The relationship between surface drug distribution of Dox-loaded microbubbles and drug release/cavitation behaviors with ultrasound.

Ultrasound (US)-triggered microbubbles (MBs) drug delivery is a promising tool for noninvasive and localized therapy. Several studies have shown the potential of drug-loaded MBs to boost the delivery of therapeutic substances to target tissue effectively. Nevertheless, little is known about the surface payload distribution affecting the cavitation activity and drug release behavior of the drug-loaded MBs. In this study, we designed a common chemodrug (Doxorubicin, Dox)-loaded MB (Dox-MBs) and regulated the payload distribution as uniform or cluster onto the outer surface of MBs. The Dox distribution on the MB shells was assessed by confocal fluorescence microscopic imaging. The acoustic properties of the Dox-MBs with different Dox distributions were evaluated by their acoustic stability and cavitation activities. The payload release and the fragments from Dox-MBs in response to different US parameters were measured and visualized by column chromatography and cryo-electron microscopy, respectively. By amalgamating these methodologies, we found that stable cavitation was sufficient for triggering uniform-loaded MBs to release their payload, but stable cavitation and inertial cavitation were required for cluster-loaded MBs. The released substances included free Dox and Dox-containing micelle/liposome; their portions depended on the payload distribution, acoustic pressure, cycle number, and sonication duration. Furthermore, we also revealed that the Dox-containing micelle/liposome in cluster-loaded MBs had the potential for multiple drug releases upon US sonication. This study compared uniform-loaded MBs and cluster-loaded MBs to enhance our comprehension of drug-loaded MBs mediated drug delivery.

Enhancing Boron Uptake in Brain Glioma by a Boron-Polymer/Microbubble Complex with Focused Ultrasound.

Boron neutron capture therapy (BNCT) is a promising radiotherapy for treating glioblastoma multiforme (GBM). However, the penetration of drugs (e.g., sodium borocaptate and BSH) for BNCT into brain tumors is limited by cerebral vesicular protective structures, the blood-brain barrier, and the blood-brain tumor barrier (BTB). Although BSH has been reported to be selectively taken up by tumors, it is rapidly excreted from the body and cannot achieve a high tumor-to-normal brain ratio (T/N ratio) and tumor-to-blood ratio (T/B ratio). Despite the development of large-molecular weight boron compounds, such as polymers and nanoparticles, to enhance the permeation and retention effect, their effects remain insufficient for clinical use. To improve the efficiency of boron delivery to the tumor site, we propose combinations of self-assembled boron-containing polyanion [polyethylene glycol- b-poly(( closo-dodecaboranyl)thiomethylstyrene) (PEG- b-PMBSH)] nanoparticles (295 ± 2.3 nm in aqueous media) coupled with cationic microbubble (B-MB)-assisted focused ultrasound (FUS) treatment. Upon FUS sonication (frequency = 1 MHz, pressure = 0.3-0.7 MPa, duty cycle = 0.5%, sonication = 1 min), B-MBs can simultaneously achieve safe BTB opening and boron drug delivery into tumor tissue. Compared with the MBs of the PEG- b-PMBSH mixture group (B + MBs), B-MBs showed 3- and 2.3-fold improvements in the T/N (4.4 ± 1.4 vs 1.3 ± 0.1) and T/B ratios (1.4 ± 0.6 vs 0.1 ± 0.1), respectively, after 4 min of FUS sonication. The spatial distribution of PEG- b-PMBSH was also improved by the complex of PEG- b-PMBSH with MBs. The findings presented herein, in combination with the expanding clinical application of FUS, may improve BNCT and treatment of GBM.

Enhanced delivery of paclitaxel liposomes using focused ultrasound with microbubbles for treating nude mice bearing intracranial glioblastoma xenografts.

