_:b42921916 _:b42921940 . _:b42922004 . _:b585248167 . _:b42921916 _:b42921941 . _:b42921916 _:b42921942 . _:b42921929 . _:b42921916 _:b42921943 . _:b42921916 _:b42921936 . _:b42922024 . _:b42922005 . _:b585248166 . _:b42921916 _:b42921937 . _:b42921916 _:b42921938 . _:b42921916 _:b42921939 . _:b42921916 _:b42921948 . _:b42922006 . _:b585248161 . _:b42921903 "increase in inhibition rateTinkov et al. (2010)295/KDRDOX-loaded MBLipid1 MHz1 W/cm250%20 s40% decrease in cell viabilityYan et al. (2013)4T1PTX-liposome loaded MBLipid1 MHz1.0 MPa50%1 min20\u201330% decrease in viabilityDeng et al. (>>2014<<)MCF7/ADRDOX-liposome loaded MBLipid1 MHz1.65 W/cm220%15 sIncreased cellular accumulation and retention, 30% decrease in viability10-HCPT, 10-hydroxycamptothecin; ND, non-defined; PNP, peak-negative-pressure." . _:b42921916 _:b42921949 . _:b585248176 "2"^^ . _:b42921916 _:b42921950 . _:b42921879 . _:b42921916 _:b42921951 . _:b42921916 _:b42921944 . _:b42922007 . _:b585248160 . _:b42921923 "Microbubble disruption can be accompanied by generation of shock waves in the medium close to the microbubbles (Junge et al., >>2003<<; Ohl and Wolfrum, 2003)." . _:b42921916 _:b42921945 . _:b42921916 _:b42921946 . _:b585248123 . _:b42921916 _:b42921947 . _:b42922059 "For example, Tinkov et al. (>>2010<<) demonstrated that the exposure of pancreas carcinoma in rats to ultrasound (1.3 MHz, 1.2 MPa PNP, four frames of ultrasound every four cardiac cycles) after i." . _:b42921916 _:b42921924 . _:b42922018 . _:b42922000 . _:b585248163 . _:b42921899 "doxorubicin (DOX)Vevo, BR14, SonoVue1 MHz400\u2013800 kPa40%30 s30\u201340% decrease in viability, depending on cell lineSorace et al. (2012)2LMPFree paclitaxel (PTX)Definity1 MHz1.0 MPa PNP20%5 min50% increase in cell deathHu et al. (>>2012<<)BEL-7402Free 10-HCPT (free)Polymer3.5 MHz22.57 mW/cm2ND10 min20\u201330% decrease in viabilityRen et al. (2013)DLD-1Docetaxel-loaded MBLipid800 kHz2.56 W/cm250%10 min40% increase in inhibition rateTinkov et al. (2010)295/KDRDOX-loaded MBLipid1" . _:b42921916 _:b42921925 . _:b42921916 _:b42921926 . _:b42921932 "extravasation by stimulating paracellular (i.e., disruption of tight junctions) and transcellular pathways (i.e., transcytosis), both in vitro as well as in vivo (Figure 2; Price et al., 1998; Sheikov et al., 2008; Juffermans et al., >>2009<<; Kooiman et al., 2010)." . _:b42921916 _:b42921927 . _:b42921916 _:b42921920 . _:b42922001 . . _:b42921916 _:b42921921 . _:b585248162 . _:b42921916 _:b42921922 . _:b42921916 _:b42921923 . _:b42921916 _:b42921932 . _:b42922002 . _:b42921889 "such as blood vessel walls (i.e., drug extravasation) and cellular membranes (i.e., cellular uptake of drugs), thus enhancing the local delivery of therapeutic molecules across these barriers in the targeted region (Lentacker et al., >>2014<<). Nowadays, the great potential of this modality for cancer therapy is clearly shown in an increasing number of publications on in vitro and in vivo drug delivery using microbubble-assisted ultrasound (Tables 1 and 2 respectively)." . _:b585248173 . _:b42921916 _:b42921933 . _:b42921916 _:b42921934 . _:b42921916 _:b42921935 . _:b42921916 _:b42921928 . _:b42922021 "Intracerebral BCNU delivery using VEGFR2-targeted and BCNU-loaded microbubbles with focused ultrasound for the glioma treatment (Adapted with permission from Fan et al., >>2013<< \u2013 Copyright \u00A9 2012 Elsevier Ltd.). (A) Antiangiogenic-targeting BCNU-loaded microbubbles combined with focused ultrasound for glioma treatment." . _:b42922003 . _:b42921986 "of the mechanism of uptake, the duration of microbubble-assisted ultrasound-mediated uptake is dependent on the plasma membrane recovery time, which is a few seconds to a few hours (van Wamel et al., 2006; Lammertink et al., >>2015<<). The different kinetics depends on the ultrasound conditions, the model drug size and the cell physiology." . _:b585248172 . _:b42921969 . _:b42921916 _:b42921929 . _:b42921916 _:b42921930 . _:b42922055 . _:b42921916 _:b42921931 . _:b42921916 _:b42921972 . _:b42922047 . _:b42922012 . _:b42921971 "ARFs can push circulating microbubbles toward the endothelial wall, thereby improving microbubble\u2013cell contact, which might enhance cavitation-mediated extravasation of intravascular compounds (Rychak et al., 2005; Wang et al., >>2014<<). Using ultrasound imaging, Frinking et al. (2012) reported that ARF (38 kPa PNP, 95% DC) induced a sevenfold increase in the binding of VEGFR2-targeted microbubbles (also known as BR-55) on the endothelial wall in a prostate" . _:b585248175 . _:b42921968 . _:b42921919 . _:b42921916 _:b42921973 . _:b42921916 _:b42921974 . _:b42921931 "increased (model-) drug extravasation by stimulating paracellular (i.e., disruption of tight junctions) and transcellular pathways (i.e., transcytosis), both in vitro as well as in vivo (Figure 2; Price et al., 1998; Sheikov et al., >>2008<<; Juffermans et al., 2009; Kooiman et al., 2010)." . _:b42921916 _:b42921975 . _:b42921936 "an acoustical pressure threshold ranging from 0.1 to 0.75 MPa was required to enhance the extravasation of intravascular agents (e.g., red blood cells, imaging tracers, fluorescent dyes, or drugs) in skeletal muscle (Price et al., >>1998<<), brain (Raymond et al., 2007; Sheikov et al., 2008), liver (Gao et al., 2012), and tumor (Bohmer et al., 2010; Hu et al., 2012) tissues." . _:b42921916 _:b42921968 . _:b42922013 . _:b42921897 "cycleTimeIwanaga et al. (2007)Ca9-22Free bleomycinOptison1 MHz1.0 W/cm210%20 s2.5-fold increase in apoptosisHeath et al. (2012)SCC-1, SCC-5, Cal27Free cisplatinDefinity1 MHz0.5 MI20%5 min\u224850% increase in apoptosisEscoffre et al. (>>2011<<)U87MG, MDA-231Free doxorubicin (DOX)Vevo, BR14, SonoVue1 MHz400\u2013800 kPa40%30 s30\u201340% decrease in viability, depending on cell lineSorace et al. (2012)2LMPFree paclitaxel (PTX)Definity1 MHz1.0 MPa PNP20%5 min50% increase in cell deathHu et" . _:b585248174 . _:b42921916 _:b42921969 . . _:b42922035 "Ultrasound transducers used in the literature can be focused or unfocused (Sanches et al., >>2011<<). Focused beams are created using spherically curved transducers, which greatly increase the ultrasound intensity in a small region of interest, e.g., a tumor. Due to a lack of standardized calibration methods concerning the applied" . _:b42922025 "For deep-seated tumors, most protocols recommend injection of drugs and microbubbles via blood flow, providing better access to deeper tumors (Treat et al., 2012; Yan et al., >>2013<<; Burke et al., 2014). The i.v. route is a relatively easy and safe way to be used in the clinic for the administration of therapeutics and microbubbles." . _:b42921916 _:b42921970 . _:b42921916 _:b42921971 . _:b42921916 _:b42921980 . _:b42922014 . _:b585248169 . _:b42921916 _:b42921981 . _:b42921916 _:b42921982 . _:b42921916 _:b42921983 . _:b42921916 _:b42921976 . _:b42922015 . _:b585248168 . _:b42921916 _:b42921977 . _:b42921916 _:b42921978 . _:b42921916 _:b42921979 . _:b585248130 . _:b42922008 . _:b42921916 _:b42921956 . _:b585248171 . _:b42921916 _:b42921957 . _:b585248143 . _:b42921916 _:b42921958 . . _:b42921916 _:b42921959 . _:b42921916 _:b42921952 . _:b42922009 . _:b585248170 . _:b42921904 "ReferenceTumor (site, animal)DrugMicrobubbleAdministration routeUS parametersOutcome vs drug aloneFrequencyIntensityDuty cycleTimeYan et al. (>>2013<<)4T1 (s.c., mouse)PTX-liposome loaded MBLipidintravenous (i.v.)2.25 MHz1.9 MPa1%10 minFourfold increase it PTX accumulation, 2.5-fold decrease in tumor volume compared to PTX-loaded MB aloneBurke et al. (2014)C6 (s." . _:b42921916 _:b42921953 . . _:b42921916 _:b42921954 . _:b42921916 _:b42921955 . _:b42921916 _:b42921964 . _:b42922010 . _:b42921916 _:b42921965 . _:b42921916 _:b42921966 . . _:b42921916 _:b42921967 . . _:b42922011 . _:b42921916 _:b42921960 . _:b42921877 . . _:b42921977 . _:b42921916 _:b42921961 . _:b42921916 _:b42921962 . _:b42921916 _:b42921963 . _:b42922020 . . _:b42922021 . _:b42922034 . _:b42921876 "In addition to challenges related to the physicochemical properties of drugs, tumors also possess physiological barriers (Jain, >>2001<<). Contrary to healthy tissues, tumor tissues have a high interstitial fluid pressure (IFP), which is related to the lack of functional lymphatics and the leaky tumor vasculature (Boucher et al., 1990). These high pressures establish an" . _:b42921881 . _:b42922022 . _:b42921951 . _:b42922028 . _:b42922023 . . _:b585248176 . . _:b42921947 . _:b42922016 . _:b42922043 . _:b42922066 "For example, Burke et al. (>>2011<<) demonstrated that the mechanical effect of low duty cycle ultrasound (1 MHz, 1 MPa PNP) in combination with microbubbles could inhibit glioma growth by blocking tumor perfusion." . _:b42922017 . . _:b42921965 . . _:b42922018 . _:b42922019 . . . . _:b42922028 . _:b585248104 . _:b42922029 . _:b42922040 . . _:b42922030 . _:b42921990 . . _:b42922031 . _:b42921925 . _:b42921927 . _:b42921885 "years, research in the field of microbubble-assisted ultrasound (also known as sonoporation) aimed at delivering therapeutic molecules in vitro and in vivo has grown rapidly (Aryal et al., 2014; Azagury et al., 2014; Kiessling et al., >>2014<<; Rychak and Klibanov, 2014; Unga and Hashida, 2014; Unger et al., 2014)." . _:b42922024 . . _:b42921918 "a second generation of microbubbles was developed, which were filled with heavy-weight hydrophobic gas (e.g., perfluorocarbon, sulfur hexafluoride) encapsulated by a biocompatible shell (e.g., lipids, polymer; Hernot and Klibanov, >>2008<<; Sirsi and Borden, 2014; Figure 1A). In studies on drug delivery by microbubble-assisted ultrasound, the bubbles are mixed with cells in vitro or injected in vivo intravascularly or directly into the