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Ultrasound focused toward tumours 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 tumours. Therefore, there is a need to optimise the ultrasound parameters and determine the properties of the tumour 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 tumours. 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 50kPa). 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 to their diffusion coefficient (close to 0.2). The model can be used to predict which tumour 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.

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. Copyright © 2021 The Author(s). Published by Elsevier Inc. on behalf of World Federation for Ultrasound in Medicine & Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Acoustic radiation force (ARF) might improve the distribution of nanoparticles (NPs) in tumors. To study this, tumors growing subcutaneously in mice were exposed to focused ultrasound (FUS) either 15 min or 4 h after the injection of NPs, to investigate the effect of ARF on the transport of NPs across the vessel wall and through the extracellular matrix. Quantitative analysis of confocal microscopy images from frozen tumor sections was performed to estimate the displacement of NPs from blood vessels. Using the same experimental exposure parameters, ARF was simulated and compared to the experimental data. Enhanced interstitial transport of NPs in tumor tissues were observed when FUS (10 MHz, acoustic power 234 W/cm2, 3.3 % duty cycle) was given either 15 min or 4 h after NP administration. According to acoustic simulations, the FUS generated an ARF per unit volume of 2.0 x 106 N/m3. The displacement of NPs was larger when FUS was applied 4h after NP injection compared to after 15 min. This study shows that ARF might contribute to a modest improved distribution of NPs into the tumor interstitium. © 2020 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other uses, in any current or future media, including reprinting/republishing this material for advertising or promotional purposes, creating new collective works, for resale or redistribution to servers or lists, or reuse of any copyrighted component of this work in other works.

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain. Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method. publishedVersion

Systemic injections of chemotherapeutics often deliver only minor fractions of the administered dose to the targeted pathology, resulting in unwanted toxicity towards healthy tissue. In brain tissue, drug delivery is impaired by the highly selective nature of the blood brain barrier impeding the influx of most substances. Acoustic Cluster Therapy (ACT®) is a platform for targeted therapeutic enhancement, facilitating increased local drug transfer. The platform is comprised of ACT® clusters, a mix of microbubbles and microdroplets and low intensity (diagnostic) ultrasound insonation. Intravenously injected ACT® clusters circulate freely through the body before activation by localised insonation at the targeted pathology. In the ultrasound field, microbubbles transfer energy to microdroplets, which undergo a liquid-to-gas phase transition, forming larger ACT® bubbles that transiently deposit in the targeted microvasculature. Further exposure to ultrasound results in controlled volume oscillation of the ACT® bubbles, inducing a range of biomechanical effects. The effects lead to enhanced extravasation across the endothelial barrier, facilitating the transport of co-administered chemotherapeutics to the targeted tissue. Proof of concept studies showed an increased therapeutic efficacy of standard of care drugs, when combined with ACT® treatment. Furthermore, ACT® treatment induced a controlled and temporal opening of the blood brain barrier observed by the extravasation of fluorescent macromolecules into brain tissue.

Background:The transgenic adenocarcinoma of the mouse prostate (TRAMP) is a widelyused genetically engineered spontaneous prostate cancer model. However, both thedegree of malignancy and time of cancer onset vary. While most mice display slowlyprogressing cancer, a subgroup develops fast‐growing poorly differentiated (PD) tumors,making the model challenging to use. We investigated the feasibility of using ultrasound(US) imaging to screen for PD tumors and compared the performances of US and magneticresonance imaging (MRI) in providing reliable measurements of disease burden.Methods:TRAMP mice (n = 74) were screened for PD tumors with US imaging andfindings verified with MRI, or in two cases with gross pathology. PD tumor volumewas estimated with US and MR imaging and the methods compared (n = 11). For non‐PD mice, prostate volume was used as a marker for disease burden and estimatedwith US imaging, MRI, and histology (n = 11). The agreement between themeasurements obtained by the various methods and the intraobserver variability(IOV) was assessed using Bland‐Altman analysis.Results:US screening showed 81% sensitivity, 91% specificity, 72% positivepredictive value, and 91% negative predictive value. The smallest tumor detectedby US screening was 14 mm3and had a maximum diameter of 2.6 mm. MRI had thelowest IOV for both PD tumor and prostate volume estimation. US IOV was almost aslow as MRI for PD tumor volumes but was considerably higher for prostate volumes.Conclusions:US imaging was found to be a good screening method for detecting PDtumors and estimating tumor volume in the TRAMP model. MRI had betterrepeatability than US, especially when estimating prostate volumes. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. © 2019 The Authors. The Prostate Published by Wiley Periodicals, Inc.

