Ultrasound and Microbubble-Induced Reduction of Functional Vasculature Depends on the Microbubble, Tumor Type and Time After Treatment.

OBJECTIVE: Ultrasound in combination with microbubbles can enhance accumulation and improve the distribution of various therapeutic agents in tumor tissue, leading to improved efficacy. Understanding the impact of treatment on the tumor microenvironment, concurrently with how microenvironment attributes affect treatment outcome, will be important for selecting appropriate patient cohorts in future clinical trials. The main aim of this work was to investigate the influence of ultrasound and microbubbles on the functional vasculature of cancer tissue. METHODS: Four different tumor models in mice (bone, pancreatic, breast and colon cancer) were characterized with respect to vascular parameters using contrast-enhanced ultrasound imaging. The effect of treatment with microbubbles and ultrasound was then investigated using immunohistochemistry and confocal microscopy, quantifying the total amount of vasculature and fraction of functional vessels. Two different microbubbles were used, the clinical contrast agent SonoVue and the large bubbles generated by Acoustic Cluster Therapy (ACT), tailored for therapeutic purposes. RESULTS: The colon cancer model displayed slower flow but a higher vascular volume than the other models. The pancreatic model showed the fastest flow but also the lowest vascular volume. Ultrasound and SonoVue transiently reduced the amount of functional vasculature in breast and colon tumors immediately after treatment. No reduction was observed for ACT, likely due to shorter ultrasound pulses and lower pressures applied. CONCLUSION: Variation between tumor models due to tissue characteristics emphasizes the importance of evaluating treatment suitability in the specific tissue of interest, as altered perfusion could have a large impact on drug delivery and therapeutic outcome.

Real-Time Multiphoton Intravital Microscopy of Drug Extravasation in Tumours during Acoustic Cluster Therapy.

Optimising drug delivery to tumours remains an obstacle to effective cancer treatment. A prerequisite for successful chemotherapy is that the drugs reach all tumour cells. The vascular network of tumours, extravasation across the capillary wall and penetration throughout the extracellular matrix limit the delivery of drugs. Ultrasound combined with microbubbles has been shown to improve the therapeutic response in preclinical and clinical studies. Most studies apply microbubbles designed as ultrasound contrast agents. Acoustic Cluster Therapy (ACT®) is a novel approach based on ultrasound-activated microbubbles, which have a diameter 5-10 times larger than regular contrast agent microbubbles. An advantage of using such large microbubbles is that they are in contact with a larger part of the capillary wall, and the oscillating microbubbles exert more effective biomechanical effects on the vessel wall. In accordance with this, ACT® has shown promising therapeutic results in combination with various drugs and drug-loaded nanoparticles. Knowledge of the mechanism and behaviour of drugs and microbubbles is needed to optimise ACT®. Real-time intravital microscopy (IVM) is a useful tool for such studies. This paper presents the experimental setup design for visualising ACT® microbubbles within the vasculature of tumours implanted in dorsal window (DW) chambers. It presents ultrasound setups, the integration and alignment of the ultrasound field with the optical system in live animal experiments, and the methodologies for visualisation and analysing the recordings. Dextran was used as a fluorescent marker to visualise the blood vessels and to trace drug extravasation and penetration into the extracellular matrix. The results reveal that the experimental setup successfully recorded the kinetics of extravasation and penetration distances into the extracellular matrix, offering a deeper understanding of ACT's mechanisms and potential in localised drug delivery.

A Comparative Analysis of Orthotopic and Subcutaneous Pancreatic Tumour Models: Tumour Microenvironment and Drug Delivery.