tissue of interest." . _:b42921966 . _:b42922025 . . _:b42922011 . . _:b42922026 . _:b42921963 . . _:b42922027 . _:b42921937 . _:b42922046 "at different time points following the exposure of tumor to microbubble-assisted ultrasound to assess the duration of enhanced permeability (few seconds \u2013 few hours, depending on the particle size; Marty et al., 2012; Tzu-Yin et al., >>2014<<; Lammertink et al., 2015). Other investigations recommend waiting for the peak concentration of drug in the blood before the administration of microbubbles and the subsequent exposure of tumors to ultrasound." . _:b42922036 . _:b585248159 . _:b42922024 "For deep-seated tumors, most protocols recommend injection of drugs and microbubbles via blood flow, providing better access to deeper tumors (Treat et al., >>2012<<; Yan et al., 2013; Burke et al., 2014). The i.v. route is a relatively easy and safe way to be used in the clinic for the administration of therapeutics and microbubbles." . _:b42922037 . . _:b42922038 . _:b42921911 . _:b42922039 . _:b42921915 "MHz0.5\u20130.7 MPa5%1 min / sonication siteFivefold increase in circulatory half-life of BCNU, fivefold decrease in liver accumulation, 13-fold decrease in tumor volume, 12% increase in median survival compared to free BCNUTinkov et al. (>>2010<<)DSL6A (s.c., rat)DOX-loaded MBLipidi.v. (perfusion)1.3 MHz1.2 MPaNDFour ultrasound frames every four cardiac cycles10-fold i." . _:b42921920 . _:b42921962 . . _:b42921975 "Based on the uptake or release of non-permeant dyes (Meijering et al., 2009; Kaddur et al., 2010) and by measuring changes in membrane electrophysiology (Tran et al., >>2007<<; Juffermans et al., 2008), previous studies showed that microbubble-assisted ultrasound induced a transient increase in membrane permeability through the generation of transient hydrophilic pores." . . _:b42922032 . . . _:b42921948 . _:b42921925 "The ultrasound-induced collapse of the microbubble can be asymmetrical, leading to the formation of high velocity jets (Postema et al., >>2005<<; Ohl et al., 2006)." . _:b585248127 . _:b585248160 . _:b42922033 . _:b42921975 . _:b42921917 "contrast agents (i.e., consisting of gas microbubbles) was introduced as a promising method in improving the therapeutic efficacy of drugs by increasing local delivery, while minimizing side effects to healthy tissues (Price et al., >>1998<<). In this paper, we refer to this combination as microbubble-assisted ultrasound." . _:b42922001 . _:b42922034 . _:b42922037 . _:b42922040 "Increasing the ultrasound dose further enhanced drug delivery in the target tissue but was also accompanied by hemorrhage and tissue injuries (Kang et al., >>2010<<; Lu et al., 2011)." . . . _:b42922035 . _:b42922000 . _:b42921959 "In addition, local heating can act as an external trigger for drug release from a carrier, e.g., thermosensitive nanoparticles (Yatvin et al., 1978; Lindner et al., >>2004<<; Manzoor et al., 2012; Hijnen et al., 2014; Al Sabbagh et al., 2015)." . . _:b42922020 . _:b42922044 . _:b42921978 . _:b42922045 . . _:b42922046 . _:b42922045 . _:b42921981 . _:b42922047 . _:b42921949 "The microbubble-assisted ultrasound enhanced transcellular pathways (e.g., transcytosis) have been mainly investigated on the brain vasculature (Raymond et al., 2007; Sheikov et al., >>2008<<; Deng et al., 2012)." . _:b42922040 . _:b42921995 . _:b42922022 "t. injection (Sonoda et al., >>2007<<; Sasaki et al., 2014). The advantages of i.t. administration over systemic injection include the circumvention of the transvascular barrier and the generation of transient interstitial pressure gradients." . . _:b585248172 . _:b42922041 . . _:b42921939 "to enhance the extravasation of intravascular agents (e.g., red blood cells, imaging tracers, fluorescent dyes, or drugs) in skeletal muscle (Price et al., 1998), brain (Raymond et al., 2007; Sheikov et al., 2008), liver (Gao et al., >>2012<<), and tumor (Bohmer et al., 2010; Hu et al., 2012) tissues. This extravasation occurs through tight junctions between endothelial cells (0.2\u2013200 \u03BCm; Price et al., 1998; Song et al., 2002; Stieger et al., 2007)." . _:b42921953 . _:b42921973 "Based on the uptake or release of non-permeant dyes (Meijering et al., >>2009<<; Kaddur et al., 2010) and by measuring changes in membrane electrophysiology (Tran et al., 2007; Juffermans et al., 2008), previous studies showed that microbubble-assisted ultrasound induced a transient increase in membrane permeability" . _:b42922042 . . _:b585248122 . _:b42922030 . _:b42922043 . . . _:b42922029 . _:b42922052 . _:b585248147 . _:b42921993 . _:b42922053 . _:b42922054 . _:b42921930 "ultrasound increased (model-) drug extravasation by stimulating paracellular (i.e., disruption of tight junctions) and transcellular pathways (i.e., transcytosis), both in vitro as well as in vivo (Figure 2; Price et al., >>1998<<; Sheikov et al., 2008; Juffermans et al., 2009; Kooiman et al., 2010)." . _:b42922055 . _:b42921876 . _:b42921974 "Based on the uptake or release of non-permeant dyes (Meijering et al., 2009; Kaddur et al., >>2010<<) and by measuring changes in membrane electrophysiology (Tran et al., 2007; Juffermans et al., 2008), previous studies showed that microbubble-assisted ultrasound induced a transient increase in membrane permeability through the" . _:b42921999 . _:b42922048 . _:b42921877 . _:b42921998 . _:b42922049 . _:b42921878 . . _:b42921892 . _:b42922050 . _:b42921879 . "10.3389%2Ffphar.2015.00138" . _:b585248158 . _:b42922051 . _:b42922060 . _:b585248145 . _:b42922061 . _:b585248150 . _:b42921874 . _:b42921929 "Cavitating microbubbles close to the endothelial wall can result in several bio-effects including vascular disruption, vasoconstriction, or even shutdown of the vessels (Goertz, >>2015<<). Several studies observed that microbubble-assisted ultrasound increased (model-) drug extravasation by stimulating paracellular (i.e., disruption of tight junctions) and transcellular pathways (i.e., transcytosis), both in vitro as well" . _:b42921907 "<40 days) compared to untargeted BCNU-loaded MBIwanaga et al. (>>2007<<)Caco-9 (s.c., mouse)Free BleomycinOptisonIntratumoral (i." . _:b42922062 . _:b42921875 . _:b42921973 . _:b585248164 . . _:b42922063 . _:b42921884 . _:b42921928 "Microbubbles are intravascular contrast agents, which do not cross the vascular endothelium (Wilson and Burns, >>2010<<). Cavitating microbubbles close to the endothelial wall can result in several bio-effects including vascular disruption, vasoconstriction, or even shutdown of the vessels (Goertz, 2015)." . . _:b42922056 . _:b42921885 . _:b42922007 "Nevertheless, preclinical and clinical studies have reported a good tolerance with 100- and 1000-fold higher doses of these microbubbles in non-human primates and patients (Grauer et al., 1996; Bokor et al., >>2001<<). Consequently, the injection of a high dose of drug-loaded microbubbles may not be a limitation for clinical use, but further preclinical studies might be necessary to identify any potential toxicity of high concentrations of liposome" . _:b42922057 . _:b42921886 . _:b42922048 "For example, Escoffre et al. (2013b) succeeded to optimize therapeutic efficacy of irinotecan using microbubble-assisted ultrasound in subcutaneous glioblastoma." . . _:b42922058 . _:b42921956 "of drugs by acting on tumor hemodynamics (Figure 3): (i) by increasing tumor perfusion, thus enhancing drug bioavailability in tumor tissue (Song, 1984); (ii) by increasing vascular permeability (Lefor et al., 1985; Kong et al., >>2001<<) and reducing tumor interstitial pressure (Vaupel and Kelleher, 2012), leading to better drug penetration within tumor tissue." . _:b42921887 . _:b42922057 "For example, Deng et al. (>>2014<<) showed enhanced intracellular Dox levels (Figure 5A) and increased retention due to a down-regulation of P-glycoprotein following ultrasound exposure in the presence of Dox-liposome loaded microbubbles." . . _:b42922061 "Among the studies that do measure this, Yan et al. (>>2013<<) reported that the application of ultrasound (2.25 MHz, 1.9 MPa, 10 min, three treatments:" . _:b42922059 . _:b42921880 . _:b585248125 . _:b42921881 . _:b42921983 . . _:b42921882 . . _:b42921883 . _:b585248133 . _:b42921937 "ranging from 0.1 to 0.75 MPa was required to enhance the extravasation of intravascular agents (e.g., red blood cells, imaging tracers, fluorescent dyes, or drugs) in skeletal muscle (Price et al., 1998), brain (Raymond et al., >>2007<<; Sheikov et al., 2008), liver (Gao et al., 2012), and tumor (Bohmer et al., 2010; Hu et al., 2012) tissues." . _:b42922050 . _:b42921892 . _:b42921968 "Using optical imaging, Shortencarier et al. (>>2004<<) showed that the application of ARF induced visible aggregates of fluorescent dye-loaded gas lipospheres in the direction of the beam on the far vessel wall." . _:b42921980 . . _:b42922064 . _:b42921893 . _:b585248173 . _:b42922065 . _:b42921894 . _:b42921921 . _:b42922066 . _:b42921895 . . _:b42921985 "Regardless of the mechanism of uptake, the duration of microbubble-assisted ultrasound-mediated uptake is dependent on the plasma membrane recovery time, which is a few seconds to a few hours (van Wamel et al., >>2006<<; Lammertink et al., 2015)." . . _:b42922067 . _:b42921893 "to treat superficial (e.g., skin) as well as deep organs (e.g., brain, liver, prostate), under the guidance of medical imaging modalities (magnetic resonance imaging, ultrasound imaging; Kinoshita et al., 2006; Deckers and Moonen, >>2010<<; Lammers et al., 2015)." . _:b42921888 . _:b42921912 "(co-injection)1 MHz2 W/cm250%4 minTumor eradication compared to free BLMTreat et al. (>>2012<<)9L (i.c., rat)Free DoxilDefinityi.v.1.7 MHz1.2 MPa1%1\u20132 min1.5-fold