Ultrasound-Triggered Delivery of Iproplatin from Microbubble-Conjugated Liposomes.

The PtIV prodrug iproplatin has been actively loaded into liposomes using a calcium acetate gradient, achieving a 3-fold enhancement in drug concentration compared to passive loading strategies. A strain-promoted cycloaddition reaction (azide- dibenzocyclooctyne) was used to attach iproplatin-loaded liposomes L(Pt) to gas-filled microbubbles (M), forming an ultrasound-responsive drug delivery vehicle [M-L(Pt)]. Ultrasound-triggered release of iproplatin from the microbubble-liposome construct was evaluated in cellulo. Breast cancer (MCF-7) cells treated with both free iproplatin and iproplatin-loaded liposome-microbubbles [M-L(Pt)] demonstrated an increase in platinum concentration when exposed to ultrasound. No appreciable platinum uptake was observed in MCF-7 cells following treatment with L(Pt) only or L(Pt)+ultrasound, suggesting that microbubble-mediated ultrasonic release of platinum-based drugs from liposomal carriers enables greater control over drug delivery.

Scaleable production of microbubbles using an ultrasound-modulated microfluidic device.

Surfactant-coated gas microbubbles are widely used as contrast agents in ultrasound imaging and increasingly in therapeutic applications. The response of microbubbles to ultrasound can be strongly influenced by their size and coating properties, and hence the production method. Ultrasonic emulsification (sonication) is the most commonly employed method and can generate high concentrations of microbubbles rapidly, but with a broad size distribution, and there is a risk of contamination and/or degradation of sensitive components. Microfluidic devices provide excellent control over microbubble size, but are often challenging or costly to manufacture, offer low production rates (<106s-1), and are prone to clogging. In this study, a hybrid sonication-microfluidic or "sonofluidic" device was developed. Bubbles of ∼180 µm diameter were produced rapidly in a T-junction and subsequently exposed to ultrasound (71-73 kHz) within a microchannel, generating microbubbles (mean diameter: 1-2 µm) at a rate of >108s-1 using a single device. Microbubbles were prepared using either the sonofluidic device or conventional sonication, and their size, concentration, and stability were comparable. The mean diameter, concentration, and stability were found to be comparable between techniques, but the microbubbles produced by the sonofluidic device were all <5 µm in diameter and thus did not require any post-production fractionation.

Combining sonodynamic therapy with chemoradiation for the treatment of pancreatic cancer.

Treatment options for patients with pancreatic cancer are limited and survival prospects have barely changed over the past 4 decades. Chemoradiation treatment (CRT) has been used as neoadjuvant therapy in patients with borderline resectable disease to reduce tumour burden and increase the proportion of patients eligible for surgery. Antimetabolite drugs such as gemcitabine and 5-fluorouracil are known to sensitise pancreatic tumours to radiation treatment. Likewise, photodynamic therapy (PDT) has also been shown to enhance the effect of radiation therapy. However, PDT is limited to treating superficial lesions due to the attenuation of light by tissue. The ability of the related technique, sonodynamic therapy (SDT), to enhance CRT was investigated in two murine models of pancreatic cancer (PSN-1 and BxPC-3) in this study. SDT uses low intensity ultrasound to activate an otherwise non-toxic sensitiser, generating toxic levels of reactive oxygen species (ROS) locally. It is applicable to greater target depths than PDT due to the ability of ultrasound to propagate further than light in tissue. Both CRT and the combination of CRT plus SDT delayed tumour growth in the two tumour models. In the PSN-1 model, but not the BxPC-3 model, the combination treatment caused an increase in survival relative to CRT alone (p = 0.038). The improvement in survival conferred by the addition of SDT in this model may be related to differences in tumour architecture between the two models. MRI and US images showed that PSN-1 tumours were less well perfused and vascularised than BxPC-3 tumours. This poor vascularisation may explain why PSN-1 tumours were more susceptible to the effects of vascular damage exerted by SDT treatment.

Evaluation of Loading Strategies to Improve Tumor Uptake of Gemcitabine in a Murine Orthotopic Bladder Cancer Model Using Ultrasound and Microbubbles.