Pancreatic ductal adenocarcinoma (PDAC) remains a challenging malignancy, mainly due to its resistance to chemotherapy and its complex tumour microenvironment characterised by stromal desmoplasia. There is a need for new strategies to improve the delivery of drugs and therapeutic response. Relevant preclinical tumour models are needed to test potential treatments. This paper compared orthotopic and subcutaneous PDAC tumour models and their suitability for drug delivery studies. A novel aspect was the broad range of tumour properties that were studied, including tumour growth, histopathology, functional vasculature, perfusion, immune cell infiltration, biomechanical characteristics, and especially the extensive analysis of the structure and the orientation of the collagen fibres in the two tumour models. The study unveiled new insights into how these factors impact the uptake of a fluorescent model drug, the macromolecule called 800CW. While the orthotopic model offered a more clinically relevant microenvironment, the subcutaneous model offered advantages for drug delivery studies, primarily due to its reproducibility, and it was characterised by a more efficient drug uptake facilitated by its collagen organisation and well-perfused vasculature. The tumour uptake seemed to be influenced mainly by the structural organisation and the alignment of the collagen fibres and perfusion. Recognising the diverse characteristics of these models and their multifaceted impacts on drug delivery is crucial for designing clinically relevant experiments and improving our understanding of pancreatic cancer biology.

Nanoparticle Dynamics in Composite Hydrogels Exposed to Low-Frequency Focused Ultrasound.

Pulsed focused ultrasound (FUS) in combination with microbubbles has been shown to improve delivery and penetration of nanoparticles in tumors. To understand the mechanisms behind this treatment, it is important to evaluate the contribution of FUS without microbubbles on increased nanoparticle penetration and transport in the tumor extracellular matrix (ECM). A composite agarose hydrogel was made to model the porous structure, the acoustic attenuation and the hydraulic conductivity of the tumor ECM. Single-particle tracking was used as a novel method to monitor nanoparticle dynamics in the hydrogel during FUS exposure. FUS exposure at 1 MHz and 1 MPa was performed to detect any increase in nanoparticle diffusion or particle streaming at acoustic parameters relevant for FUS in combination with microbubbles. Results were compared to a model of acoustic streaming. The nanoparticles displayed anomalous diffusion in the hydrogel, and FUS with a duty cycle of 20% increased the nanoparticle diffusion coefficient by 23%. No increase in diffusion was found for lower duty cycles. FUS displaced the hydrogel itself at duty cycles above 10%; however, acoustic streaming was found to be negligible. In conclusion, pulsed FUS alone cannot explain the enhanced penetration of nanoparticles seen when using FUS and microbubbles for nanoparticle delivery, but it could be used as a tool to enhance diffusion of particles in the tumor ECM.

Treatment of Liver Metastases With Focused Ultrasound and Microbubbles in Patients With Colorectal Cancer Receiving Chemotherapy.

OBJECTIVE: Pre-clinical trials have obtained promising results that focused ultrasound (FUS) combined with microbubbles (MBs) increases tumor uptake and the therapeutic effect of drugs. The aims of the study described here were to investigate whether FUS and MBs could improve the effect of chemotherapy in patients with liver metastases from colorectal cancer and to investigate the safety and feasibility of using FUS + MBs. METHODS: We included 17 patients with liver metastases from colorectal cancer, selected two lesions in each patient's liver and randomized the lesions for, respectively, treatment with FUS + MBs or control. After chemotherapy (FOLFIRI or FOLFOXIRI), the lesions were treated with FUS (frequency = 1.67 MHz, mechanical index = 0.5, pulse repetition frequency = 0.33 Hz, 33 oscillations, duty cycle = 0.2%-0.4% and MBs (SonoVue) for 35 min). Nine boluses of MBs were injected intravenously at 3.5 min intervals. Patients were scheduled for four cycles of treatment. Changes in the size of metastases were determined from computed tomography images. RESULTS: Treatment with FUS + MBs is safe at the settings used. There was considerable variation in treatment response between lesions and mixed response between lesions receiving only chemotherapy. There is a tendency toward larger-volume reduction in lesions treated with FUS + MBs compared with control lesions, but a mixed response to chemotherapy and lesion heterogeneity make it difficult to interpret the results. CONCLUSION: The combination of FUS and MBs is a safe, feasible and available strategy for improving the effect of chemotherapy in cancer patients. Therapeutic effect was not demonstrated in this trial. Multicenter trials with standardized protocols should be performed.

