Hyperthermia-triggered release of hypoxic cell radiosensitizers from temperature-sensitive liposomes improves radiotherapy efficacy in vitro.

Hypoxia is a characteristic feature of solid tumors and an important cause of resistance to radiotherapy. Hypoxic cell radiosensitizers have been shown to increase radiotherapy efficacy, but dose-limiting side effects prevent their widespread use in the clinic. We propose the encapsulation of hypoxic cell radiosensitizers in temperature-sensitive liposomes (TSL) to target the radiosensitizers specifically to tumors and to avoid unwanted accumulation in healthy tissues. The main objective of the present study is to develop and characterize TSL loaded with the radiosensitizer pimonidazole (PMZ) and to evaluate the in vitro efficacy of free PMZ and PMZ encapsulated in TSL in combination with hyperthermia and radiotherapy. PMZ was actively loaded into TSL at different drug/lipid ratios, and the physicochemical characteristics and the stability of the resulting TSL-PMZ were evaluated. PMZ release was determined at 37 °C and 42 °C in HEPES buffer saline and fetal bovine serum. The concentration-dependent radiosensitizing effect of PMZ was investigated by exposing FaDu cells to different PMZ concentrations under hypoxic conditions followed by exposure to ionizing irradiation. The efficacy of TSL-PMZ in combination with hyperthermia and radiotherapy was determined in vitro, assessing cell survival and DNA damage by means of the clonogenic assay and histone H2AX phosphorylation, respectively. All TSL-PMZ formulations showed high encapsulation efficiencies and were stable for 30 d upon storage at 4 °C and 20 °C. Fast PMZ release was observed at 42 °C, regardless of the drug/lipid ratio. Increasing the PMZ concentration significantly enhanced the effect of ionizing irradiation. Pre-heated TSL-PMZ in combination with radiotherapy caused a 14.3-fold increase in cell death as compared to radiotherapy treatment alone. In conclusion, our results indicate that TSL-PMZ in combination with hyperthermia can assist in improving the efficacy of radiotherapy under hypoxic conditions.

Microbubbles-Assisted Ultrasound Triggers the Release of Extracellular Vesicles.

Microbubbles-assisted ultrasound (USMB) has shown promise in improving local drug delivery. The formation of transient membrane pores and endocytosis are reported to be enhanced by USMB, and they contribute to cellular drug uptake. Exocytosis also seems to be linked to endocytosis upon USMB treatment. Based on this rationale, we investigated whether USMB triggers exocytosis resulting in the release of extracellular vesicles (EVs). USMB was performed on a monolayer of head-and-neck cancer cells (FaDu) with clinically approved microbubbles and commonly used ultrasound parameters. At 2, 4, and 24 h, cells and EV-containing conditioned media from USMB and control conditions (untreated cells, cells treated with microbubbles and ultrasound only) were harvested. EVs were measured using flow cytometric immuno-magnetic bead capture assay, immunogold electron microscopy, and western blotting. After USMB, levels of CD9 exposing-EVs significantly increased at 2 and 4 h, whereas levels of CD63 exposing-EVs increased at 2 h. At 24 h, EV levels were comparable to control levels. EVs released after USMB displayed a heterogeneous size distribution profile (30-1200 nm). Typical EV markers CD9, CD63, and alix were enriched in EVs released from USMB-treated FaDu cells. In conclusion, USMB treatment triggers exocytosis leading to the release of EVs from FaDu cells.

Influence of water and fat heterogeneity on fat-referenced MR thermometry.

PURPOSE: To investigate the effect of the aqueous and fatty tissue magnetic susceptibility distribution on absolute and relative temperature measurements as obtained directly from the water/fat (w/f) frequency difference. METHODS: Absolute thermometry was investigated using spherical phantoms filled with pork and margarine, which were scanned in three orthogonal orientations. To evaluate relative fat referencing, multigradient echo scans were acquired before and after heating pork tissue via high-intensity focused ultrasound (HIFU). Simulations were performed to estimate the errors that can be expected in human breast tissue. RESULTS: The sphere experiment showed susceptibility-related errors of 8.4 °C and 0.2 °C for pork and margarine, respectively. For relative fat referencing measurements, fat showed pronounced phase changes of opposite polarity to aqueous tissue. The apparent mean temperature for a numerical breast model assumed to be 37 °C was 47.2 ± 21.6 °C. Simulations of relative fat referencing for a HIFU sonication (ΔT = 29.7 °C) yielded a maximum temperature error of 6.6 °C compared with 2.5 °C without fat referencing. CONCLUSION: Variations in the observed frequency difference between water and fat are largely due to variations in the w/f spatial distribution. This effect may lead to considerable errors in absolute MR thermometry. Additionally, fat referencing may exacerbate rather than correct for proton resonance frequency shift-temperature measurement errors.