decrease in tumor volume and median survival compared to free DoxilEscoffre et al. (2013b)U-87 MG (s.c., mouse)Free IrinotecanMM1i.v.1 MHz0.4 MPa (PNP)40%3 minThreefold" . _:b42921889 . _:b42922031 . _:b42921886 . _:b42921890 . _:b42921941 "(e.g., red blood cells, imaging tracers, fluorescent dyes, or drugs) in skeletal muscle (Price et al., 1998), brain (Raymond et al., 2007; Sheikov et al., 2008), liver (Gao et al., 2012), and tumor (Bohmer et al., 2010; Hu et al., >>2012<<) tissues. This extravasation occurs through tight junctions between endothelial cells (0.2\u2013200 \u03BCm; Price et al., 1998; Song et al., 2002; Stieger et al., 2007)." . _:b42921891 . _:b42921900 . _:b585248107 . . . _:b42921901 . . . . _:b42921908 "; co-injection)1 MHz2 W/cm250%2 minTwofold decrease in tumor volume compared to free BLMKang et al. (>>2010<<)VX2 (liver, rabbit)Docetaxel-loaded MBLipidi." . _:b42921902 . _:b42922045 "drug administration at different time points following the exposure of tumor to microbubble-assisted ultrasound to assess the duration of enhanced permeability (few seconds \u2013 few hours, depending on the particle size; Marty et al., >>2012<<; Tzu-Yin et al., 2014; Lammertink et al., 2015). Other investigations recommend waiting for the peak concentration of drug in the blood before the administration of microbubbles and the subsequent exposure of tumors to ultrasound." . _:b42921903 . _:b42921923 . _:b42921961 "In addition, local heating can act as an external trigger for drug release from a carrier, e.g., thermosensitive nanoparticles (Yatvin et al., 1978; Lindner et al., 2004; Manzoor et al., 2012; Hijnen et al., >>2014<<; Al Sabbagh et al., 2015)." . _:b42921896 . _:b42922016 "For example, Fan et al. (>>2013<<) designed targeted BCNU-loaded microbubbles, which bind the VEGF-R2 overexpressed on tumor microvasculature (VEGFR2-BCNU-loaded microbubbles; Figure 4A)." . _:b42921898 . _:b42921938 . _:b42921897 . _:b42921880 . _:b585248120 . _:b585248119 . _:b42922002 . _:b42921898 . _:b42921876 . _:b42922063 . . _:b42922014 . _:b42921899 . _:b42921956 . _:b42922033 . _:b42921908 . _:b42921947 "However, Marty et al. (>>2012<<) showed that the duration of extravasation after ultrasound exposure depends on the particle size." . _:b42921909 . _:b42922036 . _:b42921949 . _:b42921910 . . . _:b42921910 . . _:b42922009 . _:b42921911 . _:b42921942 "This extravasation occurs through tight junctions between endothelial cells (0.2\u2013200 \u03BCm; Price et al., >>1998<<; Song et al., 2002; Stieger et al., 2007)." . . . _:b42921904 . _:b42921979 . _:b585248121 . . _:b42921905 . _:b42921992 . _:b42921962 "In addition, local heating can act as an external trigger for drug release from a carrier, e.g., thermosensitive nanoparticles (Yatvin et al., 1978; Lindner et al., 2004; Manzoor et al., 2012; Hijnen et al., 2014; Al Sabbagh et al., >>2015<<). Ultrasound can also generate directional ARF on molecules along its propagation path (Sarvazyan et al., 2010; Figure 3)." . _:b42921920 "Microbubble behavior in an ultrasound field has been widely studied, which led to more understanding and subsequent control of the induced bio-effects that can be used for drug delivery (Kooiman et al., >>2014<<). The response of a microbubble to ultrasound waves depends on the acoustic parameters used, such as frequency, pressure levels, and pulse duration. In short, microbubbles stably oscillate over time upon exposure to a low acoustic" . _:b42921945 . _:b585248113 . _:b42921906 . _:b42921952 "In addition, Hu et al. (>>2012<<) showed that the destruction of microbubbles with a high acoustic pressure (5 MHz, 2 MPa) decreased the tumor blood flow for 30 min before it returned back to normal, without an increase in hemorrhage." . _:b42922011 "Finally, several preclinical studies reported the use of repeated sonochemotherapy treatments (Kang et al., 2010; Tinkov et al., 2010; Li et al., 2012; Ting et al., >>2012<<). For example, Li et al. (2012) reported that the repetitive treatment (i.e., once a day for seven consecutive days) of subcutaneous hepatic tumor using 10-hydroxycamptothecin-loaded microbubbles (4 mg/kg) induced twofold stronger" . _:b42921907 . _:b585248154 . _:b42922065 "Paclitaxel (PTX) delivery by PTX-loaded microbubble with ultrasound for breast cancer treatment (Adapted with permission from Yan et al., >>2013<< \u2013 Copyright \u00A9 2013 Elsevier Ltd.)." . _:b42922042 . _:b42921950 . _:b585248169 . _:b42921916 . _:b585248111 . _:b42921917 . _:b42921957 . . _:b42921918 . _:b42921998 "Kotopoulis et al. (>>2014<<) coadministered commercially available microbubbles and gemcitabine i." . _:b42921919 . . _:b42921912 . _:b42921913 . _:b42922067 "The anti-vascular action of microbubble-assisted ultrasound (1 MHz, 1.6 MPa PNP) was also adopted by Todorova et al. (>>2013<<) who subsequently injected an anti-angiogenic agent to prevent the formation of new vessels." . . . _:b42921914 . _:b42921948 "The microbubble-assisted ultrasound enhanced transcellular pathways (e.g., transcytosis) have been mainly investigated on the brain vasculature (Raymond et al., >>2007<<; Sheikov et al., 2008; Deng et al., 2012)." . _:b585248129 . _:b42921915 . _:b42921972 . _:b42921924 . . . _:b42921925 . _:b585248118 . _:b42922061 . _:b42921917 . _:b42921909 . _:b42922046 . _:b42921926 . . _:b42922062 . _:b42922008 "Finally, several preclinical studies reported the use of repeated sonochemotherapy treatments (Kang et al., >>2010<<; Tinkov et al., 2010; Li et al., 2012; Ting et al., 2012)." . _:b42921894 "(e.g., skin) as well as deep organs (e.g., brain, liver, prostate), under the guidance of medical imaging modalities (magnetic resonance imaging, ultrasound imaging; Kinoshita et al., 2006; Deckers and Moonen, 2010; Lammers et al., >>2015<<)." . . _:b42921887 "ultrasound (also known as sonoporation) aimed at delivering therapeutic molecules in vitro and in vivo has grown rapidly (Aryal et al., 2014; Azagury et al., 2014; Kiessling et al., 2014; Rychak and Klibanov, 2014; Unga and Hashida, >>2014<<; Unger et al., 2014)." . _:b585248128 . _:b585248138 . _:b42921927 . _:b585248161 . _:b42922022 . _:b42922000 "By applying this approach, Burke et al. (>>2014<<) found that the application of ultrasound (1 MHz, 1.2 MPa, every 5 s for 60 min) on subcutaneous C6 glioma tumor following the i." . _:b42921931 . _:b42921920 . _:b42922003 "The binding of drug-loaded nanoparticles on microbubbles may not be necessary for polymer-based microbubbles, as significant amounts of (model) drug can be loaded into the polymer-based shell (Fokong et al., >>2012<<). Cochran et al. (2011) showed that the loading capacity is higher for hydrophobic drugs compared to hydrophilic drugs, and that the acoustic properties of the microbubbles were unaffected (Cochran et al., 2011)." . _:b42921921 . _:b42922064 . _:b42921935 "In addition, Juffermans et al. (>>2009<<) showed that microbubble-assisted ultrasound significantly affected the integrity of in vitro endothelial monolayers by the destabilization of the tight junctions." . _:b42921922 . _:b42921893 . _:b42921955 . _:b42921923 . . _:b42921921 "oscillations generate fluid flows surrounding the bubble, known as acoustic micro-streaming, and when in close contact with cells, result in shear stress on the cell membrane, leading to cellular uptake of drugs (Leighton, 1994; Wu, >>2002<<; Doinikov and Bouakaz, 2010). At higher acoustic pressures, microbubbles oscillate more rigorously, leading to their violent collapse and destruction, i.e., inertial cavitation (Figure 2)." . . _:b42921932 . . _:b42922039 "Drug delivery requires a minimum MI known as the permeabilization threshold, which is typically lower than 1 (Choi et al., >>2007<<). Exposure of tumor tissues above, but near the cavitation threshold has so far yielded the most promising results of drug delivery without significant side effects. Increasing the ultrasound dose further enhanced drug delivery in the" . _:b42921933 . . . . _:b42921934 . "PMC0" . _:b42921935 . _:b42922007 . _:b42921928 . _:b42921929 . . . _:b585248146 . _:b42921930 . _:b42921944 . _:b585248137 . _:b42921922 . _:b42921931 . _:b42922019 "For imaging, several groups have reported on the in vivo accumulation of targeted microbubbles in the tumor microvasculature by binding inflammation markers overexpressed on tumor endothelial cells (Deshpande et al., >>2010<<). Although these microbubbles were designed as ultrasound contrast agents for molecular imaging, it might be possible to develop optimal tissue- or organ-selective drug delivery agents by combining targeting capacities and drug loading of" . . _:b42921940 . _:b42921941 . _:b42921878 . _:b42921928 . _:b42921942 . _:b42921889 . _:b42921896 . _:b42921943 . . _:b42922049 "administration of microbubbles (Escoffre et al., 2013b). This delay is required to reach the maximal systemic concentration of SN-38, the active metabolite of irinotecan, in the blood." . _:b42921978 "The intracellular delivery of molecules through membrane pores is likely governed by passive diffusion or by ultrasound-mediated propulsion (i.e., microstreaming, ARF; Shortencarier et al., 2004; Lum et al., >>2006<<). The size of these ultrasound induced pores depend on the acoustic parameters used, ranging from 1 to 94 nm at 0.19 MPa PSP and from 2 to 4 \u03BCm at 0.48 MPa PSP (Yang et al., 2008)." . _:b42921960 . _:b42921936 . _:b42921906 . _:b42921937 . _:b42921994 "The coadministration approach seems to be the best strategy for in vitro purposes (Escoffre et al., >>2011<<; Sorace et al., 2012) or, in vivo, i." . _:b42922065 . _:b42922049 . _:b42922035 . _:b42921938 . _:b42922048 . _:b42921939 . _:b42921991 "The simplest method for drug delivery