"mendeley":{"formattedCitation":"[19]","plainTextFormattedCitation":"[19]","previouslyFormattedCitation":"[18]"},"properties":{"noteIndex":0},"schema":""}[19] and this was the combination selected for the present study.To enable the loading of both the RB and Gem on the MB surface, a novel therapeutic was formulated with a single biotin anchor connected to both drugs. The methods used to prepare this complex are described in this section following Scheme 1.Scheme. SEQ Scheme \* ARABIC 1. Synthetic scheme for the production of biotin-RB-Gem (8) and biotin-RB (9).2.2.1 Synthesis of N-(2-(bis(2-aminoethyl)amino)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (3)To a stirred solution of tris(2-aminoethyl)amine (2) (0.22 g, 1.5 mmol) and trimethylamine (TEA) (catalytic amount) in anhydrous dimethylformamide (DMF) (5 mL), Biotin-NHS (1) (0.5 g, 1.5 mmol) was added and the reaction mixture was stirred at 0oC under a nitrogen atmosphere for 30 min. The solvent was removed under reduced pressure and the residue was triturated with diethyl ether. The crude product was purified by column chromatography on basic (TEA) silica gel (methanol: dichloromethane 1:9 to 3:7) to give 3 (0.33 g, 61% yield) as a white semi solid. 1H NMR (DMSO-d6): δ 7.94 (brs, 1H, NH), 6.42 (brs, 1H, NH), 6.35 (brs, 1H, NH), 4.49 (brs, 4H, NH2 X 2), 4.29 (s, 1H, CH), 4.12 (s, 1H, CH), 3.07-3.02 (m, 6H, CH2 X 3), 2.88-2.82 (m, 1H, CH), 2.44-2.06 (m, 10H, CH2 X 5), 1.59-1.48 (m, 4H, CH2 X 2), 1.47-1.29 (m, 2H, CH2). ESI-MS: calculated for C16H32N6O2S, 372.23; found 373.31 (M +H). 2.2.2 Synthesis of bis(2,5-dioxopyrrolidin-1-yl) 8,8'-((((2-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl)azanediyl)bis(ethane-2,1-diyl))bis(azanediyl))bis(8-oxooctanoate) (5)To a stirred solution of compound 3 (0.5 g, 1.3 mmol) and TEA (catalytic amount) in 10 mL anhydrous DMF was added disuccinimidyl suberate (4, 1 g, 2.7mmol) and the reaction mixture stirred at room temperature for 6 hrs under a nitrogen atmosphere. After completion of the reaction, excess diethyl ether (200 mL) was added to the reaction mixture. The white precipitate thus obtained was filtered and washed 3 times with diethyl ether (50 mL X 3). The crude product was purified by column chromatography on basic (TEA) silica gel (methanol : chloroform 2:8 to 5:5 v/v) to give 5 (0.83 g, 71% yield) as low melting white solid. 1H NMR (DMSO-d6): δ 7.94 (brs, 2H, NH X 2), 7.67 (brs, 1H, NH), 6.41 (brs, 1H, NH), 6.34 (brs, 1H, NH), 4.29 (s, 1H, CH), 4.12 (s, 1H, CH), 3.06-3.04 (m, 3H, CH and CH2), 2.88-2.72 (m, 6H, CH2 X 3), 2.71-2.63 (m, 8H, CH2 X 4), 2.45-2.34 (m, 6H, CH2 X 3), 2.20-2.06 (m, 10H, CH2 X 5), 1.60-1.21 (m, 22H, CH2 X 11). 13C NMR (DMSO-d6): 172.5 (C=O), 170.7 (C=O), 163.1 (C=O), 162.7 (C=O), 61.4 (CH), 59.6 (CH), 55.8 (CH2), 53.9 (NCH2), 39.9 (CH2), 39.8(CH2), 39.6(CH2), 37.3(CH2), 36.2(CH2), 35.6(CH2), 31.2 (CH2), 28.7(CH2), 28.5(CH2), 25.8(CH2), 25.7(CH2), 25.6(CH2). ESI-MS: calculated for C40H62N8O12S, 878.4; found 901.3 (M +Na salt).2.2.3 Synthesis of ((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methyl 4,11,19-trioxo-15-(2-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl)-1-((2,3,4,5-tetrachloro-6-(6-hydroxy-2,4,5,7-tetraiodo-3-oxo-3H-xanthen-9-yl)benzoyl)oxy)-3,12,15,18-tetraazahexacosan-26-oate (8)To a stirred solution of 5 (0.4 g, 0.45 mmol) in anhydrous DMF (5 mL) was added gemcitabine hydrochloride (6, 0.136 g, 0.45 mmol) and TEA (0.5 mL) and the reaction was stirred at 22oC for 24 hrs under a nitrogen atmosphere. After completion of the reaction, Rose Bengal amine (7) (prepared separately according to ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1039/c2cc33913g","ISBN":"1364-548X (Electronic)\\r1359-7345 (Linking)","ISSN":"1364-548X","PMID":"22790600","abstract":"A Rose Bengal sonosensitiser has been covalently attached to a lipid microbubble and the resulting conjugate shown to produce higher levels of singlet oxygen, enhanced cytotoxicity in a cancer cell line and a greater reduction in tumour growth than the sonosensitiser alone.","author":[{"dropping-particle":"","family":"Nomikou","given":"Nikolitsa","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Fowley","given":"Colin","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Byrne","given":"Niall M","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"McCaughan","given":"Bridgeen","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"McHale","given":"Anthony P","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"John F","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Chemical communications (Cambridge, England)","id":"ITEM-1","issue":"67","issued":{"date-parts":[["2012"]]},"page":"8332-4","title":"Microbubble-sonosensitiser conjugates as therapeutics in sonodynamic therapy.","type":"article-journal","volume":"48"},"uris":[""]}],"mendeley":{"formattedCitation":"[22]","plainTextFormattedCitation":"[22]","previouslyFormattedCitation":"[21]"},"properties":{"noteIndex":0},"schema":""}[22]), (0.43 g, 0.45 mmol) in anhydrous DMF (5 mL) and TEA (0.5 mL) were added to the reaction mixture and continued to stir for 24 hrs. Once the reaction was complete, excess diethyl ether (200 mL) was added to the solution and stirred for 30 min. The pink red precipitate thus obtained was triturated with diethyl ether (100 mL), ethyl acetate (100 mL), acetone-water mixture (10%, v/v, 100 mL) and finally with ethyl acetate-hexane mixture (50%, v/v, 100 mL) respectively to afford a pink powder of compound 8 (0.26 g, 30% yield). 1H NMR (DMSO-d6), Fig. S1: δ 7.95 (brs, 2H, NH2), 7.69 (s, 1H, CH, aromatic proton), 7.68 (s, 1H, CH, aromatic proton), 7.37(s, 1H, CH), 7.32 (brs, 4H, NH X 4), 6.89 (s, 1H, CH), 6.42 (brs, 1H, NH), 6.35 (brs, 1H, NH), 6.22 (d, J = 5.5 Hz, 1H, CH), 6.13 (brs, 1H, NH), 5. 78-5.77 (m, 1H, CH), 5.19 (s, 1H, CH X 2), 4.9 (brs, 1H, OH), 4.30 (s, 2H, -OCH2), 4.13 (s, 2H, -OCH2), 3.79-3.60 (m, 3H, CH, CH2), 3.39-3.32 (m, 2H, CH2), 3.07 (brs, 6H, N-NHCH2 X 3), 2.94-2.84 (m, 6H, NCH2 X 3), 2.81 (brs, 1H, OH), 2.45-2.46 (m, 3H, CH, CH2), 2.17-2.06 (m, 10H, CH2 X 5), 1.60- 1.10 (m, 22H, CH2 X 11). 13C NMR (DMSO-d6), (Fig. S2) 171.8 (C=O, C), 165.98 (C=O), 163.2 (C=O), 162.7 (C=O), 159.3 (CH), 155.0 (C=O), 150.5 (C), 145.8 (C), 141.2 (CH), 131.0 (C), 128.7 (C), 123.5 (C), 116.2 (C), 95.0 (CH), 80.9 (C), 69.3 (CH), 61.5 (C), 59.6 (CH2), 59.4 (CH), 55.8 (CH), 51.7 (CH2), 45.8 (CH2), 40.2 (CH2), 37.3 (CH2), 37.05 (CH2), 36.2 (CH2), 35.5 (CH2), 31.0 (CH2), 28.7 (CH2), 28.5 (CH2), 28.2 (CH2), 25.6 (CH2). ESI-MS, (Fig. S3): calculated for C63H72Cl4F2I4 N10O15S, 1925.98; found 1925.90 (M -H).2.3 In vitro treatment of BxPC-3 and Mia-PaCa-2 cells with biotin-RB-Gem and Gemcitabine The human primary pancreatic adenocarcinoma cell line BxPC-3 was maintained in RPMI 1640 medium which was supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (FBS) in a humidified 5% CO2 atmosphere at 37oC. The Mia-PaCa-2 cell line was maintained using Dulbecco’s Modified Eagle’s Medium (DMEM) containing 1 g/L glucose and supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (FBS) in a humidified 5% CO2 atmosphere at 37oC. These cells were seeded into 96-well plates at a density of 5000 cells per well. The plates were then incubated for 24 hrs followed by the addition of 100 μL of media spiked with Gem or 8 at concentrations ranging from 0.001-1000 μM. The cells were then further incubated for 48 hrs before the cell viability was determined by an MTT assay.2.4 Preparation of drug-loaded magnetic MBsAvidin functionalised magnetically responsive MBs (MagMBs) were prepared through the sonication of a lipid mixture containing DBPC:DSPE-PEG2000:DSPE-PEG2000-biotin at a ratio of (82:9:9) as previously described in ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.jconrel.2017.07.040","ISSN":"01683659","abstract":"Magnetically responsive microbubbles (MagMBs), consisting of an oxygen gas core and a phospholipid coating functionalised with Rose Bengal (RB) and/or 5-fluorouracil (5-FU), were assessed as a delivery vehicle for the targeted treatment of pancreatic cancer using combined antimetabolite and sonodynamic therapy (SDT). MagMBs delivering the combined 5-FU/SDT treatment produced a reduction in cell viability of over 50% when tested against a panel of four pancreatic cancer cell lines in vitro. Intravenous administration of the MagMBs to mice bearing orthotopic human xenograft BxPC-3 tumours yielded a 48.3% reduction in tumour volume relative to an untreated control group (p < 0.05) when the tumour was exposed to both external magnetic and ultra- sound fields during administration of the MagMBs. In contrast, application of an external ultrasound field alone resulted in a 27% reduction in tumour volume. In addition, activated caspase and BAX protein levels were both observed to be significantly elevated in tumours harvested from animals treated with the MagMBs in the pre- sence of magnetic and ultrasonic fields when compared to expression of those proteins in tumours from either the control or ultrasound field only groups (p < 0.05). These results suggest MagMBs have considerable po- tential as a platform to enable the targeted delivery of combined sonodynamic/antimetabolite therapy in pan- creatic cancer.","author":[{"dropping-particle":"","family":"Sheng","given":"Yingjie","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Beguin","given":"Estelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nesbitt","given":"Heather","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kamila","given":"Sukanta","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Owen","given":"Joshua","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Barnsley","given":"Lester C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"Bridgeen","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"O'Kane","given":"Christopher","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nomikou","given":"Nikolitsa","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hamoudi","given":"Rifat","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Taylor","given":"Mark A.