Paclitaxel liposomes (PTX-LIPO) are a clinically promising antineoplastic drug formulation for the treatment of various extracranial cancers, excluding glioblastoma. A main reason for this is the presence of the blood-brain barrier (BBB) or blood-tumor barrier (BTB), preventing liposomal drugs from crossing at a therapeutically meaningful level. Focused ultrasound (FUS) in conjunction with microbubbles (MBs) has been suggested in many studies to be an effective approach to increase the BBB or BTB permeability. In this study, we investigated the feasibility of enhancing the delivery of PTX-LIPO in intracranial glioblastoma-bearing nude mice using pulsed low-intensity FUS exposure in the presence of MBs. Our results showed that the delivery efficiency of PTX-LIPO could be effectively improved in terms of the penetration of both the BBB in vitro and BTB in vivo by pulsed FUS sonication with a 10 ms pulse length and 1 Hz pulse repetition frequency at 0.64 MPa peak-rarefactional pressure in the presence of MBs. Quantitative analysis showed that a 2-fold higher drug concentration had accumulated in the glioblastoma 3 h after FUS treatment, with 7.20±1.18 µg PTX per g glioma tissue. Longitudinal magnetic resonance imaging analysis illustrated that the intracranial glioblastoma progression in nude mice treated with PTX-LIPO delivered via FUS with MBs was suppressed consistently for 4 weeks compared to the untreated group. The medium survival time of these tumor-bearing nude mice was significantly prolonged by 20.8%, compared to the untreated nude mice. Immunohistochemical analysis further confirmed the antiproliferation effect and cell apoptosis induction. Our study demonstrated that noninvasive low-intensity FUS with MBs can be used as an effective approach to deliver PTX-LIPO in order to improve their chemotherapy efficacy toward glioblastoma.

Improving Nanoparticle Penetration in Tumors by Vascular Disruption with Acoustic Droplet Vaporization.

Drug penetration influences the efficacy of tumor therapy. Although the leaky vessels of tumors can improve the penetration of nanodrugs via the enhanced permeability and retention (EPR) effect, various aspects of the tumor microenvironment still restrict this process. This study investigated whether vascular disruption using the acoustic vaporization of micro- or nanoscale droplets (MDs or NDs) induced by ultrasound sonication can overcome the limitations of the EPR effect to allow drug diffusion into extensive regions. The intravital penetration of DiI-labeled liposomes (as a drug model with red fluorescence) was observed using an acousto-optical integrated system comprising a 2-MHz focused ultrasound transducer (transmitting a three-cycle single pulse and a peak negative pressure of 10 MPa) in a window-chamber mouse model. Histology images of the solid tumor were also used to quantify and demonstrate the locations where DiI-labeled liposomes accumulated. In the intravital image analyses, the cumulative diffusion area and fluorescence intensity at 180 min were 0.08±0.01 mm(2) (mean±standard deviation) and 8.5±0.4%, respectively, in the EPR group, 0.33±0.01 mm(2) and 13.1±0.4% in the MD group (p<0.01), and 0.63±0.01 mm(2) and 18.9±1.1% in the ND group (p<0.01). The intratumoral accumulations of DiI-labeled liposomes were 1.7- and 2.3-fold higher in the MD and ND groups, respectively, than in the EPR group. These results demonstrate that vascular disruption induced by acoustic droplet vaporization can improve drug penetration more than utilizing the EPR effect. The NDs showed longer lifetime in vivo than MDs and provided potential abilities of long periods of treatment, intertissue ND vaporization, and intertissue NDs-converted bubble cavitation to improve the drug penetration and transport distance.

Non-invasive, neuron-specific gene therapy by focused ultrasound-induced blood-brain barrier opening in Parkinson's disease mouse model.