In this study we compared three different microbubble-based approaches to the delivery of a widely used chemotherapy drug, gemcitabine: (i) co-administration of gemcitabine and microbubbles (Gem+MB); (ii) conjugates of microbubbles and gemcitabine-loaded liposomes (GemlipoMB); and (iii) microbubbles with gemcitabine directly bound to their surfaces (GembioMB). Both in vitro and in vivo investigations were carried out, respectively, in the RT112 bladder cancer cell line and in a murine orthotopic muscle-invasive bladder cancer model. The in vitro (in vivo) ultrasound exposure conditions were a 1 (1.1) MHz centre frequency, 0.07 (1.0) MPa peak negative pressure, 3000 (20,000) cycles and 100 (0.5) Hz pulse repetition frequency. Ultrasound exposure produced no significant increase in drug uptake either in vitro or in vivo compared with the drug-only control for co-administered gemcitabine and microbubbles. In vivo, GemlipoMB prolonged the plasma circulation time of gemcitabine, but only GembioMB produced a statistically significant increase in cleaved caspase 3 expression in the tumor, indicative of gemcitabine-induced apoptosis.

Ultrasound-Mediated Gemcitabine Delivery Reduces the Normal-Tissue Toxicity of Chemoradiation Therapy in a Muscle-Invasive Bladder Cancer Model.

PURPOSE: Chemoradiation therapy is the standard of care in muscle-invasive bladder cancer (MIBC). Although agents such as gemcitabine can enhance tumor radiosensitivity, their side effects can limit patient eligibility and treatment efficacy. This study investigates ultrasound and microbubbles for targeting gemcitabine delivery to reduce normal-tissue toxicity in a murine orthotopic MIBC model. MATERIALS AND METHODS: CD1-nude mice were injected orthotopically with RT112 bladder tumor cells. Conventional chemoradiation involved injecting gemcitabine (10 mg/kg) before 6 Gy targeted irradiation of the bladder area using the Small Animal Radiation Research Platform (SARRP). Ultrasound-mediated gemcitabine delivery (10 mg/kg gemcitabine) involved either coadministration of microbubbles with gemcitabine or conjugating gemcitabine onto microbubbles followed by exposure to ultrasound (1.1 MHz center frequency, 1 MPa peak negative pressure, 1% duty cycle, and 0.5 Hz pulse repetition frequency) before SARRP irradiation. The effect of ultrasound and microbubbles alone was also tested. Tumor volumes were measured by 3D ultrasound imaging. Acute normal-tissue toxicity from 12 Gy to the lower bowel area was assessed using an intestinal crypt assay in mice culled 3.75 days posttreatment. RESULTS: A significant delay in tumor growth was observed with conventional chemoradiation therapy and both microbubble groups (P < .05 compared with the radiation-only group). Transient weight loss was seen in the microbubble groups, which resolved within 10 days posttreatment. A positive correlation was found between weight loss on day 3 posttreatment and tumor growth delay (P < .05; R2 = 0.76). In contrast with conventional chemoradiation therapy, ultrasound-mediated drug delivery methods did not exacerbate the acute intestinal toxicity using the crypt assay. CONCLUSIONS: Ultrasound and microbubbles offer a promising new approach for improving chemoradiation therapy for muscle-invasive bladder cancer, maintaining a delay in tumor growth but with reduced acute intestinal toxicity compared with conventional chemoradiation therapy.

Spontaneous Appearance and Transmission of Polydactyly in Dexter Cattle.

A 3-yr-old Dexter cow and her yearling Dexter heifer calf exhibited polydactyly. Neither animal was linebred within 5 generations. This cow-calf pair represented the first reported occurrence of polydactyly in Dexter cattle in the US or abroad. Based upon external examination, the cow was classified as having a spontaneous unilateral case of polydactyly with an extra digit along the medial digit of the right front limb and the heifer was classified as having bilateral polydactyly because both front limbs exhibited an extra digit along the medial digit. Radiographic examination confirmed bilateral status of the heifer and revealed bilateral status of the cow. The front feet of the cow and heifer had extra bone formation consistent with an extra digit along the medial digit. Neither animal suffered from limited mobility to date or required hoof treatments. The cow produced a second calf from a different sire, a bull calf that did not appear polydactylous per external examination and was not examined radiographically. The two polydactylous animals will remain in the breeding herd to produce more study calves unless their fitness becomes compromised. Genetic aspects of the cases are discussed.