using microbubble-assisted ultrasound is to use coadministration (Heath et al., >>2012<<; Unga and Hashida, 2014). This approach includes drugs that are administered in patients anyway in current clinical practice, with the addition of an injection of (clinically approved) microbubbles." . _:b42921997 . . _:b585248140 . _:b42921948 . _:b42921996 . . _:b42921949 . _:b42922050 "In most therapeutic protocols using the coadministration approach or drug-loaded microbubbles, ultrasound was applied to the tumors immediately (5\u201310 s) after microbubble injection (Sonoda et al., >>2007<<; Matsuo et al., 2011). This strategy supposes that drugs and microbubbles are sufficiently accumulated in the target tissue during the few seconds following their administration." . _:b42921959 . _:b585248151 . _:b42921950 . _:b42921951 . _:b42921943 "This extravasation occurs through tight junctions between endothelial cells (0.2\u2013200 \u03BCm; Price et al., 1998; Song et al., >>2002<<; Stieger et al., 2007)." . . _:b42921944 . _:b42921902 . . _:b42921945 . _:b42922006 . . _:b42921946 . . _:b42921993 "(ii) instead of mixing microbubbles and drug before injection, two separate injections of the constituents can also be performed, thus allowing drugs to reach plasma peak levels before injecting microbubbles (Escoffre et al., 2013b). Microbubbles have a short circulation time and therefore need to be exposed to ultrasound within minutes after injection, otherwise they will be degraded and unable to induce bio-effects." . . _:b42921947 . _:b42922006 "However, the recommended diagnostic doses of microbubbles currently approved for contrast-enhanced ultrasound imaging (e.g., SonoVue\u00AE, Definity\u00AE) are between 109 and 1010 microbubbles for an 80-kg adult (Wilson and Burns, >>2010<<). Nevertheless, preclinical and clinical studies have reported a good tolerance with 100- and 1000-fold higher doses of these microbubbles in non-human primates and patients (Grauer et al., 1996; Bokor et al., 2001)." . _:b42921956 . _:b585248142 . _:b42921957 . _:b42922019 . _:b42921958 . _:b585248109 . _:b585248170 . . _:b42921984 "However, De Cock et al. (>>2015<<) showed that increasing the acoustic pressures (1 MHz, 0.5 MPa, PNP) induced the intracellular delivery of large fluorescent dextrans (2 MDa) to shift from uptake by endocytosis to uptake via the membrane pores." . _:b42921905 . _:b42921959 . _:b42922037 "b., ISPTA 0.0003 \u2013 0.9 W/cm2 for ultrasound-based diagnostics) have been applied in recent studies to deliver drugs in tumor tissue without injuries (Kang et al., >>2010<<; Lu et al., 2011)." . _:b42921952 . . _:b42922013 "microbubbles (4 mg/kg) induced twofold stronger decrease in tumor volume in a subcutaneous hepatic tumor model (1 MHz, 2 W/cm2, 6 min) compared to the 10-hydroxycamptothecin-based chemotherapy alone (Li et al., >>2012<<)." . _:b42921953 . _:b42921954 . _:b42921890 . _:b42921955 . _:b42921984 . . _:b42921964 . _:b42922039 . _:b585248144 . _:b42921965 . _:b42922005 "Cochran et al. (2011) showed that the loading capacity is higher for hydrophobic drugs compared to hydrophilic drugs, and that the acoustic properties of the microbubbles were unaffected (Cochran et al., >>2011<<)." . . _:b42921955 "the therapeutic efficacy of drugs by acting on tumor hemodynamics (Figure 3): (i) by increasing tumor perfusion, thus enhancing drug bioavailability in tumor tissue (Song, 1984); (ii) by increasing vascular permeability (Lefor et al., >>1985<<; Kong et al., 2001) and reducing tumor interstitial pressure (Vaupel and Kelleher, 2012), leading to better drug penetration within tumor tissue." . _:b42921966 . _:b42921903 . _:b42921976 . _:b42921967 . _:b585248153 . _:b42921939 . _:b42921960 . . _:b42921914 . _:b42921877 "Contrary to healthy tissues, tumor tissues have a high interstitial fluid pressure (IFP), which is related to the lack of functional lymphatics and the leaky tumor vasculature (Boucher et al., >>1990<<). These high pressures establish an outward fluid motion from the core of the solid tumor to the periphery and reduce fluid infiltration across the vascular wall. Thus, even if the leaky vasculature permits drug extravasation," . _:b42921982 . . _:b42921961 . _:b42921897 . _:b42921962 . _:b42921904 . _:b42922017 "injection of VEGFR2-BCNU-loaded microbubbles (1.25 mg BCNU) resulted in 1.75-fold decrease in tumor volume compared to the untargeted BCNU-loaded microbubbles (Figure 4B; Fan et al., >>2013<<). The use of microbubbles targeting overexpressed markers on the tumor cells themselves is limited to in vitro drug delivery, i.t. or intraperitoneal (i.p.) injection of microbubbles and drugs, primarily because the microbubbles, when" . _:b42921933 "paracellular (i.e., disruption of tight junctions) and transcellular pathways (i.e., transcytosis), both in vitro as well as in vivo (Figure 2; Price et al., 1998; Sheikov et al., 2008; Juffermans et al., 2009; Kooiman et al., >>2010<<). In an in vitro endothelial barrier model, Kooiman et al. (2010) showed that microbubble-assisted ultrasound induced a 40% decrease in transendothelial electric resistance showing a loss of endothelial barrier integrity." . _:b42921963 . . _:b42921972 . . . _:b42921973 . _:b585248134 . _:b42921935 . _:b42921974 . _:b42921999 "They showed that ultrasound exposure (1 MHz, 0.2 MPa PNP) decreased the tumor volume twofold compared to gemcitabine alone (Kotopoulis et al., >>2014<<). Opposed to the advantages of coadministration using clinically approved microbubbles and drugs that allow clinical translation, there are also disadvantages. The main limitations of the i.v. injection of microbubble/drug mixture" . _:b42921975 . _:b42922052 "therapeutic efficacy vs. safety: from in vitro to preclinical studies" . . _:b42921954 "heating of a tumor (41 \u2013 43\u00B0C for 10 \u2013 60 min) may improve the therapeutic efficacy of drugs by acting on tumor hemodynamics (Figure 3): (i) by increasing tumor perfusion, thus enhancing drug bioavailability in tumor tissue (Song, >>1984<<); (ii) by increasing vascular permeability (Lefor et al., 1985; Kong et al., 2001) and reducing tumor interstitial pressure (Vaupel and Kelleher, 2012), leading to better drug penetration within tumor tissue." . _:b42921968 . _:b42921932 . _:b42921969 . _:b585248148 . _:b42921974 . _:b42921970 . . _:b42921971 . . _:b42921971 . _:b585248149 . _:b42921938 "to 0.75 MPa was required to enhance the extravasation of intravascular agents (e.g., red blood cells, imaging tracers, fluorescent dyes, or drugs) in skeletal muscle (Price et al., 1998), brain (Raymond et al., 2007; Sheikov et al., >>2008<<), liver (Gao et al., 2012), and tumor (Bohmer et al., 2010; Hu et al., 2012) tissues." . _:b42921980 . _:b42921981 . _:b585248135 . _:b42921934 "In an in vitro endothelial barrier model, Kooiman et al. (>>2010<<) showed that microbubble-assisted ultrasound induced a 40% decrease in transendothelial electric resistance showing a loss of endothelial barrier integrity." . _:b42921982 . _:b585248139 . _:b42921924 . . . _:b42921983 . _:b42921950 "The microbubble-assisted ultrasound enhanced transcellular pathways (e.g., transcytosis) have been mainly investigated on the brain vasculature (Raymond et al., 2007; Sheikov et al., 2008; Deng et al., >>2012<<). They reported that low (1 MHz, 0.2 MPa) and high (1.63 MHz, 1-3 MPa) acoustic pressures increased the number of transcytotic vesicles on both the luminal and abluminal surface of the endothelium. Sheikov et al. (2004) hypothesized that" . _:b42921964 "This enhances the extravasation of free drug or drug-loaded nanoparticles into tumor tissue by causing tissue shear stress and opening of endothelial tight junctions (Seidl et al., >>1994<<; Mesiwala et al., 2002)." . _:b42921976 . . _:b42921977 . _:b42921978 . _:b42921989 . _:b42921912 . _:b42922033 "Hence, home-made and commercial therapeutic ultrasound devices have been designed to control many ultrasound parameters, which can subsequently be optimized for drug delivery (Zhao et al., 2011; Lin et al., >>2012<<; Escoffre et al., 2013a)." . _:b42921913 "1.7 MHz1.2 MPa1%1\u20132 min1.5-fold decrease in tumor volume and median survival compared to free DoxilEscoffre et al. (2013b)U-87 MG (s." . . _:b42922053 . _:b42922029 "injection may be useful for drug delivery using microbubble-assisted ultrasound for the treatments of primary peritoneal cancers or cancers with i.p. metastases. Pu et al. (>>2014<<) investigated the i.p." . _:b42921979 . . _:b42922059 . _:b42922038 "b., ISPTA 0.0003 \u2013 0.9 W/cm2 for ultrasound-based diagnostics) have been applied in recent studies to deliver drugs in tumor tissue without injuries (Kang et al., 2010; Lu et al., >>2011<<). The MI used for in vivo drug delivery ranges from 0.2 to 2 (n.b., MI threshold for clinical diagnosis is 1.9). Drug delivery requires a minimum MI known as the permeabilization threshold, which is typically lower than 1 (Choi et al.," . . _:b42922051 "In most therapeutic protocols using the coadministration approach or drug-loaded microbubbles, ultrasound was applied to the tumors immediately (5\u201310 s) after microbubble injection (Sonoda et al., 2007; Matsuo et al., >>2011<<). This strategy supposes that drugs and microbubbles are sufficiently accumulated in the target tissue during the few seconds following their administration." . _:b42921905 "2.25 MHz1.9 MPa1%10 minFourfold increase it PTX accumulation, 2.5-fold decrease in tumor volume compared to PTX-loaded MB aloneBurke et al. (>>2014<<)C6 (s.c., rat)5FU-NPs loaded MBAlbumini.v.1 MHz1.2 MPa (PNP)NDEvery 5 s for 60 minTwofold decrease in tumor volume, increase in median survival (34 days vs. 26 days) compared to free 5FUFan et