Ultrasound and Microbubbles Increase the Uptake of Platinum in Murine Orthotopic Pancreatic Tumors.

OBJECTIVE: Currently available cytotoxic treatments have limited effect on pancreatic ductal adenocarcinoma (PDAC) because desmoplastic stroma limits drug delivery. Efforts have been made to overcome these barriers by drug targeting the tumor microenvironment. Results so far are promising, but without clinical impact. Our aim was to investigate whether ultrasound and microbubbles could improve the uptake and therapeutic response of conventional chemotherapy. METHODS: Orthotopic pancreatic tumors growing in mice were treated with commercially available FOLFIRINOX (fluorouracil, irinotecan, oxaliplatin and calcium folinate) and SonoVue microbubbles combined with focused ultrasound. Tumor uptake of platinum (Pt) was measured by inductively coupled plasma mass spectroscopy (ICP-MS), and tumor volumes were measured by ultrasound imaging. DISCUSSION: Uptake of Pt, the active ingredient of oxaliplatin, was significantly increased after ultrasound treatment of orthotopic PDAC tumors. Multiple injections with FOLFIRONOX increased the amount of Pt in tumors. However, the enhanced accumulation did not improve therapeutic response. Increased uptake of Pt confirms that ultrasound and microbubbles have potential in clinical practice with existing drugs. CONCLUSION: The lack of therapeutic response, despite increased uptake in tumor tissue, emphasizes the importance of studying how to overcome stromal barriers.

Intraperitoneal administration of cabazitaxel-loaded nanoparticles in peritoneal metastasis models.

Colorectal and ovarian cancers frequently develop peritoneal metastases with few treatment options. Intraperitoneal chemotherapy has shown promising therapeutic effects, but is limited by rapid drug clearance and systemic toxicity. We therefore encapsulated the cabazitaxel taxane in poly(alkyl cyanoacrylate) (PACA) nanoparticles (NPs), designed to improve intraperitoneal delivery. Toxicity of free and encapsulated cabazitaxel was investigated in rats by monitoring clinical signs, organ weight and blood hematological and biochemical parameters. Pharmacokinetics, biodistribution and treatment response were evaluated in mice. Biodistribution was investigated by measuring both cabazitaxel and the 2-ethylbutanol NP degradation product. Drug encapsulation was shown to increase intraperitoneal drug retention, leading to prolonged intraperitoneal drug residence time and higher drug concentrations in peritoneal tumors. As a result, encapsulation of cabazitaxel improved the treatment response in two in vivo models bearing intraperitoneal tumors. Together, these observations indicate a strong therapeutic potential of NP-based cabazitaxel encapsulation as a novel treatment for peritoneal metastases.

Alginate Microsphere Encapsulation of Drug-Loaded Nanoparticles: A Novel Strategy for Intraperitoneal Drug Delivery.

Alginate hydrogels have been broadly investigated for use in medical applications due to their biocompatibility and the possibility to encapsulate cells, proteins, and drugs. In the treatment of peritoneal metastasis, rapid drug clearance from the peritoneal cavity is a major challenge. Aiming to delay drug absorption and reduce toxic side effects, cabazitaxel (CAB)-loaded poly(alkyl cyanoacrylate) (PACA) nanoparticles were encapsulated in alginate microspheres. The PACAlg alginate microspheres were synthesized by electrostatic droplet generation and the physicochemical properties, stability, drug release kinetics, and mesothelial cytotoxicity were analyzed before biodistribution and therapeutic efficacy were studied in mice. The 450 µm microspheres were stable at in vivo conditions for at least 21 days after intraperitoneal implantation in mice, and distributed evenly throughout the peritoneal cavity without aggregation or adhesion. The nanoparticles were stably retained in the alginate microspheres, and nanoparticle toxicity to mesothelial cells was reduced, while the therapeutic efficacy of free CAB was maintained or improved in vivo. Altogether, this work presents the alginate encapsulation of drug-loaded nanoparticles as a promising novel strategy for the treatment of peritoneal metastasis that can improve the therapeutic ratio between toxicity and therapeutic efficacy.