Manganese-free chow, a refined non-invasive solution to reduce gastrointestinal signal for T1-weighted magnetic resonance imaging of the mouse abdomen.

Commercial mouse chow is designed to provide a complete, nutrient-rich diet, and it can contain upwards of 100 mg/kg manganese, an essential mineral. Manganese acts as a relaxation time-shortening contrast agent for both T1 and T2, and where standard chow is hydrated in the gastrointestinal tract, bright signals are produced when using T1-weighted imaging (T1WI). As a result of peristalsis, gastrointestinal hyperintensities result in temporally unstable signals, leading to image ghosting and decreased resolution from that prescribed. To avoid the problem, various methods of gastrointestinal tract modulation, including the use of intestinal cleansing with laxatives and dietary modulation, have been reported. Here, dietary modulation has been extended to the use of a biologically innocuous, long-term change of diet. In this study, we report on the use of a commercially available manganese-free chow to improve the image quality of the gastrointestinal tract. This manganese-free chow, apart from the omitted manganese which is available in tap water, is a complete diet and readily available. We investigated the time-dependent, diet-related gastrointestinal intensities on short-TR T1WI magnetic resonance imaging; monitored body mass, food and water consumption and standard blood biochemistry analysis following diet change; and determined manganese concentration in blood plasma following a five-day change to manganese-free chow. We show that the manganese-free chow presents a refinement to other gastrointestinal tract modulation, as it avoids the need for invasive procedures for gut voiding and can be provided ad libitum so that animals can be maintained with no need for prescribed diet change before imaging.

Spectral Imaging for Microbubble Characterization.

Microbubbles stabilized by an outer lipid shell have been studied extensively for both diagnostic and therapeutic applications. The shell composition can significantly influence microbubble behavior, but performing quantitative measurements of shell properties is challenging. The aim of this study is to investigate the use of spectral imaging to characterize the surface properties of a range of microbubble formulations representing both commercial and research agents. A lipophilic dye, C-laurdan, whose fluorescence emission varies according to the properties of the local environment, was used to compare the degree and uniformity of the lipid order in the microbubble shell, and these measurements were compared with the acoustic response and stability of the different formulations. Spectral imaging was found to be suitable for performing rapid and hence relatively high throughput measurements of microbubble surface properties. Interestingly, despite significant differences in lipid molecule size and charge, all of the different formulations exhibited highly ordered lipid shells. Measurements of liposomes with the same composition and the debris generated by destroying lipid microbubbles with ultrasound (US) showed that these exhibited a lower and more varied lipid order than intact microbubbles. This suggests that the high lipid order of microbubbles is due primarily to compression of the shell as a result of surface tension and is only minimally affected by composition. This also explains the similarity in acoustic response observed between the formulations, because microbubble dynamics are determined by the diameter and shell viscoelastic properties that are themselves a function of the lipid order. Within each population, there was considerable variability in the lipid order and response between individual microbubbles, suggesting the need for improved manufacturing techniques. In addition, the difference in the lipid order between the shell and lipid debris may be important for therapeutic applications in which shedding of the shell material is exploited, for example, drug delivery.

Mouse Models of Muscle-invasive Bladder Cancer: Key Considerations for Clinical Translation Based on Molecular Subtypes.

CONTEXT: In the past few years, research has suggested that molecular subtypes in muscle-invasive bladder cancer (MIBC) may be exploited to accelerate developments in clinical disease management and novel therapeutics. OBJECTIVE: To review MIBC mouse models from a molecular subtype perspective, their advantages and limitations, and their applications in translational medicine, based on a PubMed search for publications from January 2000 to February 2018. EVIDENCE ACQUISITION: Publications relevant to MIBC mouse models and their molecular subtypes were identified in a literature review. EVIDENCE SYNTHESIS: We classified the models according to the technique used for their establishment. For xenotransplant and allograft models, the inoculated cells and inoculated locations are the major determinants of molecular subtypes. Although the cell lines used in xenotransplant models can cover most of the basal-squamous and luminal subtypes, allograft models offer a more realistic environment in which to reconstruct aspects of the associated stromal and immune features. Autochthonous models, using genetic and/or chemical stimuli to induce disease progression, can also generate models with basal-squamous and luminal subtypes, but further molecular characterisation is needed since other mutational variants may be introduced in these models. CONCLUSIONS: We identified preclinical MIBC models with different subtype specifications and assessed their promise and current limitations. These models are versatile tools that can reproduce the molecular complexity of MIBC and support novel therapeutic development. PATIENT SUMMARY: Understanding which models of muscle-invasive bladder cancer most accurately represent the clinical situation is important for the development of novel drugs and disease management strategies. We review the different models currently available and their relevance to different clinical subtypes.