al. (2013)C6 (i.c., rat)VEGFR2-BCNU- loaded" . _:b42921943 . _:b585248171 . _:b585248152 . _:b585248153 . _:b585248154 . _:b585248116 . _:b585248155 . . _:b585248156 . _:b585248157 . _:b42921951 "Sheikov et al. (>>2004<<) hypothesized that the transient vasoconstriction constitutes a potential cause for the increased transcytosis in vivo." . _:b585248158 . _:b585248159 . _:b42922060 . _:b585248144 . _:b585248145 . . _:b42922009 "Finally, several preclinical studies reported the use of repeated sonochemotherapy treatments (Kang et al., 2010; Tinkov et al., >>2010<<; Li et al., 2012; Ting et al., 2012)." . _:b585248146 . . _:b585248152 . _:b585248147 . . _:b42921882 "In order to improve the efficiency of anti-cancer chemotherapeutics, physical methods including electroporation, laser, and magnetic fields have been developed (Sersa et al., 2008; Podaru et al., 2014; Sklar et al., >>2014<<). The general principle of physical methods is based on the transient disruption of endothelial barrier and tumor cell membrane in order to facilitate the drug extravasation and the drug uptake into the endothelial and tumor cells." . _:b585248106 . _:b585248148 . _:b585248149 . _:b42921875 "Cancer presents the second leading cause of death in the European Union with 3.45 million new cases of cancer and 1.75 million deaths from cancer in 2012 (Ferlay et al., >>2013<<). Although a lot of progress has been made in the treatment of several cancers, many types of cancer are still lacking effective treatment options." . _:b585248150 . _:b585248151 . _:b585248136 . _:b585248137 . _:b585248138 . _:b585248157 . _:b585248139 . _:b585248140 . _:b585248141 . _:b585248142 . _:b42922041 "Increasing the ultrasound dose further enhanced drug delivery in the target tissue but was also accompanied by hemorrhage and tissue injuries (Kang et al., 2010; Lu et al., >>2011<<)." . _:b585248143 . . _:b585248128 . _:b585248129 . _:b585248130 . _:b585248131 . _:b585248132 . _:b42921933 . _:b585248133 . _:b42921901 "cell deathHu et al. (2012)BEL-7402Free 10-HCPT (free)Polymer3.5 MHz22.57 mW/cm2ND10 min20\u201330% decrease in viabilityRen et al. (2013)DLD-1Docetaxel-loaded MBLipid800 kHz2.56 W/cm250%10 min40% increase in inhibition rateTinkov et al. (>>2010<<)295/KDRDOX-loaded MBLipid1 MHz1 W/cm250%20 s40% decrease in cell viabilityYan et al. (2013)4T1PTX-liposome loaded MBLipid1 MHz1.0 MPa50%1 min20\u201330% decrease in viabilityDeng et al. (2014)MCF7/ADRDOX-liposome loaded MBLipid1 MHz1.65" . _:b42921884 "In recent years, research in the field of microbubble-assisted ultrasound (also known as sonoporation) aimed at delivering therapeutic molecules in vitro and in vivo has grown rapidly (Aryal et al., 2014; Azagury et al., >>2014<<; Kiessling et al., 2014; Rychak and Klibanov, 2014; Unga and Hashida, 2014; Unger et al., 2014)." . _:b585248134 . _:b585248136 . _:b42921884 . _:b585248135 . . _:b585248114 . . . _:b42921881 "In order to improve the efficiency of anti-cancer chemotherapeutics, physical methods including electroporation, laser, and magnetic fields have been developed (Sersa et al., 2008; Podaru et al., >>2014<<; Sklar et al., 2014)." . _:b585248112 . _:b585248176 . _:b42921934 . _:b42922015 . . _:b42921995 "The coadministration approach seems to be the best strategy for in vitro purposes (Escoffre et al., 2011; Sorace et al., >>2012<<) or, in vivo, i." . _:b585248168 . _:b585248169 . _:b42921944 "This extravasation occurs through tight junctions between endothelial cells (0.2\u2013200 \u03BCm; Price et al., 1998; Song et al., 2002; Stieger et al., >>2007<<). In vivo, the integrity of the blood\u2013brain barrier was restored within 1\u20134 h following ultrasound exposure (Sheikov et al., 2008; Ting et al., 2012). However, Marty et al. (2012) showed that the duration of extravasation after ultrasound" . _:b585248170 . _:b42921989 "unlike many other drug delivery strategies, sonochemotherapy does not depend on the enhanced permeability and retention (EPR) effect, which is very heterogeneous between or within tumors, and often overestimated (Lammers et al., >>2012<<). Interestingly, you could argue that the largest effect of sonochemotherapy can be expected in tissues with \u2018non-leaky\u2019 vessels, such as the brain (Ting et al., 2012), since the potential of increasing extravasation is highest." . _:b585248171 . _:b42922032 . _:b42921890 "that can be used to deliver a wide range of anticancer molecules including low molecular weight chemotherapeutic agents (sonochemotherapy), nucleic acids and monoclonal antibodies to a target site, e.g., tumor (Escoffre et al., 2013c; Ibsen et al., 2013; Unga and Hashida, 2014)." . _:b585248172 . _:b585248173 . _:b585248174 . _:b585248175 . _:b585248160 . _:b42922010 "Finally, several preclinical studies reported the use of repeated sonochemotherapy treatments (Kang et al., 2010; Tinkov et al., 2010; Li et al., >>2012<<; Ting et al., 2012)." . . _:b585248161 . _:b585248162 . _:b585248163 . _:b585248164 . . _:b42921953 "Besides cavitation, ultrasound can also induce heating and acoustic radiation force (ARF) to improve the extravasation of drugs (Deckers and Moonen, >>2010<<). Heating can result from the absorbance of acoustic energy as the ultrasound beam propagates through tissue." . _:b585248165 . _:b585248166 . . _:b585248167 . _:b42921880 "In order to improve the efficiency of anti-cancer chemotherapeutics, physical methods including electroporation, laser, and magnetic fields have been developed (Sersa et al., >>2008<<; Podaru et al., 2014; Sklar et al., 2014)." . . . _:b42922058 "\u2217p < 0.05, \u2217\u2217p < 0.01 (Adapted with permission from Deng et al., >>2014<< \u2013 Copyright \u00A9 2014 Elsevier Ltd." . _:b42921927 "are both exploited to transiently increase the permeability of biological barriers, including the vascular endothelium and plasma membrane, and therefore enhance the extravasation and the cellular uptake of drugs (Lentacker et al., >>2014<<; Figure 2)." . _:b42922018 "injection of microbubbles and drugs, primarily because the microbubbles, when administrated intravenously, cannot extravasate due to the size (Cavalieri et al., >>2010<<). For imaging, several groups have reported on the in vivo accumulation of targeted microbubbles in the tumor microvasculature by binding inflammation markers overexpressed on tumor endothelial cells (Deshpande et al., 2010). Although" . _:b42921970 "addition to lipospheres, ARFs can push circulating microbubbles toward the endothelial wall, thereby improving microbubble\u2013cell contact, which might enhance cavitation-mediated extravasation of intravascular compounds (Rychak et al., >>2005<<; Wang et al., 2014)." . _:b42922067 . . _:b42921970 . . _:b42921987 "anti-cancer drug delivery protocols" . _:b585248120 . _:b585248121 . _:b585248122 . _:b585248123 . _:b585248124 . _:b42922025 . _:b585248125 . _:b585248126 . _:b42922062 "administration of the PTX-loaded microbubbles and ultrasound exposure (Yan et al., >>2013<<). The PTX biodistribution in heart, spleen, and lung was not significantly different between mice that received PTX-loaded microbubbles treatment alone or combined with ultrasound (Figure 6A). However, the PTX delivery using" . _:b585248127 . _:b585248112 . _:b585248113 . _:b585248114 . _:b585248115 . . _:b585248116 . _:b585248117 . _:b585248117 . _:b585248118 . . _:b585248119 . _:b585248104 . _:b42921972 "Using ultrasound imaging, Frinking et al. (>>2012<<) reported that ARF (38 kPa PNP, 95% DC) induced a sevenfold increase in the binding of VEGFR2-targeted microbubbles (also known as BR-55) on the endothelial wall in a prostate adenocarcinoma rat model compared with the binding without ARF." . _:b585248105 . _:b585248106 . _:b585248107 . _:b585248108 . _:b585248110 . _:b585248109 . _:b585248110 . _:b42921991 . _:b585248111 . _:b42921915 . _:b42922023 . _:b42922034 "Hence, home-made and commercial therapeutic ultrasound devices have been designed to control many ultrasound parameters, which can subsequently be optimized for drug delivery (Zhao et al., 2011; Lin et al., 2012; Escoffre et al., 2013a). Ultrasound transducers used in the literature can be focused or unfocused (Sanches et al., 2011)." . _:b42921963 "Ultrasound can also generate directional ARF on molecules along its propagation path (Sarvazyan et al., >>2010<<; Figure 3)." . _:b585248126 . _:b585248167 . _:b585248103 . _:b42921879 "For several hydrophilic and charged drugs, e.g., bleomycin, this is a serious challenge and requires active uptake through plasma membrane transporters, which are not always present in the target cells (Pron et al., >>1999<<)." . _:b42921967 . . _:b42921979 "The size of these ultrasound induced pores depend on the acoustic parameters used, ranging from 1 to 94 nm at 0.19 MPa PSP and from 2 to 4 \u03BCm at 0.48 MPa PSP (Yang et al., >>2008<<)." . . _:b42922012 "For example, Li et al. (>>2012<<) reported that the repetitive treatment (i.e., once a day for seven consecutive days) of subcutaneous hepatic tumor using 10-hydroxycamptothecin-loaded microbubbles (4 mg/kg) induced twofold stronger decrease in tumor volume in a" . . . _:b42921908 . _:b42921980 "In addition to hydrophilic pore formation, enhancement of endocytosis has also been demonstrated following microbubble-assisted ultrasound exposure (Meijering et al., >>2009<<). Electrophysiological studies reported that microbubble-assisted ultrasound induced an influx of Ca2+, followed by an activation of BKCa channels that results in local hyperpolarization of the cell membrane (Tran et al., 2007; Juffermans" . _:b585248165 . _:b42922003 . _:b42921958 "In addition, local heating can act as an external trigger for drug release from a