Histopathology of breast cancer after magnetic resonance-guided high-intensity focused ultrasound and radiofrequency ablation.

AIMS: Magnetic resonance-guided high-intensity focused ultrasound (MR-HIFU) ablation and radiofrequency ablation (RFA) are being researched as possible substitutes for surgery in breast cancer patients. The histopathological appearance of ablated tissue has not been studied in great detail. This study aimed to compare histopathological features of breast cancer after MR-HIFU ablation and RFA. METHODS AND RESULTS: MR-HIFU ablation and RFA were performed in- and ex-vivo. Tumours in six mastectomy specimens were partially ablated with RFA or MR-HIFU. In-vivo MR-HIFU ablation was performed 3-6 days before excision; RFA was performed in the operation room. Tissue was fixed in formalin and processed to haematoxylin and eosin (H&E) and cytokeratin-8 (CK-8)-stained slides. Morphology and cell viability were assessed. Ex-vivo ablation resulted in clear morphological changes after RFA versus subtle differences after MR-HIFU. CK-8 staining was decreased or absent. H&E tended to underestimate the size of thermal damage. In-vivo MR-HIFU resulted in necrotic-like changes. Surprisingly, some ablated lesions were CK-8-positive. Histopathology after in-vivo RFA resembled ex-vivo RFA, with hyper-eosinophilic stroma and elongated nuclei. Lesion borders were sharp after MR-HIFU and indistinct after RFA. CONCLUSION: Histopathological differences between MR-HIFU-ablated tissue and RF-ablated tissue were demonstrated. CK-8 was more reliable for cell viability assessment than H&E when used directly after ablation, while H&E was more reliable in ablated tissue left in situ for a few days. Our results contribute to improved understanding of histopathological features in breast cancer lesions treated with minimally invasive ablative techniques.

First clinical experience with a dedicated MRI-guided high-intensity focused ultrasound system for breast cancer ablation.

OBJECTIVES: To assess the safety and feasibility of MRI-guided high-intensity focused ultrasound (MR-HIFU) ablation in breast cancer patients using a dedicated breast platform. METHODS: Patients with early-stage invasive breast cancer underwent partial tumour ablation prior to surgical resection. MR-HIFU ablation was performed using proton resonance frequency shift MR thermometry and an MR-HIFU system specifically designed for breast tumour ablation. The presence and extent of tumour necrosis was assessed by histopathological analysis of the surgical specimen. Pearson correlation coefficients were calculated to assess the relationship between sonication parameters, temperature increase and size of tumour necrosis at histopathology. RESULTS: Ten female patients underwent MR-HIFU treatment. No skin redness or burns were observed in any of the patients. No correlation was found between the applied energy and the temperature increase. In six patients, tumour necrosis was observed with a maximum diameter of 3-11 mm. In these patients, the number of targeted locations was equal to the number of areas with tumour necrosis. A good correlation was found between the applied energy and the size of tumour necrosis at histopathology (Pearson = 0.76, p = 0.002). CONCLUSIONS: Our results show that MR-HIFU ablation with the dedicated breast system is safe and results in histopathologically proven tumour necrosis. KEY POINTS: • MR-HIFU ablation with the dedicated breast system is safe and feasible • In none of the patients was skin redness or burns observed • No correlation was found between the applied energy and the temperature increase • The correlation between applied energy and size of tumour necrosis was good.

Bubble-Assisted Ultrasound: Application in Immunotherapy and Vaccination.