Influence of crossbreeding on meat goat doe fitness when comparing Boer F1 with base breeds in the Southeastern United States.

Understanding fitness level among various breeds is essential for sustainable meat goat production. Research on the relative fitness of Boer F1 does and straightbred base breed has been limited. Meat goat does of various genotypes (Boer, Kiko, Spanish, Boer × Kiko reciprocal F1 crosses, and Boer × Spanish reciprocal F1 crosses) were studied to evaluate breed effects on doe fitness traits and the expression of heterosis over 7 production years. The herd was semi-intensively managed under humid subtropical pasture. Doe age affected (P < 0.05) various traits. Boer × Kiko does were heavier (P < 0.05) than Boer does at fall breeding, but Boer × Spanish does did not differ (P > 0.05) from Boer does for breeding weight. The body weights of Boer × Spanish and Boer × Kiko crosses did not differ (P > 0.05) from the weights of their respective Kiko and Spanish base cohorts at breeding, kidding, or weaning. Boer does had lower (P < 0.05) kidding rate (KR) and weaning rate (WR) than the other breeds and crosses. Boer × Kiko and Kiko were similar for KR and WR. Boer × Spanish and Spanish were also similar for KR and WR. However, the combined group of Boer F1 does had lower (P < 0.01) KR and WR than the combined purebred biotype group of Kiko and Spanish does. Boer does weaned smaller (P < 0.05) litter sizes per doe exposed compared with Kiko, Spanish, Boer × Kiko, and Boer × Spanish does with the latter four doe breedtypes not differing from each other. The combined Boer F1 doe group weaned smaller (P < 0.05) litter sizes per doe exposed than the combined purebred group of Kiko and Spanish does. Boer × Kiko dams had higher (P < 0.05) fecal egg counts at parturition than Kiko dams. Significant heterosis was observed for reproductive traits within each of the 2-breed diallels. Boer F1 does exhibited reproductive output similar to or lower than Kiko and Spanish straightbred does and higher than Boer straightbred does.

Effects of creep feeding and its interactions with other factors on the performance of meat goat kids and dams when managed on pasture.

Creep feeding and its possible interactions with other influential factors (genetics, litter type, and sex) for weaning traits were studied in meat goat kids and their dams. Kids across 3 yr were creep fed (254 kids; 5 pens) or not creep fed (255 kids; 5 pens) from 30 to 90 d of age. Creep-fed kids had higher (P ≤ 0.05) preweaning average daily weight gain and weaning weights (113.1 ± 13.0 g/d; 15.0 ± 0.8 kg) than kids not creep fed (99.8 ± 13.1 g/d; 14.0 ± 0.8 kg). However, financial returns were not higher (P > 0.05) for creep-fed kids compared with kids not creep fed. There was no difference (P > 0.05) in kid conformation score or survival rates between the treatment groups. The only important interaction among kid traits was treatment × litter type (P < 0.05) for FAMACHA scores. Within noncreep pens, single kids had lower (better; P < 0.05) FAMACHA scores (2.9 ± 0.3) than twin kids (3.9 ± 0.3). There was no litter-type effect on FAMACHA scores for kids within the creep feed pens. Dams of the creep-fed (n = 175) and noncreep (n = 178) kids were also evaluated. Treatment did not affect (P > 0.05) litter weights, dam weight change, gross revenue for weaned litters, or fecal egg counts. Treatment interacted with litter type (P < 0.05) to effect packed cell volume (PCV). In the noncreep group, dams raising singles had higher (better; P < 0.05) PCV (18.7 ± 1.3%) than dams rearing twin kids (15.7 ± 1.3%). The litter-type effect on dam PCV was not evident (P > 0.05) in the creep-fed group. Creep feeding improved some kid growth traits but did not improve dam traits or financial returns. Interactions of creep treatment with other factors were minimal for doe-kid traits.