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

Therapeutic agents can benefit from encapsulation in nanoparticles, due to improved pharmacokinetics and biodistribution, protection from degradation, increased cellular uptake and sustained release. Microbubbles in combination with ultrasound have been shown to improve the delivery of nanoparticles and drugs to tumors and across the blood-brain barrier. Here, we evaluate two different microbubbles for enhancing the delivery of polymeric nanoparticles to cells in vitro: a commercially available lipid microbubble (Sonazoid) and a microbubble with a shell composed of protein and nanoparticles. Various ultrasound parameters are applied and confocal microscopy is employed to image cellular uptake. Ultrasound enhanced cellular uptake depending on the pressure and duty cycle. The responsible mechanisms are probably sonoporation and sonoprinting, followed by uptake, and to a smaller degree enhanced endocytosis. The use of commercial Sonazoid microbubbles leads to significantly lower uptake than when using nanoparticle-loaded microbubbles, suggesting that proximity between cells, nanoparticles and microbubbles is important, and that mainly nanoparticles in the shell are taken up, rather than free nanoparticles in solution.

Drug delivery to tumors is challenging due to biological barriers obstructing effective delivery. Sonopermeation with ultrasound and microbubbles has been shown to improve therapeutic effect of many classes of drugs, but the underlying mechanism is not fully understood. In this study, two subcutaneous xenograft tumor models, that differed substantially in blood vessel density and stiffness, is treated with poly(alkyl cyanoacrylate) nanoparticles and nanoparticle-stabilized microbubbles combined with ultrasound. Improved nanoparticle accumulation and extracellular matrix (ECM) penetration is found. The stiffness and solid stress in the tumors are measured and it is discovered that sonopermeation can reduce the solid stress in both models, with the highest effect in the stiffest tumor model. This suggests that sonopermeation affects not only the blood vessel wall which has been described previously, but also the ECM to reduce solid stress and increase diffusion and transport of nanomedicines.

Advanced science (2021). doi:10.1002/advs.202100370, Published by Wiley-VCH, Weinheim

The blood-brain barrier (BBB) is a major obstacle to treating several brain disorders. Focused ultrasound (FUS) in combination with intravascular microbubbles increases BBB permeability by opening tight junctions, creating endothelial cell openings, improving endocytosis and increasing transcytosis. Here we investigated whether combining FUS and microbubbles with transferrin receptor-targeting liposomes would result in enhanced delivery to the brain of post-natal rats compared with liposomes lacking the BBB-targeting moiety. For all animals, increased BBB permeability was observed after FUS treatment. A 40% increase in accumulation of transferrin receptor-targeting liposomes was observed in the FUS-treated hemisphere, whereas the isotype immunoglobulin G liposomes showed no increased accumulation. Confocal laser scanning microscopy of brain sections revealed that both types of liposomes were mainly observed in endothelial cells in the FUS-treated hemisphere. The results demonstrate that FUS and microbubble treatment combined with BBB-targeting liposomes could be a promising approach to enhance drug delivery to the brain. (E-mail: marieke.olsman@ntnu. no) © 2021 The Author(s). Published by Elsevier Inc. on behalf of World Federation for Ultrasound in Medicine & Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Penetration of nanoscale therapeutic agents into the extracellular matrix (ECM) of a tumor is a limiting factor for the sufficient delivery of drugs in tumors. Ultrasound (US) in combination with microbubbles causing cavitation is reported to improve delivery of nanoparticles (NPs) and drugs to tumors. Acoustic radiation force (ARF) could also enhance the penetration of NPs in tumor ECM. In this work, a collagen gel was used as a model for tumor ECM to study the effects of ARF on the penetration of NPs as well as the deformation of collagen gels applying different US parameters (varying pressure and duty cycle). The collagen gel was characterized, and the diffusion of water and NPs was measured. The penetration of NPs into the gel was measured by confocal laser scanning microscopy and numerical simulations were performed to determine the ARF and to estimate the penetration distance and extent of deformation. ARF had no effect on the penetration of NPs into the collagen gels for the US parameters and gel used, whereas a substantial deformation was observed. The width of the deformation on the collagen gel surface corresponded to the US beam. Comparing ARF caused by attenuation within the gel and Langevin pressure caused by reflection at the gel-water surface, ARF was the prevalent mechanism for the gel deformation. The experimental and theoretical results were consistent both with respect to the NP penetration and the gel deformation. © 2020 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other uses, in any current or future media, including reprinting/republishing this material for advertising or promotional purposes, creating new collective works, for resale or redistribution to servers or lists, or reuse of any copyrighted component of this work in other works.

