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.

Diagnostic and Therapeutic Applications of Quantum Dots in Nanomedicine.

The interest in Quantum Dots as a class of nanomaterials has grown considerably since their discovery by Ekimov and Efros in the early 1980s. Although this early work focussed primarily on CdSe-based nanocrystals, the field has now expanded to include various classes of nanoparticles with different types of core, shell or passivation chemistry. Such differences can have a profound effect on the optical properties and potential biocompatibility of the resulting constructs. Although QDs have predominantly been used for imaging and sensing applications, more examples of their use as therapeutics are beginning to emerge. In this chapter we discuss the progress made over the past decade in developing QDs for imaging and therapeutic applications.

Optical probes for the detection of protons, and alkali and alkaline earth metal cations.

Luminescent sensors and switches continue to play a key role in shaping our understanding of key biochemical processes, assist in the diagnosis of disease and contribute to the design of new drugs and therapies. Similarly, their contribution to the environment cannot be understated as they offer a portable means to undertake field testing for hazardous chemicals and pollutants such as heavy metals. From a physiological perspective, the Group I and II metal ions are among the most important in the periodic table with blood plasma levels of H(+), Na(+) and Ca(2+) being indicators of several possible disease states. In this review, we examine the progress that has been made in the development of luminescent probes for Group I and Group II ions as well as protons. The potential applications of these probes and the mechanism involved in controlling their luminescent response upon analyte binding will also be discussed.

Microwave-assisted Hantzsch thiazole synthesis of N-phenyl-4-(6-phenylimidazo[2,1-b]thiazol-5-yl)thiazol-2-amines from the reaction of 2-chloro-1-(6-phenylimidazo[2,1-b]thiazol-5-yl)ethanones and thioureas.

N-Phenyl-4-(6-phenylimidazo[2,1-b]thiazol-5-yl)thiazol-2-amines (6a-q) have been synthesized by the Hantzsch thiazole reaction of 2-chloro-1-(6-phenylimidazo[2,1-b]thiazol-5-yl)ethanones (4a-e) with suitably substituted thioureas using microwave heating. The ethanones (4a-e) were prepared by the reaction of 6-phenylimidazo[2,1-b]thiazoles (3a-e) with chloroacetylchloride in refluxing 1,4-dioxane whereas the thiazoles (3a-e) were synthesized by the reaction of 2-bromo-1-phenylethanones (2a-e) with thiazol-2-amine in refluxing acetone.

An efficient microwave assisted synthesis of novel class of Rhodanine derivatives as potential HIV-1 and JSP-1 inhibitors.

(Z)-5-(2-(1H-Indol-3-yl)-2-oxoethylidene)-3-phenyl-2-thioxothiazolidin-4-one (7a-q) derivatives have been synthesized by the condensation reaction of 3-phenyl-2-thioxothiazolidin-4-ones (3a-h) with suitably substituted 2-(1H-indol-3-yl)-2-oxoacetaldehyde (6a-d) under microwave condition. The thioxothiazolidine-4-ones were prepared from corresponding aromatic amines (1a-e) and di-(carboxymethyl)-trithiocarbonyl (2). The aldehydes (6a-h) were synthesized from the corresponding acidchlorides (5a-d) using HSnBu(3).

Microwave assisted synthesis of novel functionalized hydantoin derivatives and their conversion to 5-(Z) arylidene-4H-imidazoles.

2-(Alkyl-1-yl)-1H-imidazol-5(4H)-ones 5a-n were synthesized via nucleophilic substitution of the methylsulfanyl group of the corresponding 2-(methylthio)-1H-imidazol-5(4H)-ones 3a-c with suitably substituted secondary amines. The starting 2-thioxo- imidazolidin-4-ones 2a,2b were prepared by condensation of thiohydantoin and benzo[b]-thiophene-3-carbaldehyde or benzofuran-3-carbaldehyde under microwave irracdiation (MW) conditions. 2-Methylthio derivatives 3a-c were prepared by treatment of 2a-b with methyl iodide in the presence of aqueous sodium hydroxide.

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.

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.

Magnetic microbubble mediated chemo-sonodynamic therapy using a combined magnetic-acoustic device.

Recent pre-clinical studies have demonstrated the potential of combining chemotherapy and sonodynamic therapy for the treatment of pancreatic cancer. Oxygen-loaded magnetic microbubbles have been explored as a targeted delivery vehicle for this application. Despite preliminary positive results, a previous study identified a significant practical challenge regarding the co-alignment of the magnetic and ultrasound fields. The aim of this study was to determine whether this challenge could be addressed through the use of a magnetic-acoustic device (MAD) combining a magnetic array and ultrasound transducer in a single unit, to simultaneously concentrate and activate the microbubbles at the target site. in vitro experiments were performed in tissue phantoms and followed by in vivo treatment of xenograft pancreatic cancer (BxPC-3) tumours in a murine model. In vitro, a 1.4-fold (p < .01) increase in the deposition of a model therapeutic payload within the phantom was achieved using the MAD compared to separate magnetic and ultrasound devices. In vivo, tumours treated with the MAD had a 9% smaller mean volume 8 days after treatment, while tumours treated with separate devices or microbubbles alone were respectively 45% and 112% larger. This substantial and sustained decrease in tumour volume suggests that the proposed drug delivery approach has the potential to be an effective neoadjuvant therapy for pancreatic cancer patients.