Bubble-assisted ultrasound is a versatile technology with great potential in immunotherapy and vaccination. This technology involves the exposure of immune cells (i.e., dendritic cells, lymphocytes) in-vitro or diseased tissues (i.e., brain, tumor) in-vivo to ultrasound treatment with gas bubbles. Bubble destruction leads to physical forces that induce the direct delivery of weakly permeant immuno-stimulatory molecules either into the cytoplasm of immune cells, or through the endothelial barrier of diseased tissues. Hence, therapeutic antibodies (i.e., antibody-based immunotherapy) and cytokine-encoding nucleic acids (i.e., cytokine gene therapy) can be successfully delivered into diseased tissues, thus improving immune responses. In addition, protein antigens, as well as antigen-encoding nucleic acids (pDNA, mRNA), can be delivered into dendritic cells (i.e., dendritic cell-based vaccines), thus leading to a long-lasting prophylactic or therapeutic immunization. This chapter focuses on the state-of-the-art of bubble-assisted ultrasound in the field of immunotherapy and vaccination.

T1 and T2 temperature dependence of female human breast adipose tissue at 1.5 T: groundwork for monitoring thermal therapies in the breast.

The T1 and T2 temperature dependence of female breast adipose tissue was investigated at 1.5 T in order to evaluate the applicability of relaxation-based MR thermometry in fat for the monitoring of thermal therapies in the breast. Relaxation times T1 , T2 and T2TSE (the apparent T2 measured using a turbo spin echo readout sequence) were measured in seven fresh adipose breast samples for temperatures from 25 to 65 °C. Spectral water suppression was used to reduce the influence of the residual water signal. The temperature dependence of the relaxation times was characterized. The expected maximum temperature measurement errors based on average calibration lines were calculated. In addition, the heating-cooling reversibility was investigated for two samples. The T1 and T2TSE temperature (T) dependence could be fitted well with an exponential function of 1/T. A linear relationship between T2 and temperature was found. The temperature coefficients (mean ± inter-sample standard deviation) of T1 and T2TSE increased from 25 °C (dT1/dT = 5.35 ± 0.08 ms/°C, dT2TSE/dT = 3.82 ± 0.06 ms/°C) to 65 °C (dT1 /dT = 9.50 ± 0.16 ms/°C, dT2TSE/dT = 7.99 ± 0.38 ms/°C). The temperature coefficient of T2 was 0.90 ± 0.03 ms/°C. The temperature-induced changes in the relaxation times were found to be reversible after heating to 65 °C. Given the small inter-sample variation of the temperature coefficients, relaxation-based MR thermometry appears to be feasible in breast adipose tissue, and may be used as an adjunct to proton resonance frequency shift (PRFS) thermometry in aqueous tissue (glandular + tumor).

Mild hyperthermia influence on Herceptin(®) properties.

BACKGROUND: Mild hyperthermia (mHT) increases the tumor perfusion and vascular permeability, and reduces the interstitial fluid pressure, resulting in better intra-tumoral bioavailability of low molecular weight drugs. This approach is potentially also attractive for delivery of therapeutic macromolecules, such as antibodies. Here, we investigated the effects of mHT on the stability, immunological and pharmacological properties of Herceptin(®), a clinically approved antibody, targeting the human epidermal growth factor receptor 2 (HER-2) overexpressed in breast cancer. RESULTS: Herceptin(®) was heated to 37°C (control) and 42°C (mHT) for 1 hour. Formation of Herceptin(®) aggregates was measured using Nile Red assay. mHT did not result in additional Herceptin(®) aggregates compared to 37°C, showing the Herceptin(®) stability is unchanged. Immunological and pharmacological properties of Herceptin(®) were evaluated following mHT using HER-2 positive breast cancer cells (BT-474). Exposure of Herceptin(®) to mHT preserved recognition and binding affinity of Herceptin(®) to HER-2. Western-blot and cell proliferation assays on BT-474 cells showed that mHT left the inhibitory activities of Herceptin(®) unchanged. CONCLUSIONS: The stability, and the immunological and pharmacological properties of Herceptin(®) are not negatively affected by mHT. Further in-vivo studies are required to evaluate the influence of mHT on intra-tumoral bioavailability and therapeutic effectiveness of Herceptin(®).

In vivo T2 -based MR thermometry in adipose tissue layers for high-intensity focused ultrasound near-field monitoring.