IntroductionAcoustic 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.MethodsIn 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.ResultsResults 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.

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. Copyright © 2020 The Author(s). Published by Elsevier Inc. on behalf of World Federation for Ultrasound in Medicine & Biology. This is an open access article under the CC BY license. (http://creativecommons.org/licenses/by/4.0/)

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. This is an open access article distributed under the terms of the Creative Commons CC-BY license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 αvβ3-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. This work was supported by the Central Norway Regional Health Authority ‘Helse Midt-Norge’ [AMS: PhD stipend (90062100) and travel grant (90284100); SH: researcher grant (90262100)], the National Institutes of Health (WJMM: R01 CA220234, TR: P30 CA00574), the American Heart Association (CPM: 16SDG31390007), the Norwegian Research Council (SH: 230788/F20), and the Tromsø Research Foundation and Trond Mohn Foundation (SH: 180 °N project). Sí

Preclinical research has demonstrated that nanoparticles and macromolecules can accumulate in solid tumors due to the enhanced permeability and retention effect. However, drug loaded nanoparticles often fail to show increased efficacy in clinical trials. A better understanding of how tumor heterogeneity affects nanoparticle accumulation could help elucidate this discrepancy and help in patient selection for nanomedicine therapy. Here we studied five human tumor models with varying morphology and evaluated the accumulation of 100 nm polystyrene nanoparticles. Each tumor model was characterized in vivo using micro-computed tomography, contrast-enhanced ultrasound and diffusion-weighted and dynamic contrast-enhanced magnetic resonance imaging. Ex vivo, the tumors were sectioned for both fluorescence microscopy and histology. Nanoparticle uptake and distribution in the tumors were generally heterogeneous. Density of functional blood vessels measured by fluorescence microscopy correlated significantly (p = 0.0056) with nanoparticle accumulation and interestingly, inflow of microbubbles measured with ultrasound also showed a moderate but significant (p = 0.041) correlation with nanoparticle accumulation indicating that both amount of vessels and vessel morphology and perfusion predict nanoparticle accumulation. This indicates that blood vessel characterization using contrast-enhanced ultrasound imaging or other methods could be valuable for patient stratification for treatment with nanomedicines. © 2018. This is the authors’ accepted and refereed manuscript to the article. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

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.

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. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) License. See http://ivyspring.com/terms for full terms and conditions.

Acoustic Cluster Therapy (ACT) employs an intravenously-injected dispersion of clusters of microbubbles and oil microdroplets, in combination with ultrasound directed at the tumor, to enhance the delivery of a co-administered drug. Diagnostic ultrasound activates the clusters, causing the droplets to change phase and become large (> 20 m) bubbles which lodge in the tumor capillaries. Delivery-enhancement is then achieved using a low-frequency (e.g. 300 kHz) ultrasound field also directed at the tumor, which causes the stationary bubbles to pulsate whilst in direct contact with the vascular endothelium. ACT has been shown to significantly increase the efficacy of various chemotherapeutics in a variety of tumor models in mice. Here we demonstrate through preclinical in-vivo experiments that the efficacy of treating human triple negative breast cancer in mice with liposomal doxorubicin (Doxil®) is significantly enhanced by ACT; for example, 63 % of animals achieved complete, stable remission, in the group treated with the drug and ACT versus only 10 % for Doxil® alone (p < 0.02). We also show in the same experiments that the ACT contrast enhancement obtained under ultrasound activation of the ACT clusters, either before or during treatment, holds promise as an imaging biomarker for predicting response.