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Love","given":"Mark","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kelly","given":"Paul","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"O'Rourke","given":"Declan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"McHale","given":"Anthony P.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"John F.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Controlled Release","id":"ITEM-1","issue":"June","issued":{"date-parts":[["2017"]]},"page":"192-200","publisher":"Elsevier","title":"Magnetically responsive microbubbles as delivery vehicles for targeted sonodynamic and antimetabolite therapy of pancreatic cancer","type":"article-journal","volume":"262"},"uris":[""]}],"mendeley":{"formattedCitation":"[17]","plainTextFormattedCitation":"[17]","previouslyFormattedCitation":"[17]"},"properties":{"noteIndex":0},"schema":""}[17]. However, in the current work, 1,2-dibehenoyl-sn-glycero-3-phosphocholine coated IONPs were used (3.75 mg iron) instead of FluidMAG-Lipid nanoparticles, in order to use the same lipid as the one composing the microbubble coating. Compound 8 or 9 (1 mL, 5.2 mM in PBS with 0.5% (v/v) DMSO) was loaded onto the MagMBs following the method reported in ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.jconrel.2017.07.040","ISSN":"01683659","abstract":"Magnetically responsive microbubbles (MagMBs), consisting of an oxygen gas core and a phospholipid coating functionalised with Rose Bengal (RB) and/or 5-fluorouracil (5-FU), were assessed as a delivery vehicle for the targeted treatment of pancreatic cancer using combined antimetabolite and sonodynamic therapy (SDT). MagMBs delivering the combined 5-FU/SDT treatment produced a reduction in cell viability of over 50% when tested against a panel of four pancreatic cancer cell lines in vitro. Intravenous administration of the MagMBs to mice bearing orthotopic human xenograft BxPC-3 tumours yielded a 48.3% reduction in tumour volume relative to an untreated control group (p < 0.05) when the tumour was exposed to both external magnetic and ultra- sound fields during administration of the MagMBs. In contrast, application of an external ultrasound field alone resulted in a 27% reduction in tumour volume. In addition, activated caspase and BAX protein levels were both observed to be significantly elevated in tumours harvested from animals treated with the MagMBs in the pre- sence of magnetic and ultrasonic fields when compared to expression of those proteins in tumours from either the control or ultrasound field only groups (p < 0.05). These results suggest MagMBs have considerable po- tential as a platform to enable the targeted delivery of combined sonodynamic/antimetabolite therapy in pan- creatic cancer.","author":[{"dropping-particle":"","family":"Sheng","given":"Yingjie","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Beguin","given":"Estelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nesbitt","given":"Heather","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kamila","given":"Sukanta","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Owen","given":"Joshua","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Barnsley","given":"Lester C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"Bridgeen","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"O'Kane","given":"Christopher","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nomikou","given":"Nikolitsa","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hamoudi","given":"Rifat","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Taylor","given":"Mark A.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Love","given":"Mark","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kelly","given":"Paul","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"O'Rourke","given":"Declan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"McHale","given":"Anthony P.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"John F.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Controlled Release","id":"ITEM-1","issue":"June","issued":{"date-parts":[["2017"]]},"page":"192-200","publisher":"Elsevier","title":"Magnetically responsive microbubbles as delivery vehicles for targeted sonodynamic and antimetabolite therapy of pancreatic cancer","type":"article-journal","volume":"262"},"uris":[""]}],"mendeley":{"formattedCitation":"[17]","plainTextFormattedCitation":"[17]","previouslyFormattedCitation":"[17]"},"properties":{"noteIndex":0},"schema":""}[17]. All microbubbles evaluated in this manuscript were washed three times by centrifugation to remove excess material from the suspension. In vitro, MagMB characterisation was completed using 9 to prevent wastage of the more difficult to synthesise 8. Drug-loaded MagMBs were kept in reduced light conditions and on ice prior to use and are referred to as MagO2MB-RB-Gem (Fig. 1) or MagO2MB-RB depending on which drug product was used. If the samples were not sparged with oxygen, they are referred to as MagMB-RB-Gem and MagMB-RB.Fig. 1 Schematic representation of the MagO2MB-RB-Gem conjugate.2.5 Characterisation of MB size and concentrationThe MBs were characterised for their size and concentration following analysis of optical microscope images using a custom MATLAB script ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.ultrasmedbio.2010.09.004","ISSN":"03015629","PMID":"21030137","abstract":"Intra- and interobserver (n = 3) variability of sizing and counting microbubbles using optical microscopy (OM) was assessed. The system was calibrated using standardised mono-disperse and poly-disperse microspheres. Results of the calibration show intraobserver variations of number count (C) = 13.0% and arithmetic mean size (MS) = 0.2%, and interobserver variations of C = 18.4% and MS = 0.6%, for the mono-disperse microspheres. For the poly-disperse microspheres, intraobserver variations were: C = 6.9% and MS = 0.8%, and interobserver: C = 10.5% and MS = 0.3%. For SonoVue? the intraobserver variations were: C = 23.3% and MS = 8.0%, and interobserver C = 6.8% and MS = 3.8%. The results suggest that the higher values of the intraobserver variation for SonoVue? arise from the natural decay of microbubbles over time. This article presents a detailed protocol and outlines potential pitfalls in our approach. These results are in general agreement with those previously reported and compare well with known size distributions. ? 2010 World Federation for Ultrasound in Medicine & Biology.","author":[{"dropping-particle":"","family":"Sennoga","given":"Charles a.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Mahue","given":"Veronique","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Loughran","given":"Jonathan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Casey","given":"Jonathan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Seddon","given":"John M.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Tang","given":"Mengxing","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Eckersley","given":"Robert J.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Ultrasound in Medicine and Biology","id":"ITEM-1","issue":"12","issued":{"date-parts":[["2010"]]},"page":"2093-2096","title":"On sizing and counting of microbubbles using optical microscopy","type":"article-journal","volume":"36"},"uris":["",""]}],"mendeley":{"formattedCitation":"[23]","plainTextFormattedCitation":"[23]","previouslyFormattedCitation":"[22]"},"properties":{"noteIndex":0},"schema":""}[23]. For this, 10 μL of a diluted (1:20 v/v) sample in PBS was loaded onto a haemocytometer and imaged 30 times using an optical microscope fitted with a 40X objective, leading to approximately 1800 microbubbles examined per microbubble batch.2.6 Characterisation of MB drug loadingThe drug loading of MBs was investigated for both 8 and 9 using UV-Vis spectroscopy. As the ratio of RB to Gem in each molecule of 8 is 1:1, the concentration of both Gem and RB attached to MagO2MB-RB-Gem can be determined from the RB absorbance at 559 nm, using a previously constructed calibration graph. Similarly, for 9, the RB concentration of MagO2MB-RB was determined based on the RB absorbance at 559 nm. For each MB batch prepared, a 50 μL sample of MBs was sonicated (40 kHz ultrasound bath) for 5 seconds before diluting it (1:100 v/v) and recording the absorbance at 559 nm using a plate-reader. 