PURPOSE: During MR-guided high-intensity focused ultrasound (HIFU) therapy, ultrasound absorption in the near field represents a safety risk and limits efficient energy deposition at the target. In this study, we investigated the feasibility of using T2 mapping to monitor the temperature change in subcutaneous adipose tissue layers. METHODS: The T2 temperature dependence and reversibility was determined for fresh adipose porcine samples. The accuracy was evaluated by comparing T2 -based temperature measurements with probe readings in an ex vivo HIFU experiment. The in vivo feasibility of T2 -based thermometry was studied during HIFU ablations in the liver in pigs and of uterine fibroids in human patients. RESULTS: T2 changed linearly and reversibly with temperature with an average coefficient of 5.2 ± 0.1 ms/°C. For the ex vivo HIFU experiment, the difference between the T2 -based temperature change and the probe temperature was <0.9°C. All in vivo experiments showed temperature-related T2 changes in the near field directly after sonications. As expected, considerable intersubject variations in the cooling times were measured in the in vivo porcine experiments. CONCLUSIONS: The reversibility and linearity of the T2 -temperature dependence of adipose tissue allows for the monitoring of the temperature in the subcutaneous adipose tissue layers.

Correction of proton resonance frequency shift MR-thermometry errors caused by heat-induced magnetic susceptibility changes during high intensity focused ultrasound ablations in tissues containing fat.

PURPOSE: In this study, we aim to demonstrate the sensitivity of proton resonance frequency shift (PRFS) -based thermometry to heat-induced magnetic susceptibility changes and to present and evaluate a model-based correction procedure. THEORY AND METHODS: To demonstrate the expected temperature effect, field disturbances during high intensity focused ultrasound sonications were monitored in breast fat samples with a three-dimensional (3D) gradient echo sequence. To evaluate the correction procedure, the interface of tissue-mimicking ethylene glycol gel and fat was sonicated. During sonication, the temperature was monitored with a 2D dual flip angle multi-echo gradient echo sequence, allowing for PRFS-based relative and referenced temperature measurements in the gel and T1 -based temperature measurements in fat. The PRFS-based measurement in the gel was corrected by minimizing the discrepancy between the observed 2D temperature profile and the profile predicted by a 3D thermal model. RESULTS: The HIFU sonications of breast fat resulted in a magnetic field disturbance which completely disappeared after cooling. For the correction method, the 5th to 95th percentile interval of the PRFS-thermometry error in the gel decreased from 3.8°C before correction to 2.0-2.3°C after correction. CONCLUSION: This study has shown the effects of magnetic susceptibility changes induced by heating of breast fatty tissue samples. The resultant errors can be reduced by the use of a model-based correction procedure.

Triggered release of doxorubicin from temperature-sensitive poly(N-(2-hydroxypropyl)-methacrylamide mono/dilactate) grafted liposomes.

The objective of this study was to design temperature-sensitive liposomes with tunable release characteristics that release their content at an elevated temperature generated by high intensity focused ultrasound (HIFU) exposure. To this end, thermosensitive polymers of N-(2-hydroxypropyl)methacrylamide mono/dilactate of different molecular weights and composition with a cholesterol anchor (chol-pHPMAlac) were synthesized and grafted onto liposomes loaded with doxorubicin (DOX). The liposomes were incubated at different temperatures and their release kinetics were studied. A good correlation between the release-onset temperature of the liposomes and the cloud point (CP) of chol-pHPMAlac was found. However, release took place at significantly higher temperatures than the CP of chol-pHPMAlac, likely at the CP, the dehydration and thus hydrophobicity is insufficient to penetrate and permeabilize the liposomal membrane. Liposomes grafted with chol-pHPMAlac with a CP of 11.5 °C released 89% DOX within 5 min at 42 °C while for the liposomes grafted with a polymer with CP of 25.0 °C, a temperature of 52 °C was needed to obtain the same extent of DOX release. At a fixed copolymer composition, an increase in molecular weight from 6.5 to 14.5 kDa decreased the temperature at which DOX was released with a release-onset temperature from 52 to 42 °C. Liposomes grafted with 5% chol-pHPMAlac exhibited a rapid release to a temperature increase, while at a grafting density of 2 and 10%, the liposomes were less sensitive to an increase in temperature. Sequential release of DOX was obtained by mixing liposomes grafted with chol-pHPMAlac having different CPs. Chol-pHPMAlac grafted liposomes released DOX nearly quantitatively after pulsed wave HIFU. In conclusion, the release of DOX from liposomes grafted with thermosensitive polymers of N-(2-hydroxypropyl)methacrylamide mono/dilactate can be tuned to the characteristics and the grafting density of chol-pHPMAlac, making these liposomes attractive for local drug delivery using hyperthermia.