Acoustic cluster therapy (ACT) is a novel approach for ultrasound mediated, targeted drug delivery. In the current study, we have investigated ACT in combination with paclitaxel and Abraxane® for treatment of a subcutaneous human prostate adenocarcinoma (PC3) in mice. In combination with paclitaxel (12 mg/kg given i.p.), ACT induced a strong increase in therapeutic efficacy; 120 days after study start, 42% of the animals were in stable, complete remission vs. 0% for the paclitaxel only group and the median survival was increased by 86%. In combination with Abraxane® (12 mg paclitaxel/kg given i.v.), ACT induced a strong increase in the therapeutic efficacy; 60 days after study start 100% of the animals were in stable, remission vs. 0% for the Abraxane® only group, 120 days after study start 67% of the animals were in stable, complete remission vs. 0% for the Abraxane® only group. For the ACT + Abraxane group 100% of the animals were alive after 120 days vs. 0% for the Abraxane® only group. Proof of concept for Acoustic Cluster Therapy has been demonstrated; ACT markedly increases the therapeutic efficacy of both paclitaxel and Abraxane® for treatment of human prostate adenocarcinoma in mice. © 2016. This is the authors’ accepted and refereed manuscript to the article. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

In vitro and in vivo behavior of nanoparticles (NPs) is often studied by tracing the NPs with fluorescent dyes. This requires stable incorporation of dyes within the NPs, as dye leakage may give a wrong interpretation of NP biodistribution, cellular uptake, and intracellular distribution. Furthermore, NP labeling with trace amounts of dye should not alter NP properties such as interactions with cells or tissues. To allow for versatile NP studies with a variety of fluorescence-based assays, labeling of NPs with different dyes is desirable. Hence, when new dyes are introduced, simple and fast screening methods to assess labeling stability and NP-cell interactions are needed. For this purpose, we have used a previously described generic flow cytometry assay; incubation of cells with NPs at 4 and 37°C. Cell-NP interaction is confirmed by cellular fluorescence after 37°C incubation, and NP-dye retention is confirmed when no cellular fluorescence is detected at 4°C. Three different NP-platforms labeled with six different dyes were screened, and a great variability in dye retention was observed. Surprisingly, incorporation of trace amounts of certain dyes was found to reduce or even inhibit NP uptake. This work highlights the importance of thoroughly evaluating every dye-NP combination before pursuing NP-based applications. © 2016 International Society for Advancement of Cytometry.

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. © Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions

Ultrasound (US)and systemic administration of microbubbles (MBs)have been shown to improve the delivery of drugs and nanoparticles (NPs)to tumor tissue. A better understanding of the mechanisms is crucial for effective delivery of NPs and drugs. To elucidate the kinetics of extravasation events, and relate it to the vessel diameter and flow, we have performed real-time intravital multiphoton microscopy during US sonication of tumors growing in dorsal window chambers. We studied the effects of four different mechanical index (MI)levels (0.2 to 0.8)while injecting SonoVue or in-house made MBs with NPs in the shell (NPMB). We found that high MI of 0.8 induced a violent extravasation of both NPs and dextrans. Using lower MIs (0.2-0.6), less extravasation was detected and it occurred in vessels with larger diameters compared to sonication at MI 0.8. The rate of extravasation of both NPs and dextrans, and the displacement of NPs and dextrans from the vessel into the tumor tissue, correlated with MI. The observed extravasation events happened within milliseconds to minutes after the US exposure started. Moreover, we have observed a change of blood flow rate and direction with all the MIs tested © 2018 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other uses, in any current or future media, including reprinting/republishing this material for advertising or promotional purposes, creating new collective works, for resale or redistribution to servers or lists, or reuse of any copyrighted component of this work in other works.

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. © 2018 Baghirov et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Microbubbles (MBs) are routinely used as contrast agents for ultrasound imaging. The use of ultrasound in combination with MBs has also attracted attention as a method to enhance drug delivery. We have developed a technology platform incorporating multiple functionalities, including imaging and therapy in a single system consisting of MBs stabilized by polyethylene glycol (PEG)-coated polymeric nanoparticles (NPs). The NPs, containing lipophilic drugs and/or contrast agents, are composed of the widely used poly(butyl cyanoacrylate) (PBCA) polymer and prepared in a single step. MBs stabilized by these NPs are subsequently prepared by self-assembly of NPs at the MB air-liquid interface. Here we show that these MBs can act as contrast agents for conventional ultrasound imaging. Successful encapsulation of iron oxide NPs inside the PBCA NPs is demonstrated, potentially enabling the NP-MBs to be used as magnetic resonance imaging (MRI) and/or molecular ultrasound imaging contrast agents. By precise tuning of the applied ultrasound pulse, the MBs burst and the NPs constituting the shell are released. This could result in increased local deposit of NPs into target tissue, providing improved therapy and imaging contrast compared with freely distributed NPs.