The Role of PEG-40-stearate in the Production, Morphology, and Stability of Microbubbles.

Phospholipid coated microbubbles are currently in widespread clinical use as ultrasound contrast agents and under investigation for therapeutic applications. Previous studies have demonstrated the importance of the coating nanostructure in determining microbubble stability and its dependence upon both composition and processing method. While the influence of different phospholipids has been widely investigated, the role of other constituents such as emulsifiers has received comparatively little attention. Herein, we present an examination of the impact of polyethylene glycol (PEG) derivatives upon microbubble structure and properties. We present data using both pegylated phospholipids and a fluorescent PEG-40-stearate analogue synthesized in-house to directly observe its distribution in the microbubble coating. We examined microbubbles of clinically relevant sizes, investigating both their surface properties and population size distribution and stability. Domain formation was observed only on the surface of larger microbubbles, which were found to contain a higher concentration of PEG-40-stearate. Lipid analogue dyes were also found to influence domain formation compared with PEG-40-stearate alone. "Squeezing out" of PEG-40-stearate was not observed from any of the microbubble sizes investigated. At ambient temperature, microbubbles formulated with DSPE-PEG(2000) were found to be more stable than those containing PEG-40-stearate. At 37 °C, however, the stability in serum was found to be the same for both formulations, and no difference in acoustic backscatter was detected. This could potentially reduce the cost of PEGylated microbubbles and facilitate simpler attachment of targeting or therapeutic species. However, whether PEG-40-stearate sufficiently shields microbubbles to inhibit physiological clearance mechanisms still requires investigation.

A versatile method for the preparation of particle-loaded microbubbles for multimodality imaging and targeted drug delivery.

Microbubbles are currently in clinical use as ultrasound contrast agents and under active investigation as mediators of ultrasound therapy. To improve the theranostic potential of microbubbles, nanoparticles can be attached to the bubble shell for imaging, targeting and/or enhancement of acoustic response. Existing methods for fabricating particle-loaded bubbles, however, require the use of polymers, oil layers or chemical reactions for particle incorporation; embed/attach the particles that can reduce echogenicity; impair biocompatibility; and/or involve multiple processing steps. Here, we describe a simple method to embed nanoparticles in a phospholipid-coated microbubble formulation that overcomes these limitations. Magnetic nanoparticles are used to demonstrate the method with a range of different microbubble formulations. The size distribution and yield of microbubbles are shown to be unaffected by the addition of the particles. We further show that the microbubbles can be retained against flow using a permanent magnet, can be visualised by both ultrasound and magnetic resonance imaging (MRI) and can be used to transfect SH-SY5Y cells with fluorescent small interfering RNA under the application of a magnetic field and ultrasound field.

Drug Delivery Strategies for Platinum-Based Chemotherapy.

Few chemotherapeutics have had such an impact on cancer management as cis-diamminedichloridoplatinum(II) (CDDP), also known as cisplatin. The first member of the platinum-based drug family, CDDP's potent toxicity in disrupting DNA replication has led to its widespread use in multidrug therapies, with particular benefit in patients with testicular cancers. However, CDDP also produces significant side effects that limit the maximum systemic dose. Various strategies have been developed to address this challenge including encapsulation within micro- or nanocarriers and the use of external stimuli such as ultrasound to promote uptake and release. The aim of this review is to look at these strategies and recent scientific and clinical developments.

Electrohydrodynamic fabrication of core-shell PLGA nanoparticles with controlled release of cisplatin for enhanced cancer treatment.

Increasing the clinical efficacy of toxic chemotherapy drugs such as cisplatin (CDDP), via targeted drug delivery, is a key area of research in cancer treatment. In this study, CDDP-loaded poly(lactic-co-glycolic acid) (PLGA) polymeric nanoparticles (NPs) were successfully prepared using electrohydrodynamic atomization (EHDA). The configuration was varied to control the distribution of CDDP within the particles, and high encapsulation efficiency (>70%) of the drug was achieved. NPs were produced with either a core-shell (CS) or a matrix (uniform) structure. It was shown that CS NPs had the most sustained release of the 2 formulations, demonstrating a slower linear release post initial "burst" and longer duration. The role of particle architecture on the rate of drug release in vitro was confirmed by fitting the experimental data with various kinetic models. This indicated that the release process was a simple diffusion mechanism. The CS NPs were effectively internalized into the endolysosomal compartments of cancer cells and demonstrated an increased cytotoxic efficacy (concentration of a drug that gives half maximal response [EC50] reaching 6.2 µM) compared to free drug (EC50 =9 µM) and uniform CDDP-distributed NPs (EC50 =7.6 µM) in vitro. Thus, these experiments indicate that engineering the structure of PLGA NPs can be exploited to control both the dosage and the release characteristics for improved clinical chemotherapy treatment.