Focused ultrasound (FUS)-induced with microbubbles (MBs) is a promising technique for noninvasive opening of the blood-brain barrier (BBB) to allow targeted delivery of therapeutic substances into the brain and thus the noninvasive delivery of gene vectors for CNS treatment. We have previously demonstrated that a separated gene-carrying liposome and MBs administration plus FUS exposure can deliver genes into the brain, with the successful expression of the reporter gene and glial cell line-derived neurotrophic factor (GDNF) gene. In this study, we further modify the delivery system by conjugating gene-carrying liposomes with MBs to improve the GDNF gene-delivery efficiency, and to verify the possibility of using this system to perform treatment in the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced animal disease model. FUS-BBB opening was verified by contrast-enhanced MRI, and GFP gene expression was verified via in vivo imaging system (IVIS). Western blots as well as enzyme-linked immunosorbent assay (ELISA) were conducted to measure protein expression, and immunohistochemistry (IHC) was conducted to test the Tyrosine hydroxylase (TH)-neuron distribution. Dopamine (DA) and its metabolites as well as dopamine active transporter (DAT) were quantitatively analyzed to show dopaminergic neuronal dopamine secretion/activity/metabolism. Motor performance was evaluated by rotarod test weekly. Results demonstrated that the LpDNA-MBs (gene-liposome-MBs) complexes successfully serve as gene carrier and BBB-opening catalyst, and outperformed the separated LpDNA/MBs administration both in terms of gene delivery and expression. TH-positive IHC and measurement of DA and its metabolites DOPAC and HVA confirmed improved neuronal function, and the proposed system also provided the best neuroprotective effect to retard the progression of motor-related behavioral abnormalities. Immunoblotting and histological staining further confirmed the expression of reporter genes in neuronal cells. This study suggests that FUS exposures with the administration of LpDNA-MBs complexes synergistically can serve as an effective gene therapy strategy for MPTP-animal treatment, and may have potential for further application to perform gene therapy for neurodegenerative disease.

Focused ultrasound-induced blood-brain barrier opening for non-viral, non-invasive, and targeted gene delivery.

Focused ultrasound (FUS) exposure in the presence of microbubbles can temporally open the blood-brain barrier (BBB) and is an emerging technique for non-invasive brain therapeutic agent delivery. Given the potential to deliver large molecules into the CNS via this technique, we propose a reliable strategy to synergistically apply FUS-BBB opening for the non-invasive and targeted delivery of non-viral genes into the CNS for therapeutic purpose. In this study, we developed a gene-liposome system, in which the liposomes are designed to carry plasmid DNA (pDNA, containing luciferase reporter gene) to form a liposomal-plasmid DNA (LpDNA) complex. Pulsed FUS exposure was delivered to induce BBB opening (500-kHz, burst length=10ms, 1% duty cycle, PRF=1Hz). The longitudinal expression of luciferase was quantitated via an in vivo imaging system (IVIS). The reporter gene expression level was confirmed via immunoblotting, and histological staining was used to identify transfected cells via fluorescent microscopy. In a comparison of gene transduction efficiency, the LpDNA system showed better cell transduction than the pDNA system. With longitudinal observation of IVIS monitoring, animals with FUS treatment showed significant promotion of LpDNA release into the CNS and demonstrated enhanced expression of genes upon sonication with FUS-BBB opening, while both the luciferase and GDNF protein expression were successfully measured via Western blotting. The gene expression peak was observed at day 2, and the gene expression level was up to 5-fold higher than that in the untreated hemisphere (compared to a 1-fold increase in the direct-inject positive-control group). The transfection efficiency was also found to be LpDNA dose-dependent, where higher payloads of pDNA resulted in a higher transfection rate. Immunoblotting and histological staining confirmed the expression of reporter genes in glial cells as well as astrocytes. This study suggests that IV administration of LpDNA in combination with FUS-BBB opening can provide effective gene delivery and expression in the CNS, demonstrating the potential to achieve non-invasive and targeted gene delivery for treatment of CNS diseases.

Tumortropic monocyte-mediated delivery of echogenic polymer bubbles and therapeutic vesicles for chemotherapy of tumor hypoxia.