Enhancing carrier flux for efficient drug delivery in cancer tissues.

Ultrasound focused toward tumors in the presence of circulating microbubbles improves the delivery of drug-loaded nanoparticles and therapeutic outcomes; however, the efficacy varies among the different properties and conditions of the tumors. Therefore, there is a need to optimize the ultrasound parameters and determine the properties of the tumor tissue important for the successful delivery of nanoparticles. Here, we propose a mesoscopic model considering the presence of entropic forces to explain the ultrasound-enhanced transport of nanoparticles across the capillary wall and through the interstitium of tumors. The nanoparticles move through channels of variable shape whose irregularities can be assimilated to barriers of entropic nature that the nanoparticles must overcome to reach their targets. The model assumes that focused ultrasound and circulating microbubbles cause the capillary wall to oscillate, thereby changing the width of transcapillary and interstitial channels. Our analysis provides values for the penetration distances of nanoparticles into the interstitium that are in agreement with experimental results. We found that the penetration increased significantly with increasing acoustic intensity as well as tissue elasticity, which means softer and more deformable tissue (Young modulus lower than 50 kPa), whereas porosity of the tissue and pulse repetition frequency of the ultrasound had less impact on the penetration length. We also considered that nanoparticles can be absorbed into cells and to extracellular matrix constituents, finding that the penetration length is increased when there is a low absorbance coefficient of the nanoparticles compared with their diffusion coefficient (close to 0.2). The model can be used to predict which tumor types, in terms of elasticity, will successfully deliver nanoparticles into the interstitium. It can also be used to predict the penetration distance into the interstitium of nanoparticles with various sizes and the ultrasound intensity needed for the efficient distribution of the nanoparticles.

Ultrasound and microbubbles to beat barriers in tumors: Improving delivery of nanomedicine.

Successful delivery of drugs and nanomedicine to tumors requires a functional vascular network, extravasation across the capillary wall, penetration through the extracellular matrix, and cellular uptake. Nanomedicine has many merits, but penetration deep into the tumor interstitium remains a challenge. Failure of cancer treatment can be caused by insufficient delivery of the therapeutic agents. After intravenous administration, nanomedicines are often found in off-target organs and the tumor extracellular matrix close to the capillary wall. With circulating microbubbles, ultrasound exposure focused toward the tumor shows great promise in improving the delivery of therapeutic agents. In this review, we address the impact of focused ultrasound and microbubbles to overcome barriers for drug delivery such as perfusion, extravasation, and transport through the extracellular matrix. Furthermore, we discuss the induction of an immune response with ultrasound and delivery of immunotherapeutics. The review discusses mainly preclinical results and ends with a summary of ongoing clinical trials.

Sonopermeation Enhances Uptake and Therapeutic Effect of Free and Encapsulated Cabazitaxel.

Delivery of drugs and nanomedicines to tumors is often heterogeneous and insufficient and, thus, of limited efficacy. Microbubbles in combination with ultrasound have been found to improve delivery to tumors, enhancing accumulation and penetration. We used a subcutaneous prostate cancer xenograft model in mice to investigate the effect of free and nanoparticle-encapsulated cabazitaxel in combination with ultrasound and microbubbles with a lipid shell or a shell of nanoparticles. Sonopermeation reduced tumor growth and prolonged survival (26%-100%), whether the free drug was co-injected with lipid-shelled microbubbles or the nanoformulation was co-injected with lipid-shelled or nanoparticle-shelled microbubbles. Coherently with the improved therapeutic response, we found enhanced uptake of nanoparticles directly after ultrasound treatment that lasted several weeks (2.3 × -15.8 × increase). Neither cavitation dose nor total accumulation of nanoparticles could explain the variation within treatment groups, emphasizing the need for a better understanding of the tumor biology and mechanisms involved in ultrasound-mediated treatment.

Ultrasound-Mediated Delivery of Chemotherapy into the Transgenic Adenocarcinoma of the Mouse Prostate Model.