carrier, e.g., thermosensitive nanoparticles (Yatvin et al., >>1978<<; Lindner et al., 2004; Manzoor et al., 2012; Hijnen et al., 2014; Al Sabbagh et al., 2015)." . _:b42921954 . _:b42921875 . _:b42921926 . . _:b42922021 . _:b42921918 . _:b42921987 _:b42922048 . _:b42921987 _:b42922049 . . _:b42921987 _:b42922050 . _:b42921987 _:b42922051 . _:b42921957 "tumor perfusion, thus enhancing drug bioavailability in tumor tissue (Song, 1984); (ii) by increasing vascular permeability (Lefor et al., 1985; Kong et al., 2001) and reducing tumor interstitial pressure (Vaupel and Kelleher, >>2012<<), leading to better drug penetration within tumor tissue." . _:b42921878 "High tumor cell proliferation results in tumor cells forcing vessels apart, leading to a decrease in vascular density and a limitation in the access of drugs to distant tumor cells (Minchinton and Tannock, >>2006<<). In addition, the presence of high levels of extracellular matrix limits the interstitial transport of drugs (Weinberg, 2014). Altogether these barriers oppose sufficient and uniform distribution of drugs in solid tumors, thereby" . . . _:b42922016 . . _:b42921926 "The ultrasound-induced collapse of the microbubble can be asymmetrical, leading to the formation of high velocity jets (Postema et al., 2005; Ohl et al., >>2006<<). While shock waves induce shear stress to cells in close proximity, resulting in membrane permeability, the high velocity jets can pierce the cell membrane, and thereby create permeability. Stable and inertial cavitation are both" . _:b42922004 . . _:b42922017 . _:b42922063 "Moreover, Ting et al. (>>2012<<) designed a therapeutic protocol based on BCNU-loaded microbubbles (0.8 mg \u2013 1 \u00D7 1010) with focused ultrasound (1 MHz, 0.5\u20130.7 MPa, 2 sonications, 1 min/sonication) to improve BCNU-based chemotherapy for glioblastoma treatment." . _:b42922005 . . _:b42921946 "In vivo, the integrity of the blood\u2013brain barrier was restored within 1\u20134 h following ultrasound exposure (Sheikov et al., 2008; Ting et al., >>2012<<). However, Marty et al. (2012) showed that the duration of extravasation after ultrasound exposure depends on the particle size. The microbubble-assisted ultrasound enhanced transcellular pathways (e.g., transcytosis) have been mainly" . _:b42922044 . _:b42921987 . . _:b42921987 _:b42922024 . _:b42921987 _:b42922025 . _:b42921987 _:b42922026 . _:b42921899 . _:b42921987 _:b42922027 . _:b42921987 _:b42922028 . _:b42921987 _:b42922029 . _:b42921987 _:b42922030 . _:b42922002 "Recent publications reported that the binding of drug-loaded nanoparticles on the microbubble\u2019s surface could increase the amount of loaded drug (Geers et al., >>2011<<). The loading efficiency can be further improved by applying multiple layers of drug-loaded nanoparticles around the microbubble shell. The binding of drug-loaded nanoparticles on microbubbles may not be necessary for polymer-based" . _:b585248103 "7"^^ . _:b42921987 _:b42922031 . _:b42921987 _:b42922016 . _:b42921987 _:b42922017 . _:b42921987 _:b42922018 . _:b42922066 . . _:b42921987 _:b42922019 . _:b42921987 _:b42922020 . _:b42921874 "introduction" . . . _:b42921987 _:b42922021 . _:b42921987 _:b42922022 . . _:b42921987 _:b42922023 . _:b42921987 _:b42922040 . _:b42921987 _:b42922041 . _:b42921987 _:b42922042 . _:b42921987 _:b42922043 . _:b42921987 _:b42922044 . _:b42921987 _:b42922045 . _:b42921987 _:b42922046 . _:b42921987 _:b42922047 . . _:b42921987 _:b42922032 . _:b42921909 "(infusion)0.3 MHz2 W/cm250%6 minThreefold increase tumor inhibition, twofold increase in apoptosis, twofold decrease in proliferation compared to free DocetaxelLi et al. (>>2012<<)H22 (s.c., mouse)10-HCPT loaded MBLipidi.v.1 MHz2 W/cm250%6 minSixfold increase in it 10-HCPT accumulation, twofold decrease in tumor volume compared to free 10-HCPTPu et al. (2014)A2780/DDP (i.p. mouse)LHRHa-PTX loaded" . _:b42921987 _:b42922033 . _:b42921987 _:b42922034 . _:b42922057 . _:b42921919 "of microbubbles was developed, which were filled with heavy-weight hydrophobic gas (e.g., perfluorocarbon, sulfur hexafluoride) encapsulated by a biocompatible shell (e.g., lipids, polymer; Hernot and Klibanov, 2008; Sirsi and Borden, >>2014<<; Figure 1A). In studies on drug delivery by microbubble-assisted ultrasound, the bubbles are mixed with cells in vitro or injected in vivo intravascularly or directly into the tissue of interest." . _:b42921987 _:b42922035 . _:b42921901 . . _:b42921987 _:b42922036 . _:b42921987 _:b42922037 . . _:b42921987 _:b42922038 . _:b42921906 "26 days) compared to free 5FUFan et al. (>>2013<<)C6 (i.c., rat)VEGFR2-BCNU- loaded MBLipidi." . _:b42921987 _:b42922039 . _:b42921987 _:b42921992 . _:b42921961 . _:b42921987 _:b42921993 . _:b42921987 _:b42921994 . _:b585248109 "3"^^ . _:b42921987 _:b42921995 . _:b42921987 _:b42921996 . _:b42921987 _:b42921997 . _:b585248168 . _:b42921987 _:b42921998 . _:b42922058 . _:b585248108 "3"^^ . _:b42921987 _:b42921999 . _:b585248174 . _:b585248111 "3"^^ . _:b585248156 . _:b42922030 "This therapeutic protocol led to a twofold increase in apoptotic index and a 2.5-fold decrease in vessel number compared to the single injection of free PTX or PTX delivery using ultrasound alone (Pu et al., >>2014<<). Due to the microbubble size, penetration of the microbubbles by convection throughout the tumor is hindered, thereby limiting the tumor cell binding to the peripheral rim of the tumor. Nevertheless, the targeted microbubbles in this" . _:b42921987 _:b42921988 . . _:b42921987 _:b42921989 . _:b42921987 _:b42921990 . _:b585248110 "3"^^ . _:b42921987 _:b42921991 . _:b42921987 _:b42922008 . _:b42922060 "DOX accumulation compared to DOX-loaded microbubble injection alone (Tinkov et al., >>2010<<). This therapeutic protocol led to a twofold decrease in tumor volume." . _:b42921916 "microbubble-assisted ultrasound" . _:b42921987 _:b42922009 . . . _:b42921967 "ARF induces fluid streaming through the interstitium, thus improving biodistribution of intravascular dyes and drugs in the target tissue (Lum et al., 2006; Hancock et al., >>2009<<). Using optical imaging, Shortencarier et al. (2004) showed that the application of ARF induced visible aggregates of fluorescent dye-loaded gas lipospheres in the direction of the beam on the far vessel wall. The lipospheres disappeared" . _:b585248105 "3"^^ . _:b42921987 _:b42922010 . _:b42921987 _:b42922011 . _:b42921987 _:b42922012 . _:b585248163 . _:b42921987 _:b42922013 . _:b42921987 _:b42922014 . _:b42921987 _:b42922015 . . _:b585248104 "5"^^ . _:b42921987 _:b42922000 . _:b42921987 _:b42922001 . _:b42922054 . _:b42922054 "using microbubble-assisted ultrasound relies on enhancing accumulation of drugs in tumor cells or tissues and on decreasing their deposition in healthy tissues, thus reducing their side effects (Tinkov et al., 2010; Li et al., >>2012<<; Fan et al., 2013; Burke et al., 2014)." . . _:b585248107 "3"^^ . _:b42921987 _:b42922002 . _:b42921987 _:b42922003 . . _:b42921987 _:b42922004 . _:b585248176 . _:b42921887 . _:b585248108 . _:b42921987 _:b42922005 . . _:b585248106 "3"^^ . _:b42922052 . _:b42921987 _:b42922006 . _:b42921987 _:b42922007 . _:b42921914 "MHz0.4 MPa (PNP)40%3 minThreefold decrease in tumor volume, twofold decrease in tumor perfusion, threefold increase necrosis, 35% decrease in mitosis index, no acute liver toxicity compared to free irinotecanTing et al. (>>2012<<)C6 (i.c., rat)BCNU-loaded MBLipidi.v." . _:b42921997 "co-injection of microbubbles and bleomycin also resulted in a twofold decrease in tumor volume (Iwanaga et al., >>2007<<). Kotopoulis et al. (2014) coadministered commercially available microbubbles and gemcitabine i.v. in a pancreatic cancer model in mice. They showed that ultrasound exposure (1 MHz, 0.2 MPa PNP) decreased the tumor volume twofold compared" . _:b585248117 "3"^^ . _:b42922055 "ultrasound relies on enhancing accumulation of drugs in tumor cells or tissues and on decreasing their deposition in healthy tissues, thus reducing their side effects (Tinkov et al., 2010; Li et al., 2012; Fan et al., >>2013<<; Burke et al., 2014)." . _:b42922004 "Cochran et al. (>>2011<<) showed that the loading capacity is higher for hydrophobic drugs compared to hydrophilic drugs, and that the acoustic properties of the microbubbles were unaffected (Cochran et al., 2011)." . _:b585248116 "3"^^ . _:b42921982 "studies reported that microbubble-assisted ultrasound induced an influx of Ca2+, followed by an activation of BKCa channels that results in local hyperpolarization of the cell membrane (Tran et al., 2007; Juffermans et al., >>2008<<). At moderate ultrasound conditions (1 MHz, 0.15\u20130.3 MPa), the membrane hyperpolarization facilitates the molecular uptake through endocytosis and macropinocytosis." . . . . _:b585248119 "3"^^ . . _:b585248103 . _:b42921996 "Iwanaga et al. (>>2007<<) showed that the in vitro delivery of bleomycin using microbubble-assisted ultrasound induced twofold decrease in cell viability compared to the bleomycin treatment alone (Table 1)." . _:b585248118 "3"^^ . . _:b585248113 "3"^^ . _:b42921981 "Electrophysiological studies reported that microbubble-assisted ultrasound induced an influx of Ca2+, followed by an activation of BKCa channels that results in local hyperpolarization of the cell membrane (Tran et al., >>2007<<; Juffermans et al., 2008)." . _:b585248112 "3"^^ . . _:b42921976 "Based on the uptake or release of non-permeant dyes (Meijering et al., 2009; Kaddur et al., 2010) and by measuring changes in membrane electrophysiology (Tran et al., 2007; Juffermans et al., >>2008<<), previous studies showed that microbubble-assisted ultrasound induced a transient increase in membrane permeability through the generation of