Overcoming limitations often experienced in nanomedicine delivery toward hypoxia regions of malignant tumors remains a great challenge. In this study, a promising modality for active hypoxia drug delivery was developed by adopting tumortropic monocytes/macrophages as a cellular vehicle for co-delivery of echogenic polymer/C5F12 bubbles and doxorubicin-loaded polymer vesicles. Through the remote-controlled focused ultrasound (FUS)-triggered drug liberation, therapeutic monocytes show prominent capability of inducing apoptosis of cancer cells. The in vivo and ex vivo fluorescence imaging shows appreciable accumulation of cell-mediated therapeutics in tumor as compared to the nanoparticle counterpart residing mostly in liver. Inhibition of tumor recurrence with γ-ray pre-irradiated Tramp-C1-bearing mice receiving therapeutic monocytes intravenously alongside the FUS activation at tumor site was significantly observed. Immunohistochemical examination of tumor sections confirms successful cellular transport of therapeutic payloads to hypoxic regions and pronounced cytotoxic action against hypoxic cells. Following the intravenous administration, the cellular-mediated therapeutics can penetrate easily to a depth beyond 150 µm from the nearest blood vessels within pre-irradiated tumor while nanoparticles are severely limited to a depth of ca 10-15 µm. This work demonstrates the great promise of cellular delivery to carry therapeutic payloads for improving chemotherapy in hypoxia by combining external trigger for drug release.

Combining microbubbles and ultrasound for drug delivery to brain tumors: current progress and overview.

Malignant glioma is one of the most challenging central nervous system (CNS) diseases, which is typically associated with high rates of recurrence and mortality. Current surgical debulking combined with radiation or chemotherapy has failed to control tumor progression or improve glioma patient survival. Microbubbles (MBs) originally serve as contrast agents in diagnostic ultrasound but have recently attracted considerable attention for therapeutic application in enhancing blood-tissue permeability for drug delivery. MB-facilitated focused ultrasound (FUS) has already been confirmed to enhance CNS-blood permeability by temporally opening the blood-brain barrier (BBB), thus has potential to enhance delivery of various kinds of therapeutic agents into brain tumors. Here we review the current preclinical studies which demonstrate the reports by using FUS with MB-facilitated drug delivery technology in brain tumor treatment. In addition, we review newly developed multifunctional theranostic MBs for FUS-induced BBB opening for brain tumor therapy.

Controlling the size distribution of lipid-coated bubbles via fluidity regulation.

Lipid-coated bubbles exhibit oscillation responses capable of enhancing the sensitivity of ultrasound imaging by improving contrast. Further improvements in performance enhancement require control of the size distribution of bubbles to promote correspondence between their resonance frequency and the frequency of the ultrasound. Here we describe a size-controlling technique that can shift the size distribution using a currently available agitation method. This technique is based on regulating the membrane dynamic fluidity of lipid mixtures and provides a general size-controlling variable that could also be applied in other fabrication methods. Three materials (1,2-dihexadecanoyl-sn-glycero-3-phosphocholine, 1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) and polyethylene glycol 40 stearate) with distinct initial fluidities and phase behaviors were used to demonstrate the use of fluidity regulation to control bubble sizes. Bubble size distributions of different formulations were determined by electrical impedance sensing, and bubble volumes and surface areas were calculated. To confirm the relationship between regulated fluidity and mean bubble size, the membrane fluidity of each composition was determined by fluorescence anisotropy, with the results indicating linear relations in the compositions with similar main transition temperatures. Compositions with a higher molar proportion of polyethylene glycol 40 stearate showed higher fluidities and larger bubbles. B-mode ultrasound imaging was performed to investigate the echogenicity and lifetime of the fabricated bubbles, with the results indicating that co-mixing a high-transition-temperature charged lipid (i.e., 1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)) extends the tailoring range of this fluidity regulation technique, allowing refined and continuous changes in mean bubble size (from 0.93 to 2.86 µm in steps of ∼0.5 µm), and also prolongs bubble lifetime. The polydispersity of each composition was also determined to evaluate practicality in particular applications. Our study demonstrates a feasible approach to naturally controling bubble size distribution and provides a practical reference for other fabrication systems and ultrasound imaging applications.

Paclitaxel-liposome-microbubble complexes as ultrasound-triggered therapeutic drug delivery carriers.