New insights into the HIFU-triggered release from polymeric micelles.

Continuous wave (CW), low frequency, high intensity focused ultrasound (HIFU) is a promising modality to trigger release of active compounds from polymeric micelles. The aim of the present study was to investigate whether high frequency CW as well as pulsed wave (PW) HIFU can induce the release of a hydrophobic agent from non-cross-linked (NCL) and core cross-linked (CCL) poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-p(HPMAm-Lac(n))) micelles. It was shown that high frequency CW as well as PW HIFU was able to trigger the release (up to 85%) of a hydrophobic compound (i.e., nile red, NR) from NCL and CCL micelles. No changes in size distribution of the micelles after CW and PW HIFU exposure were observed and no degradation of polymer chain had occurred. We therefore hypothesize that the polymeric micelles are temporally destabilized upon HIFU exposure due to radiation force induced shear forces, leading to NR release on demand.

Ultrasound and Microbubbles Mediated Bleomycin Delivery in Feline Oral Squamous Cell Carcinoma-An In Vivo Veterinary Study.

To investigate the feasibility and tolerability of ultrasound and microbubbles (USMB)-enhanced chemotherapy delivery for head and neck cancer, we performed a veterinary trial in feline companion animals with oral squamous cell carcinomas. Six cats were treated with a combination of bleomycin and USMB therapy three times, using the Pulse Wave Doppler mode on a clinical ultrasound system and EMA/FDA approved microbubbles. They were evaluated for adverse events, quality of life, tumour response and survival. Furthermore, tumour perfusion was monitored before and after USMB therapy using contrast-enhanced ultrasound (CEUS). USMB treatments were feasible and well tolerated. Among 5 cats treated with optimized US settings, 3 had stable disease at first, but showed disease progression 5 or 11 weeks after first treatment. One cat had progressive disease one week after the first treatment session, maintaining a stable disease thereafter. Eventually, all cats except one showed progressive disease, but each survived longer than the median overall survival time of 44 days reported in literature. CEUS performed immediately before and after USMB therapy suggested an increase in tumour perfusion based on an increase in median area under the curve (AUC) in 6 out of 12 evaluated treatment sessions. In this small hypothesis-generating study, USMB plus chemotherapy was feasible and well-tolerated in a feline companion animal model and showed potential for enhancing tumour perfusion in order to increase drug delivery. This could be a forward step toward clinical translation of USMB therapy to human patients with a clinical need for locally enhanced treatment.

Microbubble-Assisted Ultrasound for Drug Delivery to the Retina in an Ex Vivo Eye Model.

Drug delivery to the retina is one of the major challenges in ophthalmology due to the biological barriers that protect it from harmful substances in the body. Despite the advancement in ocular therapeutics, there are many unmet needs for the treatment of retinal diseases. Ultrasound combined with microbubbles (USMB) was proposed as a minimally invasive method for improving delivery of drugs in the retina from the blood circulation. This study aimed to investigate the applicability of USMB for the delivery of model drugs (molecular weight varying from 600 Da to 20 kDa) in the retina of ex vivo porcine eyes. A clinical ultrasound system, in combination with microbubbles approved for clinical ultrasound imaging, was used for the treatment. Intracellular accumulation of model drugs was observed in the cells lining blood vessels in the retina and choroid of eyes treated with USMB but not in eyes that received ultrasound only. Specifically, 25.6 ± 2.9% of cells had intracellular uptake at mechanical index (MI) 0.2 and 34.5 ± 6.0% at MI 0.4. Histological examination of retinal and choroid tissues revealed that at these USMB conditions, no irreversible alterations were induced at the USMB conditions used. These results indicate that USMB can be used as a minimally invasive targeted means to induce intracellular accumulation of drugs for the treatment of retinal diseases.