transient hydrophilic pores." . _:b585248105 . _:b585248115 "3"^^ . _:b42922053 "benefit of drug delivery using microbubble-assisted ultrasound relies on enhancing accumulation of drugs in tumor cells or tissues and on decreasing their deposition in healthy tissues, thus reducing their side effects (Tinkov et al., >>2010<<; Li et al., 2012; Fan et al., 2013; Burke et al., 2014)." . . . . _:b42921966 "ARF induces fluid streaming through the interstitium, thus improving biodistribution of intravascular dyes and drugs in the target tissue (Lum et al., >>2006<<; Hancock et al., 2009)." . _:b585248114 "3"^^ . . . _:b585248125 "2"^^ . _:b42921900 "on cell lineSorace et al. (2012)2LMPFree paclitaxel (PTX)Definity1 MHz1.0 MPa PNP20%5 min50% increase in cell deathHu et al. (2012)BEL-7402Free 10-HCPT (free)Polymer3.5 MHz22.57 mW/cm2ND10 min20\u201330% decrease in viabilityRen et al. (>>2013<<)DLD-1Docetaxel-loaded MBLipid800 kHz2.56 W/cm250%10 min40% increase in inhibition rateTinkov et al. (2010)295/KDRDOX-loaded MBLipid1 MHz1 W/cm250%20 s40% decrease in cell viabilityYan et al. (2013)4T1PTX-liposome loaded MBLipid1 MHz1.0" . _:b42922032 "Hence, home-made and commercial therapeutic ultrasound devices have been designed to control many ultrasound parameters, which can subsequently be optimized for drug delivery (Zhao et al., >>2011<<; Lin et al., 2012; Escoffre et al., 2013a)." . _:b585248109 . _:b585248124 "2"^^ . _:b585248108 . . _:b585248127 "2"^^ . . _:b585248111 . _:b42922064 "encapsulation of BCNU in microbubbles prolonged its circulatory half-life fivefold and intrahepatic accumulation of BCNU was reduced fivefold due to the slow reticuloendothelial system uptake of BCNU-loaded microbubbles (Ting et al., >>2012<<). These microbubbles alone or in combination with focused ultrasound were associated with lower levels of aspartate- and alanine-aminotransferases compared to free BCNU, suggesting that these microbubbles may effectively reduce liver" . _:b42921946 . _:b42921896 "drug aloneFrequencyIntensityDuty cycleTimeIwanaga et al. (2007)Ca9-22Free bleomycinOptison1 MHz1.0 W/cm210%20 s2.5-fold increase in apoptosisHeath et al. (>>2012<<)SCC-1, SCC-5, Cal27Free cisplatinDefinity1 MHz0.5 MI20%5 min\u224850% increase in apoptosisEscoffre et al. (2011)U87MG, MDA-231Free doxorubicin (DOX)Vevo, BR14, SonoVue1 MHz400\u2013800 kPa40%30 s30\u201340% decrease in viability, depending on cell" . . _:b42921891 . _:b585248126 "2"^^ . _:b585248110 . _:b585248121 "2"^^ . . _:b585248105 . _:b42921892 "method offers the possibility to treat superficial (e.g., skin) as well as deep organs (e.g., brain, liver, prostate), under the guidance of medical imaging modalities (magnetic resonance imaging, ultrasound imaging; Kinoshita et al., >>2006<<; Deckers and Moonen, 2010; Lammers et al., 2015)." . _:b585248120 "2"^^ . _:b42922041 . _:b42921910 "1 MHz2 W/cm250%6 minSixfold increase in it 10-HCPT accumulation, twofold decrease in tumor volume compared to free 10-HCPTPu et al. (>>2014<<)A2780/DDP (i." . _:b585248104 . _:b585248123 "2"^^ . . _:b585248107 . . _:b585248122 "2"^^ . . _:b585248106 . _:b585248133 "2"^^ . _:b42921940 . _:b585248117 . _:b585248132 "2"^^ . _:b42922056 . _:b585248116 . _:b585248135 "2"^^ . _:b42921874 _:b42921900 . _:b42921874 _:b42921901 . _:b42921895 "MB-loaded)MicrobubbleUltrasound (US) parametersOutcome vs. drug aloneFrequencyIntensityDuty cycleTimeIwanaga et al. (>>2007<<)Ca9-22Free bleomycinOptison1 MHz1.0 W/cm210%20 s2.5-fold increase in apoptosisHeath et al. (2012)SCC-1, SCC-5, Cal27Free cisplatinDefinity1 MHz0.5 MI20%5 min\u224850% increase in apoptosisEscoffre et al. (2011)U87MG, MDA-231Free doxorubicin" . _:b585248119 . _:b585248103 . _:b42921874 _:b42921902 . _:b42921988 "to the specific properties of each tumor tissue, such as differences in tissue organization, extracellular matrix, presence of necrosis and hypoxia, cell density, and the endothelial lining of the tumor vasculature (Chauhan et al., >>2011<<). To the best of our knowledge, no comparative study between tumor tissues with different properties has been reported using microbubble-assisted ultrasound for drug delivery." . _:b585248134 "2"^^ . . _:b42921874 _:b42921903 . _:b42921874 _:b42921896 . _:b42921874 _:b42921897 . _:b585248118 . . _:b42921874 _:b42921898 . _:b585248129 "2"^^ . . _:b42921874 _:b42921899 . _:b42921874 _:b42921892 . _:b42921874 _:b42921893 . _:b42922027 "Therefore, drugs can be loaded on microbubbles to overcome these shortcomings (Ting et al., >>2012<<; Sirsi and Borden, 2014)." . _:b585248113 . _:b42921874 _:b42921894 . _:b585248128 "2"^^ . _:b42921874 _:b42921895 . _:b42921874 _:b42921888 . _:b42922043 "However, exposure times of 1\u20135 min are recommended to prevent tissue injuries (e.g., hemorrhages; Mei et al., >>2009<<; Yan et al., 2013)." . _:b42921874 _:b42921889 . _:b585248112 . _:b42921874 _:b42921890 . _:b585248131 "2"^^ . _:b42921874 _:b42921891 . _:b42921985 . . _:b585248115 . _:b42921895 . _:b42921874 . _:b585248130 "2"^^ . _:b585248131 . _:b42921874 _:b42921912 . _:b42921874 _:b42921913 . _:b585248114 . _:b42921874 _:b42921914 . _:b42921960 "In addition, local heating can act as an external trigger for drug release from a carrier, e.g., thermosensitive nanoparticles (Yatvin et al., 1978; Lindner et al., 2004; Manzoor et al., >>2012<<; Hijnen et al., 2014; Al Sabbagh et al., 2015)." . _:b585248141 "2"^^ . _:b42921874 _:b42921915 . . _:b42921874 _:b42921908 . _:b42921874 _:b42921909 . _:b42921924 "Microbubble disruption can be accompanied by generation of shock waves in the medium close to the microbubbles (Junge et al., 2003; Ohl and Wolfrum, >>2003<<). The ultrasound-induced collapse of the microbubble can be asymmetrical, leading to the formation of high velocity jets (Postema et al., 2005; Ohl et al., 2006). While shock waves induce shear stress to cells in close proximity," . _:b585248125 . _:b42921874 _:b42921910 . _:b42922042 "To prevent thermal tissue damage, low duty cycles are used when high ultrasound intensities are applied and vice versa (Lin et al., >>2012<<; Wei et al., 2013)." . _:b585248140 "2"^^ . _:b42921874 _:b42921911 . _:b42921874 _:b42921904 . _:b42921874 _:b42921905 . _:b585248124 . _:b42921874 _:b42921906 . _:b42921940 "intravascular agents (e.g., red blood cells, imaging tracers, fluorescent dyes, or drugs) in skeletal muscle (Price et al., 1998), brain (Raymond et al., 2007; Sheikov et al., 2008), liver (Gao et al., 2012), and tumor (Bohmer et al., >>2010<<; Hu et al., 2012) tissues. This extravasation occurs through tight junctions between endothelial cells (0.2\u2013200 \u03BCm; Price et al., 1998; Song et al., 2002; Stieger et al., 2007)." . _:b585248143 "2"^^ . _:b42921874 _:b42921907 . _:b585248127 . . _:b585248142 "2"^^ . _:b42921916 _:b42921984 . _:b585248126 . . _:b42922038 . _:b585248137 "2"^^ . _:b42921916 _:b42921985 . _:b42921916 _:b42921986 . _:b585248121 . . _:b585248136 "2"^^ . . _:b585248141 . _:b42921916 . _:b585248120 . _:b42921965 "This enhances the extravasation of free drug or drug-loaded nanoparticles into tumor tissue by causing tissue shear stress and opening of endothelial tight junctions (Seidl et al., 1994; Mesiwala et al., >>2002<<). ARF induces fluid streaming through the interstitium, thus improving biodistribution of intravascular dyes and drugs in the target tissue (Lum et al., 2006; Hancock et al., 2009). Using optical imaging, Shortencarier et al. (2004)" . _:b585248139 "2"^^ . _:b42922001 "injection of 5-FU-loaded microbubbles (1 \u00D7 105 microbubbles/g body weight) led to twofold decrease in tumor volume compared to 5-FU treatment alone (Burke et al., >>2014<<). While these approaches seem to be promising, the low drug loading capacity of microbubbles is a major drawback. Consequently, the use of drug-loaded microbubbles requires either enhancement of the drug loading efficiency, administration" . _:b42921888 . _:b42921874 _:b42921884 . _:b42921874 _:b42921885 . _:b42921900 . _:b585248123 . _:b42921874 _:b42921886 . _:b585248138 "2"^^ . _:b42921874 _:b42921887 . _:b42921994 . _:b42921874 _:b42921880 . _:b42922015 "LHR, luteinizing hormone receptor) or tumor microvasculature (VEGF-R2, vascular endothelial growth factor receptor -2) through attachment of targeting ligands or antibodies onto the microbubble\u2019s shell (Kiessling et al., 2012, >>2014<<; Novell et al., 2013). This may lead to enhanced accumulation of the microbubbles in the target tumor cells or tissues." . _:b42921913 . _:b42921874 _:b42921881 . _:b585248122 . _:b42921874 _:b42921882 . _:b585248149 "2"^^ . _:b42921874 _:b42921883 . _:b42921990 "Interestingly, you could argue that the largest effect of sonochemotherapy can be expected in tissues with \u2018non-leaky\u2019 vessels, such as the brain (Ting et al., >>2012<<), since the potential of increasing extravasation is highest." . _:b42921874 _:b42921876 . . _:b585248133 . . _:b42921874 _:b42921877 . _:b42921874 _:b42921878 . _:b585248148 "2"^^ . . _:b42921874 _:b42921879 . . _:b585248132 . _:b585248151 "2"^^ . . _:b42921874 _:b42921875 . _:b585248135 . _:b42921907 . _:b585248150 "2"^^ . _:b42922056 "relies on enhancing accumulation of drugs in tumor cells or tissues and on decreasing their deposition in healthy tissues, thus reducing their side effects (Tinkov et al., 2010; Li et al., 2012; Fan et al., 2013; Burke et al., >>2014<<). Using the coadministration approach or drug-loaded microbubbles, microbubble-assisted ultrasound enhances in vitro the therapeutic efficacy of clinically approved chemotherapeutics including doxorubicin (Dox), cisplatin, bleomycin, PTX," . . _:b42921958 . _:b585248134 . _:b585248145 "2"^^ . _:b42921911 "<40 days) compared to free PTXSonoda et