2.7 Characterisation of MB iron loadingAs DBPC-IONPs were incorporated within the MB coating, the functionalisation of MBs with drug products on its outer surface is unlikely to affect the iron loading of the MBs. The iron content of MBs was therefore measured without the addition of drugs to prevent wastage of synthesised ligands, and was determined by ICP-OES measurements of samples diluted in 2% nitric acid at a wavelength of 238 nm. 2.8 Production of singlet oxygen from MagO2MB-RB exposed to USThe production of singlet oxygen (1O2) from activated RB exposed to US was determined using SOSG. A sample of 9 mL degassed PBS, ± 5x107 MB/mL, ± 541 μM biotin-RB, and 1.25 μM SOSG was exposed to 1.17 MHz, 0.70 MPa peak negative pressure, 30% duty cycle (DC), 100 Hz pulse repetition frequency (PRF) US for 3.5 minutes. Sample exposure was undertaken using a custom-made tank with the built-in transducer driven at 1.17 MHz. The sample was injected into a holder placed in the pre-focal region of the transducer to ensure a uniform pressure field at the top of the sample chamber. The fluorescence intensity of SOSG (Ex: 490 nm / Em: 520 nm) was measured for each sample with and without US exposure. To minimise the scattering from MBs in the sample without US exposure, increased hydrostatic pressure was applied to the sample using a sealed syringe to destroy the bubbles prior to fluorescence measurement. The generation of 1O2 was then calculated as a percent change in SOSG fluorescence intensity at 520 nm for a sample exposed to US compared to a control sample for each experimental run. In addition to measuring 1O2 generation, the characterisation of MB acoustic emissions during US exposure were recorded using PCD focused on the top of the sample chamber. A 2 MHz analog high-pass filter was used to remove the drive frequency from the recorded signal before pre-amplification, digitization, and storage onto a computer drive. The power spectral density was calculated for each PCD signal acquisition. These results were used to quantify cavitation activity during each experiment (3.5 minutes exposure) by determining the cumulative energy at ultraharmonic frequencies (f0*(n+0.5), with f0 = 1.17 MHz and n = 2,3...9), which are indicative of nonlinear bubble oscillations.2.9 Magnetic-Acoustic-Device (MAD) and control deviceThe MAD was designed as described by Barnsley et al. and assembled as shown in Fig. 2 ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1002/admt.201800081","ISSN":"2365709X","author":[{"dropping-particle":"","family":"Barnsley","given":"Lester C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gray","given":"Michael D.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Beguin","given":"Estelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Carugo","given":"Dario","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Advanced Materials Technologies","id":"ITEM-1","issue":"7","issued":{"date-parts":[["2018"]]},"page":"1800081","title":"A Combined Magnetic-Acoustic Device for Simultaneous, Coaligned Application of Magnetic and Ultrasonic Fields","type":"article-journal","volume":"3"},"uris":["",""]}],"mendeley":{"formattedCitation":"[20]","plainTextFormattedCitation":"[20]","previouslyFormattedCitation":"[19]"},"properties":{"noteIndex":0},"schema":""}[20]. Briefly, the magnetic body consisted of N52 grade NdFeB permanent magnet material whose geometry was optimized to have a maximum magnetic field of 0.2 T at a distance of 10 mm from the body’s leading edge. An integrated ultrasonic element with a focal distance also of 10 mm provided a pressure field that spatially overlapped with the magnetic field peak, with sufficient amplitude to cause inertial cavitation of MBs used in this study. An aluminium-bodied copy of the MAD (hereafter referred to as “aMAD”) was produced to provide an US-only control for in vitro and in vivo experimentation. Fig. 2. (A) MAD configuration illustration and (B) photograph as tested, with Perspex holder.In order to span the gap between the US element and the delivery site of interest in the present work, a coupling cone (Fig. 2) was cast from paraffin wax and secured with US gel. The cone material was chosen for its ease of casting and minimal transmission loss in the 1 MHz frequency range as determined by through-transmission measurements. Since the acoustic boundary conditions for this configuration were different from those used in the initial characterization ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1002/admt.201800081","ISSN":"2365709X","author":[{"dropping-particle":"","family":"Barnsley","given":"Lester C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gray","given":"Michael D.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Beguin","given":"Estelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Carugo","given":"Dario","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Advanced Materials Technologies","id":"ITEM-1","issue":"7","issued":{"date-parts":[["2018"]]},"page":"1800081","title":"A Combined Magnetic-Acoustic Device for Simultaneous, Coaligned Application of Magnetic and Ultrasonic Fields","type":"article-journal","volume":"3"},"uris":["",""]}],"mendeley":{"formattedCitation":"[20]","plainTextFormattedCitation":"[20]","previouslyFormattedCitation":"[19]"},"properties":{"noteIndex":0},"schema":""}[20], both the MAD and the aluminium copy were recalibrated (Fig. S4).2.10 Drug delivery comparison in agar between devicesDrug delivery was quantified in vitro by flowing MagO2MB-RB through a tissue mimicking agar phantom (Fig. 3). The phantom body was formed within a Delrin frame filled with 1.25% agar gel. The cured agar phantom was 7 mm thick and covered in clear and acoustically transparent Mylar films on each side. Each phantom contained at least one straight channel of 1.2 mm diameter, with fittings on the frame for connection to a syringe pump and drain tubing. During testing, the phantom was partially immersed in a water bath heated to 36 ± 1°C, with the upper phantom surface in air so that acoustic boundary conditions would be similar to those in the in vivo experiments. The phantom assembly was free of ferrous metal parts in order to minimize the likelihood of secondary magnetic fields influencing the results.For acoustic treatments (MAD or aMAD), the device was held in place with a lab clamp, and the cone tip was coupled to the phantom face with water. Acoustic drive pulses (3000 cycles of 1.17 MHz, 30% duty cycle) were provided so that the peak negative pressure 10 mm in front of the device would be 0.7 MPa. This level matched the spatial peak value from a separate device that had been used in previous SDT experiments ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.jconrel.2015.02.004","ISSN":"1873-4995","PMID":"25660073","abstract":"Tumour hypoxia represents a major challenge in the effective treatment of solid cancerous tumours using conventional approaches. As oxygen is a key substrate for Photo-/Sono-dynamic Therapy (PDT/SDT), hypoxia is also problematic for the treatment of solid tumours using these techniques. The ability to deliver oxygen to the vicinity of the tumour increases its local partial pressure improving the possibility of ROS generation in PDT/SDT. In this manuscript, we investigate the use of oxygen-loaded, lipid-stabilised microbubbles (MBs), decorated with a Rose Bengal sensitiser, for SDT-based treatment of a pancreatic cancer model (BxPc-3) in vitro and in vivo. We directly compare the effectiveness of the oxygen-loaded MBs with sulphur hexafluoride (SF6)-loaded MBs and reveal a significant improvement in therapeutic efficacy. The combination of oxygen-carrying, ultrasound-responsive MBs, with an ultrasound-responsive therapeutic sensitiser, offers the possibility of delivering and activating the MB-sensitiser conjugate at the tumour site in a non-invasive manner, providing enhanced sonodynamic activation at that site.","author":[{"dropping-particle":"","family":"McEwan","given":"Conor","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Owen","given":"Joshua","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Fowley","given":"Colin","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nesbitt","given":"Heather","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Cochrane","given":"David","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Coussios","given":"Constantin C","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Borden","given":"M","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nomikou","given":"Nikolitsa","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"McHale","given":"Anthony P","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"John F","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Controlled Release","id":"ITEM-1","issued":{"date-parts":[["2015","4","10"]]},"note":"We investigate the use of oxygen-loaded, lipid-stabilised microbubbles(MBs), decorated with a Rose Bengal sensitiser, for SDT-based treatment of a pancreatic cancermodel (BxPc-3) in vitro and in vivo.