Targeted chemo-sonodynamic therapy treatment of breast tumours using ultrasound responsive microbubbles loaded with paclitaxel, doxorubicin and Rose Bengal.

Mastectomy is a common surgical treatment used in the management of breast cancer but has associated physical and psychological consequences for the patient. Breast conservation surgery (BCS) is an alternative to mastectomy but is only possible when the tumour is of an appropriate size. Neo-adjuvant chemotherapy has been successfully used to downstage tumours and increase the number of patients eligible for BCS. However, the chemotherapies used in this approach are non-targeted and often result in significant side effects to the patient. In this manuscript, we evaluate the potential of ultrasound targeted microbubble destruction (UTMD) to deliver Rose Bengal-mediated sonodynamic therapy (SDT) in combination with paclitaxel (PTX) and doxorubicin (Dox) chemotherapy as a potential treatment for breast cancer. Efficacy of the combined treatment was determined in a three-dimensional (3D) spheroid model of human breast cancer and in a murine model of the disease bearing subcutaneous MCF-7 tumours. The results demonstrated a significant reduction in both the cell viability of spheroids and tumour volume following treatment with the drug loaded microbubbles and ultrasound compared to targets treated with the drug loaded microbubbles alone or a Cremophor EL suspension of PTX and Dox. In addition, the weight of animals that received the microbubble treatment was unchanged throughout the study while a reduction of 12.1% was observed for animals treated with a Cremophor suspension of PTX/Dox. These results suggest that UTMD-mediated chemo-sonodynamic therapy is an efficacious and well tolerated approach for the treatment of breast cancer.

Synthesis and structure-activity relationship studies of 3-substituted indolin-2-ones as effective neuroprotective agents.

Neurodegenerative diseases are a major health problem particularly among the elderly. Drugs to prevent or slow down the death of neurons are urgently needed but are currently unavailable. We previously reported that the c-Raf inhibitor, GW5074 {5-iodo-3-[(3',5'-dibromo-4'-hydroxyphenyl) methylene]-2-indolinone}, is protective in tissue culture and in vivo paradigms of neurodegeneration. However, at doses slightly higher than those at which it is protective, GW5074 displays toxicity when tested in neuronal cultures. We report herein the synthesis, biological evaluation, and structure-activity relationship (SAR) studies of novel 3-substituted indolin-2-one compounds that are highly neuroprotective but lack the toxicity of GW5074. Of the 45 analogs tested in this study, compounds 7, 37, 39, and 45 were found to be the most potent neuroprotective and thus represent promising lead compounds for preclinical development for the treatment of neurodegenerative disorders.

Gemcitabine loaded microbubbles for targeted chemo-sonodynamic therapy of pancreatic cancer.

Pancreatic cancer remains one of the most lethal forms of cancer with a 10-year survival of <1%. With little improvement in survival rates observed in the past 40 years, there is a significant need for new treatments or more effective strategies to deliver existing treatments. The antimetabolite gemcitabine (Gem) is the most widely used form of chemotherapy for pancreatic cancer treatment, but is known to produce significant side effects when administered systemically. We have previously demonstrated the benefit of combined chemo-sonodynamic therapy (SDT), delivered using oxygen carrying microbubbles (O2MB), as a targeted treatment for pancreatic cancer in a murine model of the disease. In this manuscript, we report the preparation of a biotin functionalised Gem ligand for attachment to O2MBs (O2MB-Gem). We demonstrate the effectiveness of chemo-sonodynamic therapy following ultrasound-targeted-microbubble-destruction (UTMD) of the O2MB-Gem and a Rose Bengal loaded O2MB (O2MB-RB) as a targeted treatment for pancreatic cancer. Specifically, UTMD using the O2MB-Gem and O2MB-RB conjugates reduced the viability of MIA PaCa-2, PANC-1, BxPC3 and T110299 pancreatic cancer cells by >60% (p < 0.001) and provided significant tumour growth delay (>80%, p < 0.001) compared to untreated animals when human xenograft MIA PaCa-2 tumours were treated in SCID mice. The toxicity of the O2MB-Gem conjugate was also determined in healthy non-tumour bearing MF1 mice and revealed no evidence of renal or hepatic damage. Therefore, the results presented in this manuscript suggest that chemo-sonodynamic therapy using the O2MB-Gem and O2MB-RB conjugates, is potentially an effective targeted and safe treatment modality for pancreatic cancer.

Iodinated cyanine dyes: a new class of sensitisers for use in NIR activated photodynamic therapy (PDT).