al. (>>2007<<)B16 (s.c., mouse)Free BLMOptisoni." . . _:b585248175 . _:b585248129 . _:b585248124 . _:b585248144 "2"^^ . _:b585248128 . _:b585248147 "2"^^ . _:b585248115 . _:b585248131 . _:b585248146 "2"^^ . _:b585248130 . _:b585248157 "2"^^ . _:b42922047 "following the exposure of tumor to microbubble-assisted ultrasound to assess the duration of enhanced permeability (few seconds \u2013 few hours, depending on the particle size; Marty et al., 2012; Tzu-Yin et al., 2014; Lammertink et al., >>2015<<). Other investigations recommend waiting for the peak concentration of drug in the blood before the administration of microbubbles and the subsequent exposure of tumors to ultrasound." . _:b585248141 . _:b42922052 _:b42922064 . _:b42922052 _:b42922065 . _:b585248156 "2"^^ . _:b42922052 _:b42922066 . _:b42922052 _:b42922067 . _:b585248140 . _:b585248159 "2"^^ . _:b585248143 . . _:b585248158 "2"^^ . . _:b42921952 . _:b42921883 "In recent years, research in the field of microbubble-assisted ultrasound (also known as sonoporation) aimed at delivering therapeutic molecules in vitro and in vivo has grown rapidly (Aryal et al., >>2014<<; Azagury et al., 2014; Kiessling et al., 2014; Rychak and Klibanov, 2014; Unga and Hashida, 2014; Unger et al., 2014)." . _:b585248142 . _:b42922052 _:b42922053 . _:b42921894 . _:b585248153 "2"^^ . _:b42922052 _:b42922054 . _:b42922052 _:b42922055 . _:b42921969 "The lipospheres disappeared when the ARF pulses were turned off (Shortencarier et al., >>2004<<). In addition to lipospheres, ARFs can push circulating microbubbles toward the endothelial wall, thereby improving microbubble\u2013cell contact, which might enhance cavitation-mediated extravasation of intravascular compounds (Rychak et al.," . _:b585248137 . _:b42921945 "In vivo, the integrity of the blood\u2013brain barrier was restored within 1\u20134 h following ultrasound exposure (Sheikov et al., >>2008<<; Ting et al., 2012)." . _:b585248152 "2"^^ . _:b585248136 . _:b42922052 _:b42922060 . _:b42922052 _:b42922061 . _:b42921992 "The simplest method for drug delivery using microbubble-assisted ultrasound is to use coadministration (Heath et al., 2012; Unga and Hashida, >>2014<<). This approach includes drugs that are administered in patients anyway in current clinical practice, with the addition of an injection of (clinically approved) microbubbles." . _:b585248155 "2"^^ . _:b42922052 _:b42922062 . _:b42922052 _:b42922063 . _:b42921983 "Similar to pore formation, the contribution of endocytosis processes depends strongly on the marker size and the acoustic pressures. Meijering et al. (>>2009<<) reported that low acoustic pressures (1 MHz, 0.22 MPa PNP) resulted in the cellular uptake of 4.4 and 70 kDa fluorescent dextrans through membrane pores while the entrance of 155 and 500 kDa fluorescent dextrans is dominated by" . _:b585248139 . _:b42922052 _:b42922056 . . _:b42922052 _:b42922057 . _:b585248154 "2"^^ . _:b42922052 _:b42922058 . . _:b42922026 . _:b42922052 _:b42922059 . _:b585248138 . _:b585248165 "2"^^ . _:b42922028 "Therefore, drugs can be loaded on microbubbles to overcome these shortcomings (Ting et al., 2012; Sirsi and Borden, >>2014<<). The success of i.v. drug delivery relies on sufficient tumor vascularization, thus restricting the application of this administration route to hypervascularized tumors. Next to extravasation, microbubble-assisted ultrasound can also" . _:b42922027 . _:b42921930 . _:b42922044 "However, exposure times of 1\u20135 min are recommended to prevent tissue injuries (e.g., hemorrhages; Mei et al., 2009; Yan et al., >>2013<<)." . _:b585248149 . . _:b585248164 "2"^^ . _:b42921888 "as sonoporation) aimed at delivering therapeutic molecules in vitro and in vivo has grown rapidly (Aryal et al., 2014; Azagury et al., 2014; Kiessling et al., 2014; Rychak and Klibanov, 2014; Unga and Hashida, 2014; Unger et al., >>2014<<). Microbubble-assisted ultrasound transiently increases the permeability of biological barriers, such as blood vessel walls (i.e., drug extravasation) and cellular membranes (i.e., cellular uptake of drugs), thus enhancing the local" . _:b585248148 . _:b585248167 "2"^^ . _:b42922010 . _:b42921886 "of microbubble-assisted ultrasound (also known as sonoporation) aimed at delivering therapeutic molecules in vitro and in vivo has grown rapidly (Aryal et al., 2014; Azagury et al., 2014; Kiessling et al., 2014; Rychak and Klibanov, >>2014<<; Unga and Hashida, 2014; Unger et al., 2014)." . _:b42921988 . _:b585248151 . _:b42922023 "The latter can induce convection and tissue deformation, which can decrease the connectedness of the extracellular matrix and size of pores in the tumor interstitial space (Frenkel, >>2008<<). By using i.t. administration, a high drug dose can be directly delivered into the target tumor while minimizing its side effects toward healthy tissues. This administration route overcomes the drawback related to the short plasma" . _:b585248166 "2"^^ . _:b42922036 "Due to a lack of standardized calibration methods concerning the applied ultrasound parameters and the heterogeneity in equipment used, it is not straightforward to compare the results of most studies directly (ter Haar et al., >>2011<<)." . _:b42921989 . _:b585248150 . _:b585248161 "2"^^ . _:b42921964 . _:b42921990 . _:b585248145 . _:b585248160 "2"^^ . . _:b42921986 . _:b42921991 . . _:b585248144 . _:b585248163 "2"^^ . _:b42921977 "The intracellular delivery of molecules through membrane pores is likely governed by passive diffusion or by ultrasound-mediated propulsion (i.e., microstreaming, ARF; Shortencarier et al., >>2004<<; Lum et al., 2006)." . _:b42921984 . _:b585248147 . _:b585248162 "2"^^ . _:b42921891 "of anticancer molecules including low molecular weight chemotherapeutic agents (sonochemotherapy), nucleic acids and monoclonal antibodies to a target site, e.g., tumor (Escoffre et al., 2013c; Ibsen et al., 2013; Unga and Hashida, >>2014<<). In addition, this method offers the possibility to treat superficial (e.g., skin) as well as deep organs (e.g., brain, liver, prostate), under the guidance of medical imaging modalities (magnetic resonance imaging, ultrasound imaging;" . _:b42922031 "Among these studies, clinical ultrasound scanners have been used to deliver drugs using microbubble-assisted ultrasound (Tinkov et al., >>2010<<; Sasaki et al., 2014), which has the advantage of enabling both imaging of- and drug delivery to the targeted tumor." . _:b42921985 . _:b585248146 . _:b585248173 "2"^^ . _:b42922012 . _:b585248132 . _:b42921883 . _:b42921898 "cisplatinDefinity1 MHz0.5 MI20%5 min\u224850% increase in apoptosisEscoffre et al. (2011)U87MG, MDA-231Free doxorubicin (DOX)Vevo, BR14, SonoVue1 MHz400\u2013800 kPa40%30 s30\u201340% decrease in viability, depending on cell lineSorace et al. (>>2012<<)2LMPFree paclitaxel (PTX)Definity1 MHz1.0 MPa PNP20%5 min50% increase in cell deathHu et al. (2012)BEL-7402Free 10-HCPT (free)Polymer3.5 MHz22.57 mW/cm2ND10 min20\u201330% decrease in viabilityRen et al. (2013)DLD-1Docetaxel-loaded MBLipid800" . _:b42921986 . _:b585248157 . _:b585248155 . . _:b42922014 "antigen; LHR, luteinizing hormone receptor) or tumor microvasculature (VEGF-R2, vascular endothelial growth factor receptor -2) through attachment of targeting ligands or antibodies onto the microbubble\u2019s shell (Kiessling et al., >>2012<<, 2014; Novell et al., 2013). This may lead to enhanced accumulation of the microbubbles in the target tumor cells or tissues." . _:b42922013 . _:b585248172 "2"^^ . _:b42921885 . _:b585248156 . _:b42922020 "as ultrasound contrast agents for molecular imaging, it might be possible to develop optimal tissue- or organ-selective drug delivery agents by combining targeting capacities and drug loading of microbubbles (Kiessling et al., >>2012<<). However, no evidence of their use for drug delivery has been reported yet." . _:b42921987 . _:b585248175 "2"^^ . _:b42921936 . _:b42921922 "flows surrounding the bubble, known as acoustic micro-streaming, and when in close contact with cells, result in shear stress on the cell membrane, leading to cellular uptake of drugs (Leighton, 1994; Wu, 2002; Doinikov and Bouakaz, >>2010<<). At higher acoustic pressures, microbubbles oscillate more rigorously, leading to their violent collapse and destruction, i.e., inertial cavitation (Figure 2)." . _:b42921996 . _:b585248159 . _:b585248174 "2"^^ . _:b42921997 . _:b585248158 . _:b585248169 "2"^^ . _:b42922008 . _:b42921942 . _:b42921998 . _:b585248153 . _:b42921916 _:b42921917 . _:b42921916 _:b42921918 . _:b585248168 "2"^^ . _:b42921916 _:b42921919 . _:b585248162 . _:b42921999 . _:b585248152 . _:b42921902 "decrease in viabilityRen et al. (2013)DLD-1Docetaxel-loaded MBLipid800 kHz2.56 W/cm250%10 min40% increase in inhibition rateTinkov et al. (2010)295/KDRDOX-loaded MBLipid1 MHz1 W/cm250%20 s40% decrease in cell viabilityYan et al. (>>2013<<)4T1PTX-liposome loaded MBLipid1 MHz1.0 MPa50%1 min20\u201330% decrease in viabilityDeng et al. (2014)MCF7/ADRDOX-liposome loaded MBLipid1 MHz1.65 W/cm220%15 sIncreased cellular accumulation and retention, 30% decrease in viability10-HCPT," . _:b42921882 . _:b585248171 "2"^^ . _:b42921941 . . _:b42921992 . . _:b585248155 . _:b585248170 "2"^^ . _:b42921993 . . . _:b585248154 . _:b42921994 . _:b42922051 . _:b585248165 . _:b585248166 . _:b42921995 . _:b585248164 . _:b42921988 . _:b42922026 "For deep-seated tumors, most protocols recommend injection of drugs and microbubbles via blood flow, providing better access to deeper tumors (Treat et al., 2012; Yan et al., 2013; Burke et al., >>2014<<). The i.v. route is a relatively easy and safe way to be used in the clinic for the administration of therapeutics and microbubbles." .