\n\nDuring in vivo study the MBs were directly injected into the mices' tumors.\nThe average size of the MBs was 1–2 μm with a concentration of approximately 1 × 109 MB/ml","page":"51-6","title":"Oxygen carrying microbubbles for enhanced sonodynamic therapy of hypoxic tumours.","type":"article-journal","volume":"203"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1016/j.jconrel.2017.07.040","ISSN":"01683659","abstract":"Magnetically responsive microbubbles (MagMBs), consisting of an oxygen gas core and a phospholipid coating functionalised with Rose Bengal (RB) and/or 5-fluorouracil (5-FU), were assessed as a delivery vehicle for the targeted treatment of pancreatic cancer using combined antimetabolite and sonodynamic therapy (SDT). MagMBs delivering the combined 5-FU/SDT treatment produced a reduction in cell viability of over 50% when tested against a panel of four pancreatic cancer cell lines in vitro. Intravenous administration of the MagMBs to mice bearing orthotopic human xenograft BxPC-3 tumours yielded a 48.3% reduction in tumour volume relative to an untreated control group (p < 0.05) when the tumour was exposed to both external magnetic and ultra- sound fields during administration of the MagMBs. In contrast, application of an external ultrasound field alone resulted in a 27% reduction in tumour volume. In addition, activated caspase and BAX protein levels were both observed to be significantly elevated in tumours harvested from animals treated with the MagMBs in the pre- sence of magnetic and ultrasonic fields when compared to expression of those proteins in tumours from either the control or ultrasound field only groups (p < 0.05). These results suggest MagMBs have considerable po- tential as a platform to enable the targeted delivery of combined sonodynamic/antimetabolite therapy in pan- creatic cancer.","author":[{"dropping-particle":"","family":"Sheng","given":"Yingjie","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Beguin","given":"Estelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nesbitt","given":"Heather","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kamila","given":"Sukanta","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Owen","given":"Joshua","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Barnsley","given":"Lester C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"Bridgeen","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"O'Kane","given":"Christopher","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nomikou","given":"Nikolitsa","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hamoudi","given":"Rifat","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Taylor","given":"Mark A.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Love","given":"Mark","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kelly","given":"Paul","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"O'Rourke","given":"Declan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"McHale","given":"Anthony P.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"John F.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Controlled Release","id":"ITEM-2","issue":"June","issued":{"date-parts":[["2017"]]},"page":"192-200","publisher":"Elsevier","title":"Magnetically responsive microbubbles as delivery vehicles for targeted sonodynamic and antimetabolite therapy of pancreatic cancer","type":"article-journal","volume":"262"},"uris":[""]}],"mendeley":{"formattedCitation":"[17,21]","plainTextFormattedCitation":"[17,21]","previouslyFormattedCitation":"[17,20]"},"properties":{"noteIndex":0},"schema":""}[17,21]. Ultrasonic emissions from the channel were recorded using a single PCD. Signal conditioning and post-processing procedures were performed using the same procedures and instrumentation as in Section 2.8. Initial positioning of the agar flow channel and the acoustic instrumentation was guided by a crosshair laser to ensure proper alignment. Data were collected for six groups as indicated in Table 1, with three separate phantoms tested per group. The MAD was used for: (1) its co-aligned magnetic and acoustic fields (group: “MAD” and shown in Fig. 3A, (2) its magnetic field only (US off) (group: “Mag”), and (3) its magnetic field (MAD US off) with the acoustic field of the (aMAD) to study the non-coaligned fields (group: “US + Mag”), and shown in Fig. 3B. The aMAD was also used on its own for the US only control (group: “US”).Fig. 3. In vitro flow phantom set-up for (A) the MAD with co-aligned fields compared to (B) the separate but simultaneous application of magnetic (MAD with US element turned off) and US (aMAD) fields. Phantom is shown cutaway to visualize the 1.2 mm diameter channel through which MMBs were flown. The underlying water bath is not shown.Table 1. In vitro drug delivery experiment groups.Group NameWaterMagMB-RB*US**Magnet***UntreatedXMBXUSXX (aMAD)MagXX (MAD)US + MagXX (aMAD)X (MAD)MADXX (MAD)X (MAD)* MagMB-RB: MB = (1.7±0.6) x108 MB/mL and [RB] = 500±50 μM** US: 1.17 MHz, 30% duty cycle, peak negative pressure 0.7 MPa, 3.5 minutes*** Magnet: 0.2 T at 10 mmFor all treatment groups, 100 μL of MagMB-RB ([MB] = (1.74±0.62) x 108 MB/mL, [RB] = 506±53 μM) were injected at a flow rate of 0.2 mL/min. US was applied for 3.5 minutes, after which the treated channel was rinsed with 1 mL deionised water. The agar channel was immediately cut out (0.7 mL volume) and reserved for analysis after the experiment. After all groups were completed in a single test day, the reserved cut out agar channels were melted, sampled onto a pre-heated 96-well plate and left to equilibrate at 45°C for 15 minutes. Absorbance spectra were acquired at that temperature using a plate reader to measure the amount of RB delivered in the agar volume. Spectra were normalised to no-treatment controls and the samples’ absorption intensities at 559 nm were compared to a standard curve for biotin-RB in melted 1.25% agar at 45°C (R2 = 0.9991).2.11 Treatment of xenograft ectopic BxPC-3 tumours in SCID miceAll animals employed in this study were treated in accordance with the licenced procedures under the UK Animals (Scienti?c Procedures) Act, 1986. BxPC-3 cells (1?×?106) in 50?μL Matrigel + 50 μL media (RPMI 1640) were subcutaneously implanted into the rear dorsum of SCID (C·B-17/IcrHan?Hsd-Prkdcscid) mice. Tumours started to form approximately two?weeks after cell implantation, and once they became palpable, their sizes were measured using the Peira TM900 tumour measuring device (Beerse, Belgium). The TM900 platform software includes a measurement function allowing visualisation of the tumour topography, allowing tumour dimensions (weight, length and height) to be automatically measured. When the tumours reached an average of 113±13.42 mm3 the animals were distributed randomly into four groups (Table 2). Subjects were anaesthetized with intraperitoneal injections of Hypnorm / Hypnovel. A 100 μL mixture of PBS with MagO2MB-RB-Gem ([MB] = (1.25±0.41) x 109 MB/mL, [biotin-RB-Gem] = 521±80 μM) was administered by tail vein injection to the subjects receiving treatment. The drive instrumentation and settings were the same as those used for the in vitro drug delivery experiments described in Section 2.10. The cone of the MAD (or aMAD) was coupled to the skin of the subject using US gel. In order to minimize acoustic field uncertainties and tissue damage risk, the subjects were treated lying prone over a mat of US absorbing material (Aptflex F28, Precision Acoustics, Dorset, UK). To improve free transmission of sound into the absorber, the abdominal hair was removed from the subjects, and their skin coupled to the mat with US gel. Using these methods, treatments were performed on days 0, 2 and 4. Subject weight and tumour size were monitored for 28 days after the first treatment. Previous data obtained from ectopic BxPC3 tumours treated with gemcitabine (120mg/kg; IP; 2 x week) have been included in the results section for reference.Table 2. In vivo drug delivery experiment groups.Group NameNo treatmentMagO2MB-RB-Gem*US**Magnet***UntreatedXMBXUS + MagXX (aMAD)X (MAD)MADXX (MAD)X (MAD)* MagO2MB-RB-Gem: [MB] = (1.3±0.4) x 109 MB/mL, [biotin-RB-Gem] = 520±80 μM** US: 1.17 MHz, 30% duty cycle, peak negative pressure 0.7 MPa, 3.5 minutes*** Magnet: 0.2 T at 10 mm2.12 StatisticsWith the exception of the tumour volumes in the in vivo experiments, results are expressed as the mean average value ± one standard deviation. Tumour volume data are reported as mean ± standard error on account of the uncertainty in the mean tumour volume measurement. Statistical significance and comparisons were established using an unpaired t-test when evaluating two groups and a 1-way ANOVA followed by Tukey’s post hoc test when comparing more than two groups using Microsoft Excel 365. 