Liposome-microbubble complexes (LMC) have become a promising therapeutic carrier for ultrasound-triggered drug delivery to treat malignant tumors. However, the efficacy for ultrasound-assisted chemotherapy in vivo and the underlying mechanisms remain to be elucidated. Here, we investigated the feasibility of using paclitaxel-liposome-microbubble complexes (PLMC) as possible ultrasound (US)-triggered targeted chemotherapy against breast cancer. PTX-liposomes (PL) were conjugated to the microbubble (MB) surface through biotin-avidin linkage, increasing the drug-loading efficiency of MBs. The significant increased release of payloads from liposome-microbubble complexes was achieved upon US exposure. We used fluorescent quantum dots (QDs) as a model drug to show that released QDs were taken up by 4T1 breast cancer cells treated with QD-liposome-microbubble complexes (QLMC) and US, and uptake depended on the exposure time and intensity of insonication. We found that PLMC plus US inhibited tumor growth more effectively than PL plus US or PLMC without US, not only in vitro, but also in vivo. Histologically, the inhibition of tumor growth appeared to result from increased apoptosis and reduced angiogenesis in tumor xenografts. In addition, a significant increase of drug concentration in tumors was observed in comparison to treatment with non-conjugated PL or PLMC without US. The significant increase in an antitumor efficacy of PLMC plus US suggests their potential use as a new targeted US chemotherapeutic approach to inhibit breast cancer growth.

Redox nanoparticle treatment protects against neurological deficit in focused ultrasound-induced intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage is reported to induce the generation of reactive oxygen species and oxidative DNA damage in the brain. AIMS: We aimed to examine whether our designed redox polymer nanoparticle could reduce intracerebral hemorrhage induced by 1-MHz focused ultrasound sonication coupled with microbubble treatment. MATERIALS & METHODS: Contrast-enhanced ultrasound imaging, frozen section, brain edema, neurologic deficit, the number of morphologically normal neurons, DNA oxidization and superoxide anion generation were used to investigate the neuroprotective effect of redox polymer nanoparticles. RESULTS: We confirmed that the 1-MHz focused ultrasound coupled with microbubble produced intracerebral hemorrhage and showed that the redox polymer nanoparticle ameliorates intracerebral hemorrhage-induced brain edema, neurological deficit and oxidative damage. CONCLUSION: These results suggest that redox polymer nanoparticle is a potential therapeutic agent for intracerebral hemorrhage induced by focused ultrasound.

Potential-well model in acoustic tweezers.

Standing-wave acoustic tweezers are popularly used for non-invasive and non-contact particle manipulation. Because of their good penetration in biological tissue, they also show promising prospects for in vivo applications. According to the concept of an optical vortex, we propose an acoustics-vortex- based trapping model of acoustic tweezers. A four-element 1-MHz planar transducer was used to generate 1-MHz sine waves at 1 MPa, with adjacent elements being driven with a pi/2-rad phase difference. Each element was a square with a side length of 5.08 mm, with kerfs initially set at 0.51 mm. An acoustic vortex constituting the spiral motion of an acoustic wave around the beam axis was created, with an axial null. Applying Gor'kov's theory in the Rayleigh regime yielded the potential energy and radiation force for use in subsequent analysis. In the transverse direction, the vortex structure behaved as a series of potential wells that tended to drive a suspended particle toward the beam axis. They were highly fragmented in the near field that is very close to the transducer where there was spiral interference, and well-constructed in the far field. We found that the significant trapping effect was only present between these two regions in the transverse direction--particles were free to move along the beam axis, and a repulsive force was observed in the outer acoustic vortex. Because the steepness of the potential gradient near an axial null dominates the trapping effect, the far field of the acoustic vortex is inappropriate for trapping. Particles too close to the transducer are not sufficiently trapped because of the fragmented potential pattern. We suggest that the ideal distance from the transducer for trapping particles is in front of one-fourth of the Rayleigh distance, based on the superposition of the wavefronts. The maximum trapping force acting on a 13-mum polystyrene sphere in the produced acoustic vortex was 50.0 pN, and it was possible to trap approximately 10(6) particles within a plane; the maximum repulsive force was 24.5 pN, and this was reduced to less than 13 pN by smoothing the outer gradient. Most stiff and dense particles can be used in this model. The presence of transverse trapping and the long working distance make the model useful for 2-D manipulation, particularly in in vivo applications. This paper details the trapping properties in the acoustic vortex and describes methods for improving the design of the transducer. The results obtained support the feasibility of the potential-well model of acoustic tweezers.