Ultrasound (US) in combination with microbubbles (MB) has had promising results in improving delivery of chemotherapeutic agents. However, most studies are done in immunodeficient mice with xenografted tumors. We used two phenotypes of the spontaneous transgenic adenocarcinoma of the mouse prostate (TRAMP) model to evaluate if US + MB could enhance the therapeutic efficacy of cabazitaxel (Cab). Cab was either injected intravenously as free drug or encapsulated into nanoparticles. In both cases, Cab transiently reduced tumor and prostate volume in the TRAMP model. No additional therapeutic efficacy was observed combining Cab with US + MB, except for one tumor. Additionally, histology grading and immunostaining of Ki67 did not reveal differences between treatment groups. Mass spectrometry revealed that nanoparticle encapsulation of Cab increased the circulation time and enhanced the accumulation in liver and spleen compared with free Cab. The therapeutic results in this spontaneous, clinically relevant tumor model differ from the improved therapeutic response observed in xenografts combining US + MB and chemotherapy.

Ultrasound-mediated delivery enhances therapeutic efficacy of MMP sensitive liposomes.

To improve therapeutic efficacy of nanocarrier drug delivery systems, it is essential to improve their uptake and penetration in tumour tissue, enhance cellular uptake and ensure efficient drug release at the tumour site. Here we introduce a tumour targeting drug delivery system based on the ultrasound-mediated delivery of enzyme sensitive liposomes. These enzyme sensitive liposomes are coated with cleavable poly(ethylene glycol) (PEG) which will be cleaved by two members of the enzyme matrix metalloproteinase family (MMP-2 and MMP-9). Cleavage of the PEG coat can increase cellular uptake and will destabilize the liposomal membrane which can result in accelerated drug release. The main aim of the work was to study the effect of focused ultrasound and microbubbles on the delivery and therapeutic efficacy of the MMP sensitive liposome. The performance of the MMP sensitive liposome was compared to a non-MMP sensitive version and Doxil-like liposomes. In vitro, the cellular uptake and cytotoxicity of the liposomes were studied, while in vivo the effect of ultrasound and microbubbles on the tumour accumulation, biodistribution, microdistribution, and therapeutic efficacy were investigated. For all tested liposomes, ultrasound and microbubble treatment resulted in an improved tumour accumulation, increased extravasation, and increased penetration of the liposomes from blood vessels into the extracellular matrix. Surprisingly, penetration depth was independent of the ultrasound intensity used. Ultrasound-mediated delivery of free doxorubicin and the Doxil-like and MMP sensitive liposome resulted in a significant reduction in tumour volume 28 days post the first treatment and increased median survival. The MMP sensitive liposome showed better therapeutic efficacy than the non-MMP sensitive version indicating that cleaving the PEG-layer is important. However, the Doxil-like liposome outcompeted the MMP and non-MMP sensitive liposome, both with and without the use of ultrasound and microbubbles.

Tumor Targeting by αvß3-Integrin-Specific Lipid Nanoparticles Occurs via Phagocyte Hitchhiking.

Although the first nanomedicine was clinically approved more than two decades ago, nanoparticles' (NP) in vivo behavior is complex and the immune system's role in their application remains elusive. At present, only passive-targeting nanoformulations have been clinically approved, while more complicated active-targeting strategies typically fail to advance from the early clinical phase stage. This absence of clinical translation is, among others, due to the very limited understanding for in vivo targeting mechanisms. Dynamic in vivo phenomena such as NPs' real-time targeting kinetics and phagocytes' contribution to active NP targeting remain largely unexplored. To better understand in vivo targeting, monitoring NP accumulation and distribution at complementary levels of spatial and temporal resolution is imperative. Here, we integrate in vivo positron emission tomography/computed tomography imaging with intravital microscopy and flow cytometric analyses to study αvß3-integrin-targeted cyclic arginine-glycine-aspartate decorated liposomes and oil-in-water nanoemulsions in tumor mouse models. We observed that ligand-mediated accumulation in cancerous lesions is multifaceted and identified "NP hitchhiking" with phagocytes to contribute considerably to this intricate process. We anticipate that this understanding can facilitate rational improvement of nanomedicine applications and that immune cell-NP interactions can be harnessed to develop clinically viable nanomedicine-based immunotherapies.