Arrhenius analysis of the relationship between hyperthermia and Hsp70 promoter activation: a comparison between ex vivo and in vivo data.

PURPOSE: Tight regulation of gene expression in the region where therapy is necessary and for the duration required to achieve a therapeutic effect and to minimise systemic toxicity is very important for clinical applications of gene therapy. Hyperthermia in combination with a temperature sensitive heat shock protein (Hsp70) promoter presents a unique approach allowing non-invasive spatio-temporal control of transgene expression. In this study we investigated the in vivo and ex vivo relationship between temperature and duration of thermal stress with respect to the resulting gene expression using an Arrhenius analysis. MATERIALS AND METHODS: A transgenic mouse expressing the luciferase reporter gene under the transcriptional control of a thermosensitive promoter was used to assure identical genotype for in vivo (mouse leg) and ex vivo (bone marrow mononuclear and embryonic fibroblast cells) studies. The mouse leg and cells were heated at different temperatures and different exposure times. Bioluminescence imaging and in vitro enzymatic assay were used to measure the resulting transgene expression. RESULTS: We showed that temperature-induced Hsp70 promoter activation was modulated by both temperature as well as duration of hyperthermia. The relationship between temperature and duration of hyperthermia and the resulting reporter gene expression can be modelled by an Arrhenius analysis for both in vivo as well as ex vivo. CONCLUSIONS: However, the increase in reporter gene expression after elevating the temperature of the thermal stress with 1°C is not comparable for in vivo and ex vivo situations. This information may be valuable for optimising clinical gene therapy protocols.

Image-guided, noninvasive, spatiotemporal control of gene expression.

Spatiotemporal control of transgene expression is of paramount importance in gene therapy. Here, we demonstrate the use of magnetic resonance temperature imaging (MRI)-guided, high-intensity focused ultrasound (HIFU) in combination with a heat-inducible promoter [heat shock protein 70 (HSP70)] for the in vivo spatiotemporal control of transgene activation. Local gene activation induced by moderate hyperthermia in a transgenic mouse expressing luciferase under the control of the HSP70 promoter showed a high similarity between the local temperature distribution in vivo and the region emitting light. Modulation of gene expression is possible by changing temperature, duration, and location of regional heating. Mild heating protocols (2 min at 43 degrees C) causing no tissue damage were sufficient for significant gene activation. The HSP70 promoter was shown to be induced by the local temperature increase and not by the mechanical effects of ultrasound. Therefore, the combination of MRI-guided HIFU heating and transgenes under control of heat-inducible HSP promoter provides a direct, noninvasive, spatial control of gene expression via local hyperthermia.

The Effect of Microbubble-Assisted Ultrasound on Molecular Permeability across Cell Barriers.

The combination of ultrasound and microbubbles (USMB) has been applied to enhance drug permeability across tissue barriers. Most studies focused on only one physicochemical aspect (i.e., molecular weight of the delivered molecule). Using an in vitro epithelial (MDCK II) cell barrier, we examined the effects of USMB on the permeability of five molecules varying in molecular weight (182 Da to 20 kDa) and hydrophilicity (LogD at pH 7.4 from 1.5 to highly hydrophilic). Treatment of cells with USMB at increasing ultrasound pressures did not have a significant effect on the permeability of small molecules (molecular weight 259 to 376 Da), despite their differences in hydrophilicity (LogD at pH 7.4 from -3.2 to 1.5). The largest molecules (molecular weight 4 and 20 kDa) showed the highest increase in the epithelial permeability (3-7-fold). Simultaneously, USMB enhanced intracellular accumulation of the same molecules. In the case of the clinically relevant anti- C-X-C Chemokine Receptor Type 4 (CXCR4) nanobody (molecular weight 15 kDa), USMB enhanced paracellular permeability by two-fold and increased binding to retinoblastoma cells by five-fold. Consequently, USMB is a potential tool to improve the efficacy and safety of the delivery of drugs to organs protected by tissue barriers, such as the eye and the brain.

Ultrasound-directed enzyme-prodrug therapy (UDEPT) using self-immolative doxorubicin derivatives.