3. Results and discussion3.1 Synthesis of biotin-RB-Gem (8) and its efficacy in pancreatic cancer cells To enable both the SDT sensitiser RB and antimetabolite Gem to be conjugated to the MB surface, MBs were surface functionalised with avidin and a tripodal ligand was designed to have a single biotin anchor connected to both RB and Gem (8). To synthesise 8, the N-hydroxysuccinimide ester of biotin (1) was first reacted with tris(2-aminoethyl)amine (2) in a 1:1 molar reaction to encourage only one of the primary amines on 2 to form an amide bond with 1. The resulting product 3, was then reacted with disuccinimidyl suberate (4) in a 1:2 molar ratio forming amide bonds with the remaining two primary amine residues of 3, yielding compound 5 that also contained two pendant active esters. The active esters of 5 were reacted in turn with gemcitabine (6) and amine derivatised Rose Bengal (7), generating ester and amide linkages respectively with 5 to form target compound 8. The structure of 8 was characterised using 1H and 13C NMR spectroscopy and positive electrospray mass spectroscopy (Figs S1-S3). The mass spectrum reveals a base peak of 1925.9 Da corresponding to the exact mass of 8. In addition, 1H NMR analysis of 8 showed the expected 1:2 integration ratio between each of the aromatic protons on the cytosine moiety of Gem (5.77 ppm and 7.37 ppm) and the two equivalent aromatic protons present on RB (7.68 ppm) as well as the characteristic urea protons of biotin at 6.38 ppm and 6.42 ppm. Following the preparation and characterisation of compound 8, the next step was to perform in vitro testing with pancreatic cancer cells, in order to ensure that derivatising Gem for incorporation within 8 did not impair its efficacy. BxPC-3 and MiaPaCa-2 cells were incubated with 8 at a range of concentrations from 0.001 ?M to 1.0 mM and cell viability determined 48 hrs later using the MTT assay. As a comparison, cells were also treated with the same concentrations of free Gem. The results are shown in Fig. 4 and reveal no significant difference in the median lethal dose (LD50) values for 8 (0.71±0.16 μM and 0.74±0.19 ?M) or gemcitabine (362.30±0.12 μM and 474.40±0.13 μM) in BxPC-3 or Mia-PaCa-2 cells respectively. These results suggest that the ester bond connecting Gem in 8 is rapidly hydrolysed by endogenous esterase enzymes to liberate free Gem. These results also suggest no additional contribution to the cytotoxicity of 8 by RB in the absence of light or US stimulation. Furthermore, while it is important for cellular cleavage of Gem to enable its activation by deoxycytidine kinase mediated phosphorylation, rapid cleavage of RB from 8 is less important, as the mechanism of action for SDT does not require the sensitiser to bind to a receptor or be metabolised for reactive oxygen species to be generated. 3349625762000287105614605(B)00(B)1346205549(A)00(A)683398571500Fig. 4. MTT assay comparing the efficacy of biotin-RB-Gem (8) (open circles) and gemcitabine (filled circles) (A) BxPC-3 and (B) Mia-PaCa-2 cell lines.3.2 MB characterisationMagO2MBs were manufactured through the sonication of a mixture of phospholipids, surfactants and phospholipid-coated IONPs under PFB gas flow which resulted in a MB concentration of 1.8±0.3 x 109 MB/mL and 1.6±0.3 μm mean MB diameter. The removal of excess material through centrifugation significantly decreased the MB concentration to an average of 7.5±4.0 x 108 MB/mL (p<0.01) and the mean diameter to 1.9±0.4 μm (Fig. 5). A comparison of the size distributions from before and after conjugation with 9 suggests that the loading and subsequent washing processes removes the smallest MBs (Fig. 5). However, the 1 - 8 μm diameter MBs appear to stable during this process and the mean hydrodynamic diameter of the population was not significantly affected by the washing procedure. Detailed characterisation of the MB stability and their magnetic and acoustic properties prior to drug loading was reported in ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"author":[{"dropping-particle":"","family":"Beguin","given":"Estelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bau","given":"Luca","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Shrivastava","given":"Shamit","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"ACS Applied Materials & Interfaces","id":"ITEM-1","issue":"2","issued":{"date-parts":[["2018"]]},"page":"1829-1840","title":"Comparing strategies for magnetic functionalisation of microbubbles","type":"article-journal","volume":"11"},"uris":[""]}],"mendeley":{"formattedCitation":"[24]","plainTextFormattedCitation":"[24]","previouslyFormattedCitation":"[23]"},"properties":{"noteIndex":0},"schema":""}[24]. The iron content measured in the present study was 0.07 pg iron per microbubble after three centrifuge steps. This is higher than the previously reported values of 0.025 pg iron / MB ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"author":[{"dropping-particle":"","family":"Beguin","given":"Estelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bau","given":"Luca","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Shrivastava","given":"Shamit","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"ACS Applied Materials & Interfaces","id":"ITEM-1","issue":"2","issued":{"date-parts":[["2018"]]},"page":"1829-1840","title":"Comparing strategies for magnetic functionalisation of microbubbles","type":"article-journal","volume":"11"},"uris":[""]}],"mendeley":{"formattedCitation":"[24]","plainTextFormattedCitation":"[24]","previouslyFormattedCitation":"[23]"},"properties":{"noteIndex":0},"schema":""}[24] but was associated with manipulation variations in the resuspension of microbubbles during cleaning. This loading was calculated to equate to approximately 10% coverage of the microbubbles (supporting information) and their response to a magnetic field was confirmed visually (Fig. S5). Analysis of the power of acoustic emissions over time from the MBs indicated that the surface addition of drugs on microbubbles significantly lengthened the time over which an increase in acoustic emissions was recorded compared to magnetic microbubbles without drug (Fig. S6). These results suggest that the surface functionalisation of microbubbles can provide a stabilising effect by dampening the oscillations of microbubbles. The drug concentration in the suspension after washing was 480±100 μM or 520±50 μM for 8 or 9 respectively. The performance of oxygen-sparged lipid-based microbubbles to enhance sonodynamic therapy of hypoxic tumours was previously demonstrated by McEwan et al. showing in vitro that oxygen was released upon ultrasound exposure of diluted oxygen sparged microbubbles and in vivo that the expression of HIF1α was significantly decreased in hypoxic tumours treated with oxygen microbubbles compared to perfluorobutane microbubbles ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.biomaterials.2015.11.033","ISSN":"0142-9612","abstract":"In this manuscript we describe the preparation of an oxygen-loaded microbubble (O2MB) platform for the targeted treatment of pancreatic cancer using both sonodynamic therapy (SDT) and antimetabolite therapy. O2MB were prepared with either the sensitiser Rose Bengal (O2MB-RB) or the antimetabolite 5- fluorouracil (O2MB-5FU) attached to the microbubble (MB) surface. The MB were characterised with respect to size, physical stability and oxygen retention. A statistically significant reduction in cell viability was observed when three different pancreatic cancer cell lines (BxPc-3, MIA PaCa-2 and PANC-1), cultured in an anaerobic cabinet, were treated with both SDT and antimetabolite therapy compared to either therapy alone. In addition, a statistically significant reduction in tumour growth was also observed when ectopic human xenograft BxPC-3 tumours in SCID mice were treated with the combined therapy compared to treatment with either therapy alone. These results illustrate not only the potential of combined SDT/antimetabolite therapy as a stand alone treatment option in pancreatic cancer, but also the capability of O2-loaded MBs to deliver O2 to the tumour microenvironment in order to enhance the efficacy of therapies that depend on O2 to mediate their therapeutic effect. Furthermore, the use of MBs to facilitate delivery of O2 as well as the sensitiser/antimetabolite, combined with the possibility to activate the sensitiser using externally applied