Theranostic Attributes of Acoustic Cluster Therapy and Its Use for Enhancing the Effectiveness of Liposomal Doxorubicin Treatment of Human Triple Negative Breast Cancer in Mice.

INTRODUCTION: Acoustic cluster therapy (ACT) comprises co-administration of a formulation containing microbubble/microdroplet clusters (PS101), together with a regular medicinal drug (e.g., a chemotherapeutic) and local ultrasound (US) insonation of the targeted pathological tissue (e.g., the tumor). PS101 is confined to the vascular compartment and, when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase-shift to produce bubbles with a median diameter of 22 µm when unconstrained by the capillary wall. In vivo these bubbles transiently lodge in the tumor's microvasculature. Low frequency ultrasound (300 kHz) at a low mechanical index (MI = 0.15) is then applied to drive oscillations of the deposited ACT bubbles to induce a range of biomechanical effects that locally enhance extravasation, distribution, and uptake of the co-administered drug, significantly increasing its therapeutic efficacy. METHODS: In this study we investigated the therapeutic efficacy of ACT with liposomal doxorubicin for the treatment of triple negative breast cancer using orthotopic human tumor xenografts (MDA-MB-231-H.luc) in athymic mice (ICR-NCr-Foxn1nu). Doxil® (6 mg/kg, i.v.) was administered at days 0 and 21, each time immediately followed by three sequential ACT (20 ml/kg PS101) treatment procedures (n = 7-10). B-mode and nonlinear ultrasound images acquired during the activation phase were correlated to the therapeutic efficacy. RESULTS: Results show that combination with ACT induces a strong increase in the therapeutic efficacy of Doxil®, with 63% of animals in complete, stable remission at end of study, vs. 10% for Doxil® alone (p < 0.02). A significant positive correlation (p < 0.004) was found between B-mode contrast enhancement during ACT activation and therapy response. These observations indicate that ACT may also be used as a theranostic agent and that ultrasound contrast enhancement during or before ACT treatment may be employed as a biomarker of therapeutic response during clinical use.

Effect of Ultrasound on the Vasculature and Extravasation of Nanoscale Particles Imaged in Real Time.

Ultrasound and microbubbles have been found to improve the delivery of drugs and nanoparticles to tumor tissue. To obtain new knowledge on the influence of vascular parameters on extravasation and to elucidate the effect of acoustic pressure on extravasation and penetration of nanoscale particles into the extracellular matrix, real-time intravital multiphoton microscopy was performed during sonication of tumors growing in dorsal window chambers. The impact of vessel diameter, vessel structure and blood flow was characterized. Fluorescein isothiocyanate-dextran (2 MDa) was injected to visualize blood vessels. Mechanical indexes (MI) of 0.2-0.8 and in-house-made, nanoparticle-stabilized microbubbles or Sonovue were applied. The rate and extent of penetration into the extracellular matrix increased with increasing MI. However, to achieve extravasation, smaller vessels required MIs (0.8) higher than those of blood vessels with larger diameters. Ultrasound changed the blood flow rate and direction. Interestingly, the majority of extravasations occurred at vessel branching points.

Therapeutic Effect of Cabazitaxel and Blood-Brain Barrier opening in a Patient-Derived Glioblastoma Model.

Treatment of glioblastoma and other diseases in the brain is especially challenging due to the blood-brain barrier, which effectively protects the brain parenchyma. In this study we show for the first time that cabazitaxel, a semi-synthetic derivative of docetaxel can cross the blood-brain barrier and give a significant therapeutic effect in a patient-derived orthotopic model of glioblastoma. We show that the drug crosses the blood-brain barrier more effectively in the tumor than in the healthy brain due to reduced expression of p-glycoprotein efflux pumps in the vasculature of the tumor. Surprisingly, neither ultrasound-mediated blood-brain barrier opening (sonopermeation) nor drug formulation in polymeric nanoparticles could increase either accumulation of the drug in the brain or therapeutic effect. This indicates that for hydrophobic drugs, sonopermeation of the blood brain barrier might not be sufficient to achieve improved drug delivery. Nonetheless, our study shows that cabazitaxel is a promising drug for the treatment of brain tumors.