Spectral imaging toolbox: segmentation, hyperstack reconstruction, and batch processing of spectral images for the determination of cell and model membrane lipid order.

BACKGROUND: Spectral imaging with polarity-sensitive fluorescent probes enables the quantification of cell and model membrane physical properties, including local hydration, fluidity, and lateral lipid packing, usually characterized by the generalized polarization (GP) parameter. With the development of commercial microscopes equipped with spectral detectors, spectral imaging has become a convenient and powerful technique for measuring GP and other membrane properties. The existing tools for spectral image processing, however, are insufficient for processing the large data sets afforded by this technological advancement, and are unsuitable for processing images acquired with rapidly internalized fluorescent probes. RESULTS: Here we present a MATLAB spectral imaging toolbox with the aim of overcoming these limitations. In addition to common operations, such as the calculation of distributions of GP values, generation of pseudo-colored GP maps, and spectral analysis, a key highlight of this tool is reliable membrane segmentation for probes that are rapidly internalized. Furthermore, handling for hyperstacks, 3D reconstruction and batch processing facilitates analysis of data sets generated by time series, z-stack, and area scan microscope operations. Finally, the object size distribution is determined, which can provide insight into the mechanisms underlying changes in membrane properties and is desirable for e.g. studies involving model membranes and surfactant coated particles. Analysis is demonstrated for cell membranes, cell-derived vesicles, model membranes, and microbubbles with environmentally-sensitive probes Laurdan, carboxyl-modified Laurdan (C-Laurdan), Di-4-ANEPPDHQ, and Di-4-AN(F)EPPTEA (FE), for quantification of the local lateral density of lipids or lipid packing. CONCLUSIONS: The Spectral Imaging Toolbox is a powerful tool for the segmentation and processing of large spectral imaging datasets with a reliable method for membrane segmentation and no ability in programming required. The Spectral Imaging Toolbox can be downloaded from https://uk.mathworks.com/matlabcentral/fileexchange/62617-spectral-imaging-toolbox .

Blepharochalasis: something to cry about.

Blepharochalasis is a rare disorder of unknown etiology defined by loose, atrophic periorbital skin following recurrent episodes of eyelid edema. Characteristic histopathology shows complete absence of elastic fibers. The current case progressed after multiple episodes of crying, which may be related to matrix metalloproteinase dysregulation. This case offers further insights into the possible pathogenesis of blepharochalasis, paving the way for more targeted, disease-modifying therapies.

Characterization of Contrast Agent Microbubbles for Ultrasound Imaging and Therapy Research.

The high efficiency with which gas microbubbles can scatter ultrasound compared with the surrounding blood pool or tissues has led to their widespread employment as contrast agents in ultrasound imaging. In recent years, their applications have been extended to include super-resolution imaging and the stimulation of localized bio-effects for therapy. The growing exploitation of contrast agents in ultrasound and in particular these recent developments have amplified the need to characterize and fully understand microbubble behavior. The aim in doing so is to more fully exploit their utility for both diagnostic imaging and potential future therapeutic applications. This paper presents the key characteristics of microbubbles that determine their efficacy in diagnostic and therapeutic applications and the corresponding techniques for their measurement. In each case, we have presented information regarding the methods available and their respective strengths and limitations, with the aim of presenting information relevant to the selection of appropriate characterization methods. First, we examine methods for determining the physical properties of microbubble suspensions and then techniques for acoustic characterization of both suspensions and single microbubbles. The next section covers characterization of microbubbles as therapeutic agents, including as drug carriers for which detailed understanding of their surface characteristics and drug loading capacity is required. Finally, we discuss the attempts that have been made to allow comparison across the methods employed by various groups to characterize and describe their microbubble suspensions and promote wider discussion and comparison of microbubble behavior.