ultrasound, provides a more targeted approach with improved efficacy and reduced side effects when compared with conventional systemic administration of antimetabolite drugs alone.","author":[{"dropping-particle":"","family":"Mcewan","given":"Conor","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kamila","given":"Sukanta","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Owen","given":"Joshua","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nesbitt","given":"Heather","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"Bridgeen","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Borden","given":"Mark","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nomikou","given":"Nikolitsa","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hamoudi","given":"Rifat A","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Taylor","given":"Mark A","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Mchale","given":"Anthony P","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Callan","given":"John F","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Biomaterials","id":"ITEM-1","issued":{"date-parts":[["2016"]]},"page":"20-32","publisher":"Elsevier Ltd","title":"Combined Sonodynamic and Antimetabolite Therapy for the Improved Treatment of Pancreatic Cancer using Oxygen Loaded Microbubbles as a Delivery Vehicle","type":"article-journal","volume":"80"},"uris":[""]}],"mendeley":{"formattedCitation":"[25]","plainTextFormattedCitation":"[25]","previouslyFormattedCitation":"[24]"},"properties":{"noteIndex":0},"schema":""}[25].Fig. 5. The effect of cleaning and drug conjugation on microbubble (A) concentration (** p < 0.01 calculated through an unpaired t-test with equal variance) and (B) mean hydrodynamic diameter; evaluated for n=4 batches per group. The concentration of MagMB-RB-Gem after washing was 7.5±4.0 x 108 MB/mL and the mean diameter was 1.9±0.4 μm. (C) Example of size distribution of MagMBs before and after loading of biotin-RB-Gem obtained from analysis of 30 optical microscope images for each batch. 3.3 In vitro activation and deliveryUsing the US parameters indicated in Table 1, the results from the in vitro activation of RB using MagO2MBs and US are shown in Fig. 6. The generation of cytotoxic 1O2 was significantly enhanced when MagO2MB-RB in degassed PBS were exposed to US, compared to MagO2MBs or RB alone (p < 0.01), thereby indicating the activation of the sensitiser RB through exposure to microbubbles and ultrasound. A similar trend was observed in the ultraharmonic energy of MB emissions recorded from the PCD, but the emissions from the two types of MB and RB alone were all significantly different (p < 0.01). The increased cavitation activity of MagO2MB-RB compared to MagO2MBs could be explained by an enhanced stabilisation of MagO2MB-RB due to the surface functionalisation of the sensitiser that prevents bubble dissolution in the degassed medium ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.colsurfa.2013.02.054","ISSN":"09277757","abstract":"Pickering emulsions are attractive formulations because they are simple and bear strong similarities with the well-known surfactant-based emulsions. Pickering emulsions have been largely ignored since their early disclosure in 1907 and arouse a renewed interest quite recently. Since this unintelligible time gap raises suspicion, the first aim of the present review is giving the simple fundamental rules as an introduction for newcomers in the topic. The basic physical chemistry of Pickering emulsions is explained and the ways to control the parameters of higher relevance with respect to development of applications are given. This first part covers the choice of the solid nanoparticles used as stabilizers and their surface properties, the control of emulsion type, droplet size, and rheology. A second part gives examples of some applications in drug delivery and manufacturing of porous nanomaterials as illustrations of the potential of such emulsions.","author":[{"dropping-particle":"","family":"Chevalier","given":"Yves","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bolzinger","given":"Marie-Alexandrine","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Colloids and Surfaces A: Physicochemical and Engineering Aspects","id":"ITEM-1","issued":{"date-parts":[["2013","12"]]},"note":"A review of Pickering Emulsions. Mostly talking about silica-coated emulsions.\nThe high resistance to coalescence is a major benefit of the stabilization by solid particles.\n\nSolid particles of nano-metric size (or sub-micron, ～100 nm) allow the stabilization of droplets as small as few micrometers diameter\n\nStabilization of emulsion droplets takes place by means of adsorption of solid particles at the surface of emulsion droplets.\n\nPartial wetting of the surface of the solid particles by water and oil is the origin of the strong anchoring of solid particles at the oil–water interface.\n\nPartial wetting of the solid by water inside an oil medium requires that the adhesion energy of water, EAdh(w/o), is positive and the spreading coefficient of water, S(w/o), is negative.\n\nThe surface coating by solid parti- cles mainly acts against coalescence.\n\nThere are several reports showing microscopy pictures of stable Pickering emulsion droplets under incomplete coverage of the droplets by solid particles.\n\nAddition of electrolytes weakens electrostatic repulsions, thus causing sup-plementary aggregation and increasing the viscosity.\n\nThe sta-bilization of submicron-sized monomer droplets is a difficult task because most monomers have a significant solubility in water that causes de-stabilization of the miniemulsion by means of an Ost-wald ripening mechanism.","page":"23-34","title":"Emulsions stabilized with solid nanoparticles: Pickering emulsions","type":"article-journal","volume":"439"},"uris":["",""]}],"mendeley":{"formattedCitation":"[26]","plainTextFormattedCitation":"[26]","previouslyFormattedCitation":"[25]"},"properties":{"noteIndex":0},"schema":""}[26]. As previously mentioned, this is further supported by the results in Fig S6.Fig. 6. Singlet oxygen production (orange, n=3) from RB activation after US exposure, with associated ultraharmonic emissions from microbubbles (blue) undergoing nonlinear oscillations. ** = p < 0.01 determined through a 1-way ANOVA with Tukey’s post hoc test. A sample was prepared with ±5x107 MB/mL, ±541 μM biotin-RB, and 1.25 μM SOSG in degassed PBS. US parameters were 1.17 MHz, 0.7 MPa peak negative pressure, 30% duty cycle, 100 Hz pulse repetition frequency for 3.5 minutes. The performance of the MAD compared to the use of two separate devices and the contributions of US and of the magnetic field individually were assessed in an agar phantom containing a cylindrical flow channel (Fig. 3). The concentration of RB delivered was determined based on the absorbance of a set volume of agar gel surrounding a channel after flowing 100 μL of MagMB-RB through it while exposed to one of the device configurations. Fig. 7 shows the concentration of RB delivered for the different groups considered, and when US was used, the associated cavitation activity is provided. A significantly higher quantity of RB was delivered (p < 0.01) using the MAD compared to all other groups; more specifically a 1.6 and 1.4-fold increase in RB was measured compared to the US only and US + Mag groups respectively. Moreover, a comparison between the MAD and US only groups indicate a significant increase in delivery (p < 0.01) from the addition of magnetic targeting, but the minimal enhancement observed for the US + Mag group compared to US highlights the difficulty in optimally aligning devices when two separate units are used. The delivery recorded for MagO2MB-RB alone (i.e no magnet or US) could be associated with residual RB in the suspension diffusing across pores in the agar gel ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1088/1742-6596/28/1/017","ISBN":"1742-6596","ISSN":"17426596","abstract":"The absorbance measurements in the wavelength range 700 nm to 800 nm were used to probe the agarose\\r gel topology evolution and extract the pore size of the trapped solvent. By following the changes in\\r absorbance and pore size, the gelation process could be clearly divided into three stages -\\r induction stage, gelation stage and pseudo-equilibrium stage. The gelation mechanism is explained as\\r a nucleation and growth process. Following the kinetics of gelation using dynamic light scattering\\r is complicated by multiple scattering (for high concentrations) and large fluctuations in intensity\\r and relaxation time. Comparatively, scanning the absorption spectrum is fast and the method is\\r suitable for a wide range of concentrations and setting temperatures. Pore size determination using\\r absorbance is a fast and non-invasive method when compared to the DNA electrophoresis measurements,\\r which extend over several hours and use probe diffusion.","author":[{"dropping-particle":"","family":"Narayanan","given":"Jaaky","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Xiong","given":"Jun Ying","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Liu","given":"Xiang Yang","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Physics: Conference Series","id":"ITEM-1","issue":"1","issued":{"date-parts":[["2006"]]},"page":"83-86","title":"Determination of agarose gel pore size: Absorbance measurements vis a vis other techniques","type":"article-journal","volume":"28"},"uris":["",""]}],"mendeley":{"formattedCitation":"[27]","plainTextFormattedCitation":"[27]","previouslyFormattedCitation":"[26]"},"properties":{"noteIndex":0},"schema":""}[27] and was found to be significant compared to the untreated group (p < 0.01). Individually, US and Mag fields enhanced delivery to a similar degree, in agreement with previous results from Stride et al. ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.ultrasmedbio.2008.11.010","ISBN":"0301-5629","ISSN":"03015629","PMID":"19282096","abstract":"It has been shown in previous studies that gene delivery can be enhanced by a variety of minimally-invasive techniques including: (1) exposure of cells to ultrasound in the presence of DNA and gas microbubbles and (2) exposure of cells to a magnetic field in the presence of DNA conjugated to magnetic nanoparticles. The aim of this work was to investigate whether it was possible to combine the advantages of both these techniques. It was found that transfection of Chinese hamster ovary cells by naked plasmid DNA was enhanced by combined exposure of the cells to ultrasound (10 s at 1 kHz pulse repetition frequency with 40 cycle 1 MHz sinusoidal pulses, 1 MPa peak to peak pressure) and a magnetic field (provided by five square cross-section N52 grade NdFeB magnets 25 ?? 10 ?? 10 mm with transversal magnetisation Br = 1.50 T arranged in a Halbach array), in the presence of one of two different microbubble/nanoparticle preparations. The first preparation consisted of phospholipid coated microbubbles mixed with micelles containing magnetic nanoparticles. The second consisted of microbubbles which were themselves magnetically active. These preparations were found to be more effective than either magnetic micelles or phospholipid coated microbubbles alone by a factor of 2.8 (total flux ???4 versus 1.4 ?? 10 6 photon/s) and the results were found to be statistically significant (p < 0.01). Two mechanisms are proposed to explain these observations: firstly, that the magnetic field facilitates close proximity between the cells and the microbubbles and hence increases the likelihood of transfection; second, that there is sensitisation of the cells, as a result of exposure to the magnetic field in the presence of the micelles, which increases their ability to be transfected upon exposure to ultrasound. Further work is in progress to determine which of these mechanisms is the most significant and the potential for other therapeutic applications. (E-mail: e_stride@meng.ucl.ac.uk). ?? 2009 World Federation for Ultrasound in Medicine & Biology.","author":[{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Porter","given":"Colin","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Prieto","given":"Ana Garcia","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Pankhurst","given":"Quentin","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Ultrasound in Medicine and Biology","id":"ITEM-1","issue":"5","issued":{"date-parts":[["2009"]]},"page":"861-868","title":"Enhancement of Microbubble Mediated Gene Delivery by Simultaneous Exposure to Ultrasonic and Magnetic Fields","type":"article-journal","volume":"35"},"uris":["",""]}],"mendeley":{"formattedCitation":"[28]","plainTextFormattedCitation":"[28]","previouslyFormattedCitation":"[27]"},"properties":{"noteIndex":0},"schema":""}[28].Fig. 7. RB drug delivery (orange, n=3) in a volume of 0.7 mL agar (1.25% w/w) with different ultrasound and magnetic device configurations as listed in Table 1. ** = p < 0.01 determined through a 1-way ANOVA with Tukey’s post hoc test. The corresponding ultraharmonic emissions of microbubbles (blue) are shown. For the treatment-receiving groups, 100 μL of MagMB-RB ([MB] = (1.74±0.62) x 108 MB/mL, [RB] = 506±53 μM) were administered and flown at 0.2 mL/min. When a magnet was used, a 0.2 T magnetic field from the MAD was applied at the US focus. When US was applied (MAD or aMAD), the parameters were: 1.17 MHz, 0.7 MPa peak negative pressure, 30% duty cycle (DC), 100 Hz PRF for 3.5 minutes. The ultraharmonic emissions plotted for the untreated group reflect the background noise recorded with water flowing in the agar channel. 3.4 In vivo resultsTo test the utility of the MAD as a platform for the delivery of combined magnetic and US fields in vivo, SCID mice were implanted with ectopic human pancreatic BxPC-3 tumours and randomly distributed into 4 groups for treatment as described in Table 2. The results in Fig. 8 indicate a 37% reduction in tumour volume relative to the pre-treatment volume 4 days after the initial treatment for animals treated with MagO2MB-RB-Gem and the MAD, compared to a 9% reduction when combined magnetic and US fields were simultaneously delivered using separate probes (U + M). This difference was maintained for four days, and 12 days following the initial treatment, tumours treated with MagO2MB-RB-Gem and the MAD were still 9% smaller than their pre-treatment volume and 54% smaller than tumours treated with MagO2MB-RB-Gem and separate probes. Beyond day 12, all groups showed tumour growth. The MAD group showed the least, but there was no statistically significant difference from the separate probe group. This indicates that further investigation of the treatment schedule with larger groups is warranted. The observed differences between treatments performed with the MAD and physically separate but simultaneous magnetic and ultrasonic field generating devices illustrate the importance of field alignment. When co-aligned as in the MAD, the tendency of bubbles to be pushed away from the US focus by radiation force is counteracted by the pulling force of the magnet. This effect, which helps maintain a population of bubbles in the US beam, is weaker and potentially becomes detrimental to bubble availability in the US focus when the ultrasonic and magnetic fields are separated by a large angle, as with the separate device tests. Fig. 8. In vivo results with (A) tumour growth over time relative to day 0. Treatments were given on days 0, 2, and 5. (B) Comparison of relative tumour volumes at days 12 and 24 (* p<0.05 determined by an unpaired t-test. n = 5 for untreated and n = 4 for MAD, MB, and U + M). Taken together, these results demonstrate an improved therapeutic effect obtained from the co-aligned application of magnetic and acoustic fields using one device compared to the use of two separate devices. Additionally, the chemo-sonodynamic therapy delivered using the MAD provided a rapid, substantial and stable reduction in tumour volume, suggesting that this approach may be useful as a neo-adjuvant treatment to downstage pancreatic tumours in advance of surgery. There is a large proportion of pancreatic cancer patients (~20%) presenting with borderline resectable lesions, and a reduction in tumour burden could make them eligible for surgical resection. As 5-year survival rates improve 5-fold when surgery is possible, effective neo-adjuvant strategies that increase resection rates are the fastest way to improve survival in a disease that has witnessed only minor improvements over the past 40 years ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.3892/ol.2017.6008","ISSN":"17921082","PMID":"28599404","abstract":"Chemotherapy for pancreatic cancer has diversified following the addition of more treatment regimens; however, in spite of this, pancreatic cancer remains a fatal disease. Preoperative (neoadjuvant) chemotherapy (NAC) or neoadjuvant chemoradiation therapy (NACRT) has been developed and implemented. For patients with borderline resectable pancreatic cancer (BRPC) and locally advanced pancreatic cancer (LAPC), a number of clinical trials have been conducted; NACRT was demonstrated to improve resectability, R0 resection rate, overall survival rate, disease-free survival rate and even an LAPC and BRPC survival advantage over NAC. However, from the knowledge obtained from resected specimens following preoperative treatment, residual pancreatic cancer tissues following NAC are rich in chemoresistant cancer stem-like cells and epithelial-mesenchymal transition (EMT) markers. Conversely, metformin, angiotensin receptor blocker, statins and low-dose paclitaxel are well-known as drugs that inhibit EMT, which is associated with cancer stem cell-like characteristics. Although clinical effectiveness is unlikely to be achieved using one of these as an anticancer agent, it is reasonable to use these