Ultrasound-mediated delivery and distribution of polymeric nanoparticles in the normal brain parenchyma of a metastatic brain tumour model.

The treatment of brain diseases is hindered by the blood-brain barrier (BBB) preventing most drugs from entering the brain. Focused ultrasound (FUS) with microbubbles can open the BBB safely and reversibly. Systemic drug injection might induce toxicity, but encapsulation into nanoparticles reduces accumulation in normal tissue. Here we used a novel platform based on poly(2-ethyl-butyl cyanoacrylate) nanoparticle-stabilized microbubbles to permeabilize the BBB in a melanoma brain metastasis model. With a dual-frequency ultrasound transducer generating FUS at 1.1 MHz and 7.8 MHz, we opened the BBB using nanoparticle-microbubbles and low-frequency FUS, and applied high-frequency FUS to generate acoustic radiation force and push nanoparticles through the extracellular matrix. Using confocal microscopy and image analysis, we quantified nanoparticle extravasation and distribution in the brain parenchyma. We also evaluated haemorrhage, as well as the expression of P-glycoprotein, a key BBB component. FUS and microbubbles distributed nanoparticles in the brain parenchyma, and the distribution depended on the extent of BBB opening. The results from acoustic radiation force were not conclusive, but in a few animals some effect could be detected. P-glycoprotein was not significantly altered immediately after sonication. In summary, FUS with our nanoparticle-stabilized microbubbles can achieve accumulation and displacement of nanoparticles in the brain parenchyma.

Ultrasound Improves the Delivery and Therapeutic Effect of Nanoparticle-Stabilized Microbubbles in Breast Cancer Xenografts.

Compared with conventional chemotherapy, encapsulation of drugs in nanoparticles can improve efficacy and reduce toxicity. However, delivery of nanoparticles is often insufficient and heterogeneous because of various biological barriers and uneven tumor perfusion. We investigated a unique multifunctional drug delivery system consisting of microbubbles stabilized by polymeric nanoparticles (NPMBs), enabling ultrasound-mediated drug delivery. The aim was to examine mechanisms of ultrasound-mediated delivery and to determine if increased tumor uptake had a therapeutic benefit. Cellular uptake and toxicity, circulation and biodistribution were characterized. After intravenous injection of NPMBs into mice, tumors were treated with ultrasound of various pressures and pulse lengths, and distribution of nanoparticles was imaged on tumor sections. No effects of low pressures were observed, whereas complete bubble destruction at higher pressures improved tumor uptake 2.3 times, without tissue damage. An enhanced therapeutic effect was illustrated in a promising proof-of-concept study, in which all tumors exhibited regression into complete remission.

Efficient Enhancement of Blood-Brain Barrier Permeability Using Acoustic Cluster Therapy (ACT).

The blood-brain barrier (BBB) is a major obstacle in drug delivery for diseases of the brain, and today there is no standardized route to surpass it. One technique to locally and transiently disrupt the BBB, is focused ultrasound in combination with gas-filled microbubbles. However, the microbubbles used are typically developed for ultrasound imaging, not BBB disruption. Here we describe efficient opening of the BBB using the promising novel Acoustic Cluster Therapy (ACT), that recently has been used in combination with Abraxane® to successfully treat subcutaneous tumors of human prostate adenocarcinoma in mice. ACT is based on the conjugation of microbubbles to liquid oil microdroplets through electrostatic interactions. Upon activation in an ultrasound field, the microdroplet phase transfers to form a larger bubble that transiently lodges in the microvasculature. Further insonation induces volume oscillations of the activated bubble, which in turn induce biomechanical effects that increase the permeability of the BBB. ACT was able to safely and temporarily permeabilize the BBB, using an acoustic power 5-10 times lower than applied for conventional microbubbles, and successfully deliver small and large molecules into the brain.