A great deal of effort has been focused on exploring the underlying molecular mechanism of osteoarthritis (OA) especially at the cellular level. We report a confocal Raman spectroscopic investigation on human osteoarthritic chondrocytes. The objective of this investigation is to identify molecular features and the stage of OA based on the spectral signatures corresponding to bio-molecular changes at the cellular level in chondrocytes. In this study, we isolated chondrocytes from human osteoarthritic cartilage and acquired Raman spectra from single cells. Major spectral differences between the cells obtained from different International Cartilage Repair Society (ICRS) grades of osteoarthritic cartilage were identified. During progression of OA, a decrease in protein content and an increase in cell death were observed from the vibrational spectra. Principal component analysis and subsequent cross-validation was able to associate osteoarthritic chondrocytes to ICRS Grade I, II and III with specificity 100.0%, 98.1%, and 90.7% respectively, while, sensitivity was 98.6%, 82.8%, and 97.5% respectively. The overall predictive efficiency was 92.2%. Our pilot study encourages further use of Raman spectroscopy as a noninvasive and label free technique for revealing molecular features associated with osteoarthritic chondrocytes.

Protein adsorption on nanoparticles (NPs) used in nanomedicine leads to opsonization and activation of the complement system in blood, which substantially reduces the blood circulation time of NPs. The most commonly used method to avoid protein adsorption is to coat the NPs with polyethylene glycol, so-called PEGylation. Although PEGylation is of utmost importance for designing the in vivo behavior of the NP, there is still a considerable lack of methods for characterization and fundamental understanding related to the PEGylation of NPs. In this work we have studied four different poly(butyl cyanoacrylate) (PBCA) NPs, PEGylated with different types of PEG-based nonionic surfactants—Jeffamine M-2070, Brij L23, Kolliphor HS 15, Pluronic F68—or combinations thereof. We evaluated the PEGylation, both quantitatively by nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and time-of-flight secondary ion mass spectrometry (ToF-SIMS) and qualitatively by studying ζ-potential, protein adsorption, diffusion, cellular interactions, and blood circulation half-life. We found that NMR and ToF-SIMS are complementary methods, while TGA is less suitable to quantitate PEG on polymeric NPs. It was found that longer PEG increases both blood circulation time and diffusion of NPs in collagen gels. This is a submitted manuscript of an article published by American Chemical Society in Molecular Pharmaceutics, February 7, 2017

Second harmonic generation and two-photon fluorescence microscopy was used for the morphological investigation of osteoarthritic human articular cartilage. High resolution optical images revealed the hidden features in osteoarthrosis that has not been observed using standard clinical imaging systems.

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. © 2017 The Authors. Published by Elsevier Inc. on behalf of World Federation for Ultrasound in Medicine & Biology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

The interaction of the promising drug carriers poly(alkyl cyanoacrylate) nanoparticles (PACA NPs) with lipid monolayers modeling the cell membrane and with RBE4 immortalized rat brain endothelial cells was compared to assess the relevance of lipid monolayer-based cell membrane models for PACA NP cellular uptake. NP properties such as size and charge of NPs and density of poly(ethylene glycol) coating (PEG) were kept in a narrow range to assess whether the type of PEG coating and the PACA monomer affected NP-monolayer and NP-cell interactions. The interaction with lipid monolayers was evaluated using surface pressure measurements and Brewster angle microscopy. NP association with and uptake by cells were assessed using flow cytometry and confocal laser scanning microscopy. The interaction between NPs and both lipid monolayers and the plasma membrane depended on the type of PEG. PEG density affected cellular uptake but not interaction with lipid monolayers. NP monomer, NPs size and charge had no effect on the interaction. This might be due to the fact that the size and charge distribution was kept rather narrow to study the effect of PACA monomer and PEG type. In conclusion, while modeling solely the passive aspect of NP-cell interactions, lipid monolayers nevertheless proved a valuable cell membrane model whose interaction with PACA NPs correlated well with NP-cell interaction. In addition, both NP-monolayer and NP-cell interactions were dependent on PEGylation type, which could be used in the design of NPs to either facilitate or hinder cellular uptake, depending on the intended purpose. This is a submitted manuscript of an article published by Elsevier Ltd in Colloids and Surfaces B: Biointerfaces, 31 October 2016.