Background: Enzyme-activatable prodrugs are extensively employed in oncology and beyond. Because enzyme concentrations and their (sub)cellular compartmentalization are highly heterogeneous in different tumor types and patients, we propose ultrasound-directed enzyme-prodrug therapy (UDEPT) as a means to increase enzyme access and availability for prodrug activation locally. Methods: We synthesized ß-glucuronidase-sensitive self-immolative doxorubicin prodrugs with different spacer lengths between the active drug moiety and the capping group. We evaluated drug conversion, uptake and cytotoxicity in the presence and absence of the activating enzyme ß-glucuronidase. To trigger the cell release of ß-glucuronidase, we used high-intensity focused ultrasound to aid in the conversion of the prodrugs into their active counterparts. Results: More efficient enzymatic activation was observed for self-immolative prodrugs with more than one aromatic unit in the spacer. In the absence of ß-glucuronidase, the prodrugs showed significantly reduced cellular uptake and cytotoxicity compared to the parent drug. High-intensity focused ultrasound-induced mechanical destruction of cancer cells resulted in release of intact ß-glucuronidase, which activated the prodrugs, restored their cytotoxicity and induced immunogenic cell death. Conclusion: These findings shed new light on prodrug design and activation, and they contribute to novel UDEPT-based mechanochemical combination therapies for the treatment of cancer.

Ultrasound and Microbubbles for the Treatment of Ocular Diseases: From Preclinical Research towards Clinical Application.

The unique anatomy of the eye and the presence of various biological barriers make efficacious ocular drug delivery challenging, particularly in the treatment of posterior eye diseases. This review focuses on the combination of ultrasound and microbubbles (USMB) as a minimally invasive method to improve the efficacy and targeting of ocular drug delivery. An extensive overview is given of the in vitro and in vivo studies investigating the mechanical effects of ultrasound-driven microbubbles aiming to: (i) temporarily disrupt the blood-retina barrier in order to enhance the delivery of systemically administered drugs into the eye, (ii) induce intracellular uptake of anticancer drugs and macromolecules and (iii) achieve targeted delivery of genes, for the treatment of ocular malignancies and degenerative diseases. Finally, the safety and tolerability aspects of USMB, essential for the translation of USMB to the clinic, are discussed.

Effects of contrast-enhanced ultrasound treatment on neoadjuvant chemotherapy in breast cancer.

Purpose: Preclinical and clinical data indicate that contrast-enhanced ultrasound can enhance tumor perfusion and vessel permeability, thus, improving chemotherapy accumulation and therapeutic outcome. Therefore, we investigated the effects of high mechanical index (MI) contrast-enhanced Doppler ultrasound (CDUS) on tumor perfusion in breast cancer. Methods: In this prospective study, breast cancer patients were randomly assigned to receive either 18 minutes of high MI CDUS during chemotherapy infusion (n = 6) or chemotherapy alone (n = 5). Tumor perfusion was measured before and after at least six chemotherapy cycles using motion-model ultrasound localization microscopy. Additionally, acute effects of CDUS on vessel perfusion and chemotherapy distribution were evaluated in mice bearing triple-negative breast cancer (TNBC). Results: Morphological and functional vascular characteristics of breast cancer in patients were not significantly influenced by high MI CDUS. However, complete clinical tumor response after neoadjuvant chemotherapy was lower in high MI CDUS-treated (1/6) compared to untreated patients (4/5) and size reduction of high MI CDUS treated tumors tended to be delayed at early chemotherapy cycles. In mice with TNBC high MI CDUS decreased the perfused tumor vessel fraction (p < 0.01) without affecting carboplatin accumulation or distribution. Higher vascular immaturity and lower stromal stabilization may explain the stronger vascular response in murine than human tumors. Conclusion: High MI CDUS had no detectable effect on breast cancer vascularization in patients. In mice, the same high MI CDUS setting did not affect chemotherapy accumulation although strong effects on the tumor vasculature were detected histologically. Thus, sonopermeabilization in human breast cancers might not be effective using high MI CDUS protocols and future applications may rather focus on low MI approaches triggering microbubble oscillations instead of destruction. Furthermore, our results show that there are profound differences in the response of mouse and human tumor vasculature to high MI CDUS, which need to be further explored and considered in clinical translation.