drugs for patients with comorbidities in the treatment of pancreatic cancer. Furthermore, gemcitabine (GEM) affects antitumor immunity by stimulating the expression of major histocompatibility complex class I-related chain A on the surface of cancer cells to enhance the cytotoxicity of natural killer cells. Considering EMT and antitumor immunity, there is a possibility that GEM and nanoparticle albumin-bound paclitaxel therapy is the most suitable regimen for treating pancreatic cancer. However, even as preoperative treatment progresses, R0 resection is the most important factor for the long-term survival of pancreatic cancer patients.","author":[{"dropping-particle":"","family":"Tajima","given":"Hidehiro","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Makino","given":"Isamu","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ohbatake","given":"Yoshinao","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nakanuma","given":"Shinichi","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hayashi","given":"Hironori","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nakagawara","given":"Hisatoshi","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Miyashita","given":"Tomoharu","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Takamura","given":"Hiroyuki","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ohta","given":"Tetsuo","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Oncology Letters","id":"ITEM-1","issue":"6","issued":{"date-parts":[["2017"]]},"page":"3975-3981","title":"Neoadjuvant chemotherapy for pancreatic cancer: Effects on cancer tissue and novel perspectives","type":"article-journal","volume":"13"},"uris":["",""]},{"id":"ITEM-2","itemData":{"DOI":"10.1016/J.CMRP.2014.03.002","ISSN":"2352-0817","abstract":"The overall survival for carcinoma pancreas is still around 5%. Surgical resection alone is associated with a low survival and high recurrence rates. Neoadjuvant therapy for carcinoma pancreas is aimed at improving survival and resectability by downsizing borderline resectable and unresectable tumors. However its role in the resectable group of patients is still not clear. This review discussed the current available evidence for use of neoadjuvant therapy in pancreatic adenocarcinoma.","author":[{"dropping-particle":"","family":"Mitra","given":"Abhishek","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sirohi","given":"Bhawna","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"V.","family":"Shrikhande","given":"Shailesh","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Current Medicine Research and Practice","id":"ITEM-2","issue":"2","issued":{"date-parts":[["2014","3","1"]]},"page":"56-61","publisher":"Elsevier","title":"Neoadjuvant therapy in pancreatic cancer","type":"article-journal","volume":"4"},"uris":["",""]}],"mendeley":{"formattedCitation":"[29,30]","plainTextFormattedCitation":"[29,30]","previouslyFormattedCitation":"[28,29]"},"properties":{"noteIndex":0},"schema":""}[29,30]. The approach outlined in this manuscript was not only effective at reducing tumour burden but was also well tolerated, as the animals remained healthy and exhibited no weight loss over the duration of the study (Fig. S10). Scaling of the MAD to human lengthscales is demonstrated in ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1002/admt.201800081","ISSN":"2365709X","author":[{"dropping-particle":"","family":"Barnsley","given":"Lester C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gray","given":"Michael D.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Beguin","given":"Estelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Carugo","given":"Dario","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Advanced Materials Technologies","id":"ITEM-1","issue":"7","issued":{"date-parts":[["2018"]]},"page":"1800081","title":"A Combined Magnetic-Acoustic Device for Simultaneous, Coaligned Application of Magnetic and Ultrasonic Fields","type":"article-journal","volume":"3"},"uris":[""]}],"mendeley":{"formattedCitation":"[20]","plainTextFormattedCitation":"[20]","previouslyFormattedCitation":"[19]"},"properties":{"noteIndex":0},"schema":""}[20]. 3.5 LimitationsAlthough the results shown in Figs. 8 and S10 are encouraging. There are several limitations of the work that need to be discussed. First, the MAD used in this study was designed for small animal experiments but, as discussed in ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1002/admt.201800081","ISSN":"2365709X","author":[{"dropping-particle":"","family":"Barnsley","given":"Lester C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gray","given":"Michael D.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Beguin","given":"Estelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Carugo","given":"Dario","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stride","given":"Eleanor","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Advanced Materials Technologies","id":"ITEM-1","issue":"7","issued":{"date-parts":[["2018"]]},"page":"1800081","title":"A Combined Magnetic-Acoustic Device for Simultaneous, Coaligned Application of Magnetic and Ultrasonic Fields","type":"article-journal","volume":"3"},"uris":[""]}],"mendeley":{"formattedCitation":"[20]","plainTextFormattedCitation":"[20]","previouslyFormattedCitation":"[19]"},"properties":{"noteIndex":0},"schema":""}[20], a compromise had to be made between peak magnetic force and acoustic pressure at the focus. This will be overcome in future development of the device; which will also include treatment monitoring capability. Second, it was not possible with the equipment available to assess tumour vascularity prior to treatment. This is, however, likely to be an important predictor of therapeutic response as it determines the quantity of microbubbles entering the target volume. Whilst ultrasound and microbubbles are able to improve the distance to which therapeutic material is delivered from the nearest blood vessel, they cannot compensate entirely for poor perfusion. This then contributes to the variance in tumour volumes which was notably higher in the results for the groups without magnetic field (Fig. 8, S7, S9). Finally, residual free RB and Gem in the microbubble suspension, suggested by the in vitro results, could also explain the decreased tumour size observed in subjects receiving microbubbles only, compared to no treatment. Further development of the therapeutic microbubbles will focus on product loading and cleaning protocols to minimise off-target accumulation and undesired side-effects. 4. ConclusionsA novel therapeutic incorporating the antimetabolite Gemcitabine, the sensitiser Rose Bengal and the attachment ligand biotin was synthesised and enabled the simultaneous loading of both drugs onto oxygen-filled magnetic microbubbles. The co-aligned application of magnetic and ultrasound fields to the target region using the MAD produced an increase in drug deposition in vitro and tumour response in vivo compared to the application of both fields using two separate devices. These results indicate the importance of co-alignment of the magnetic and ultrasound fields and provide further supporting evidence for the potential use this approach to downstage pancreatic tumours in cancer patients with borderline resectable lesions, enabling them to undergo surgery.AcknowledgementsThe authors thank James Fisk and David Salisbury for their contribution to the design and construction of the in vitro US tanks, phantom holders and the aluminium device copy used in this work. Funding for the work was provided by the Engineering and Physical Sciences Research Council (EP/I021795/1 and EP/L024012/1) and the Institute of Engineering and Technology (AF Harvey Prize). EB thanks the Research Councils UK Digital Economy Programme for the support through grant EP/G036861/1 (Oxford Centre for Doctoral Training in Healthcare Innovation). MDG was supported by the National Institute of Health Research, Oxford Biomedical Research Centre. JFC thanks Norbrook Laboratories Ltd for an endowed chair. KAL thanks the Department for the Economy in Northern Ireland for a PhD studentship. YS and SK thank Invest N.I. in Northern Ireland and the Pancreatic Cancer Research Fund respectively for postdoctoral fellowships. ReferencesADDIN Mendeley Bibliography CSL_BIBLIOGRAPHY [1]G. Dimcevski, S. Kotopoulis, T. Bj?nes, D. Hoem, J. Schj?t, B.T. Gjertsen, M. Biermann, A. Molven, H. Sorbye, E. McCormack, M. Postema, O.H. Gilja, A human clinical trial using ultrasound and microbubbles to enhance gemcitabine treatment of inoperable pancreatic cancer, J. Control. Release. (2016). doi:10.1016/j.jconrel.2016.10.007.[2]C. A., C. M., V. A., R. V., B. K., H. C., K. C., L. D., L. C., C. J.-Y., C. L., C. P., S. M., H.-X. K., D. J.-Y., Clinical trial of blood-brain barrier disruption by pulsed ultrasound, Sci. Transl. Med. 8 (2016) 343re2. doi:.[3]S.R. 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