Lipid-based nanoparticles are extensively studied for drug delivery. These nanoparticles are often surface-coated with polyethylene glycol (PEG) to improve their biodistribution. Until now, the effects of varying PEG surface density have been studied in a narrow and low range. Here, the effects of high and a broad range of PEG surface densities on the in vivo performance of lipid-based nanoparticles were studied.Oil-in-water nanoemulsions were prepared with PEG surface densities of 5-50 mol%. Confocal microscopy was used to assess intracellular disintegration in vitro. In vivo pharmacokinetics and biodistribution in tumor bearing mice were studied using a small animal optical imager.PEG surface density did not affect intracellular nanoemulsion stability. Surprisingly, circulation half-lives decreased with increasing PEG surface density. A plausible explanation was that nanoemulsion with high (50 mol%) PEG surface density activated the complement in a whole blood assay, whereas nanoemulsion with low (5 mol%) PEG density did not. In vivo, nanoemulsion with low PEG surface density was mostly confined to the tumor and organs of the mononuclear phagocyte system, whereas nanoemulsion with high PEG density accumulated throughout the mouse.Optimal PEG surface density of lipid-based nanoparticles for tumor targeting was found to be below 10 mol%.

In the past two decades advances in the development of targeted nanoparticles have facilitated their application as molecular imaging agents and targeted drug delivery vehicles. Nanoparticle-enhanced molecular imaging of the angiogenic tumor vasculature has been of particular interest. Not only because angiogenesis plays an important role in various pathologies, but also since endothelial cell surface receptors are directly accessible for relatively large circulating nanoparticles. Typically, nanoparticle targeting towards these receptors is studied by analyzing the contrast distribution on tumor images acquired before and at set time points after administration. Although several exciting proof-of-concept studies demonstrated qualitative assessment of relative target concentration and distribution, these studies did not provide quantitative information on the nanoparticle targeting kinetics. These kinetics will not only depend on nanoparticle characteristics, but also on receptor binding and recycling. In this study, we monitored the in vivo targeting kinetics of αvβ3-integrin specific nanoparticles with intravital microscopy and dynamic contrast enhanced magnetic resonance imaging, and using compartment modeling we were able to quantify nanoparticle targeting rates. As such, this approach can facilitate optimization of targeted nanoparticle design and it holds promise for providing more quantitative information on in vivo receptor levels. Interestingly, we also observed a periodicity in the accumulation kinetics of αvβ3-integrin targeted nanoparticles and hypothesize that this periodicity is caused by receptor binding, internalization and recycling dynamics. Taken together, this demonstrates that our experimental approach provides new insights in in vivo nanoparticle targeting, which may proof useful for vascular targeting in general.

In the current study we show the dissociation and tumor accumulation dynamics of dual labeled near infrared (NIR) quantum dot core self-assembled lipidic nanoparticles (SALNPs) in a mouse model upon intravenous administration. Using advanced in vivo fluorescence energy transfer imaging techniques, we observed swift exchange with plasma protein components in the blood and progressive SALNP dissociation and subsequent trafficking of individual SALNP components following tumor accumulation. Our results suggest that upon intravenous administration SALNPs quickly transform, which may affect their functionality. The presented technology provides a modular in vivo tool to visualize SALNP behavior in real time and may contribute to improving the therapeutic outcome or molecular imaging signature of SALNPs.

Proof of principle for local drug delivery with Acoustic Cluster Therapy (ACT) was demonstrated in a human prostate adenocarcinoma growing in athymic mice, using near infrared (NIR) dyes as model molecules. A dispersion of negatively charged microbubble/positively charged microdroplet clusters are injected i.v., activated within the target pathology by diagnostic ultrasound (US), undergo an ensuing liquid-to-gas phase shift and transiently deposit 20–30 μm large bubbles in the microvasculature, occluding blood flow for ~ 5–10 min. Further application of low frequency US induces biomechanical effects that increase the vascular permeability, leading to a locally enhanced extravasation of components from the vascular compartment (e.g., released or co-administered drugs). Results demonstrated deposition of activated bubbles in tumor vasculature. Following ACT treatment, a significant and tumor specific increase in the uptake of a co-administered macromolecular NIR dye was shown. In addition, ACT compound loaded with a lipophilic NIR dye to the microdroplet component was shown to facilitate local release and tumor specific uptake. Whereas the mechanisms behind the observed increased and tumor specific uptake are not fully elucidated, it is demonstrated that the ACT concept can be applied as a versatile technique for targeted drug delivery. © 2016. This is the authors’ accepted and refereed manuscript to the article. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/