A new class of iodinated cyanine dyes have been prepared for use in NIR excited photodynamic therapy (PDT) and demonstrated improved efficacy in two pancreatic cell lines as well as excellent tumour control in a murine model of the disease.

Magnetically responsive microbubbles as delivery vehicles for targeted sonodynamic and antimetabolite therapy of pancreatic cancer.

Magnetically responsive microbubbles (MagMBs), consisting of an oxygen gas core and a phospholipid coating functionalised with Rose Bengal (RB) and/or 5-fluorouracil (5-FU), were assessed as a delivery vehicle for the targeted treatment of pancreatic cancer using combined antimetabolite and sonodynamic therapy (SDT). MagMBs delivering the combined 5-FU/SDT treatment produced a reduction in cell viability of over 50% when tested against a panel of four pancreatic cancer cell lines in vitro. Intravenous administration of the MagMBs to mice bearing orthotopic human xenograft BxPC-3 tumours yielded a 48.3% reduction in tumour volume relative to an untreated control group (p<0.05) when the tumour was exposed to both external magnetic and ultrasound fields during administration of the MagMBs. In contrast, application of an external ultrasound field alone resulted in a 27% reduction in tumour volume. In addition, activated caspase and BAX protein levels were both observed to be significantly elevated in tumours harvested from animals treated with the MagMBs in the presence of magnetic and ultrasonic fields when compared to expression of those proteins in tumours from either the control or ultrasound field only groups (p<0.05). These results suggest MagMBs have considerable potential as a platform to enable the targeted delivery of combined sonodynamic/antimetabolite therapy in pancreatic cancer.

Unexpected stereorecognition in nitrilase-catalyzed hydrolysis of beta-hydroxy nitriles.

Biocatalytic enantioselective hydrolysis of beta-hydroxy nitriles to corresponding (S)-enriched beta-hydroxy carboxylic acids has been achieved for the first time by an isolated nitrilase bll6402 from Bradyrhizobium japonicum USDA110. This offers a new "green" approach to optically pure beta-hydroxy nitriles and beta-hydroxy carboxylic acids. The observed remote stereorecognition is surprising because this nitrilase shows no enantioselectivity for the hydrolysis of alpha-hydroxy nitriles such as mandelonitrile.

Ring expansion/homologation--aldehyde condensation cascade using tert-trihalomethylcarbinols.

Treatment of cyclic tert-trihalomethylcarbinols with CrCl(2) in THF/HMPA in the presence of aryl or aliphatic aldehydes initiates a cascade sequence of one carbon ring expansion-olefination affording conjugated exocyclic ketones. Acyclic tert-trihalomethylcarbinols undergo a comparable cascade of one carbon homologation-olefination.

Combined sonodynamic and antimetabolite therapy for the improved treatment of pancreatic cancer using oxygen loaded microbubbles as a delivery vehicle.

In this manuscript we describe the preparation of an oxygen-loaded microbubble (O2MB) platform for the targeted treatment of pancreatic cancer using both sonodynamic therapy (SDT) and antimetabolite therapy. O2MB were prepared with either the sensitiser Rose Bengal (O2MB-RB) or the antimetabolite 5-fluorouracil (O2MB-5FU) attached to the microbubble (MB) surface. The MB were characterised with respect to size, physical stability and oxygen retention. A statistically significant reduction in cell viability was observed when three different pancreatic cancer cell lines (BxPc-3, MIA PaCa-2 and PANC-1), cultured in an anaerobic cabinet, were treated with both SDT and antimetabolite therapy compared to either therapy alone. In addition, a statistically significant reduction in tumour growth was also observed when ectopic human xenograft BxPC-3 tumours in SCID mice were treated with the combined therapy compared to treatment with either therapy alone. These results illustrate not only the potential of combined SDT/antimetabolite therapy as a stand alone treatment option in pancreatic cancer, but also the capability of O2-loaded MBs to deliver O2 to the tumour microenvironment in order to enhance the efficacy of therapies that depend on O2 to mediate their therapeutic effect. Furthermore, the use of MBs to facilitate delivery of O2 as well as the sensitiser/antimetabolite, combined with the possibility to activate the sensitiser using externally applied ultrasound, provides a more targeted approach with improved efficacy and reduced side effects when compared with conventional systemic administration of antimetabolite drugs alone.

Modulation of ROS production in photodynamic therapy using a pH controlled photoinduced electron transfer (PET) based sensitiser.

A new sensitiser (4) for use in photodynamic therapy (PDT) has been developed to enable control of ROS production as a function of pH. This pH dependent PDT behaviour was tested in HeLa cells and in SCID mice bearing human xenograft pancreatic cancer (BxPC-3) tumours.

Development of inducible leucine-rich repeat kinase 2 (LRRK2) cell lines for therapeutics development in Parkinson's disease.

The pathogenic mechanism(s) contributing to loss of dopamine neurons in Parkinson's disease (PD) remain obscure. Leucine-rich repeat kinase 2 (LRRK2) mutations are linked, as a causative gene, to PD. LRRK2 mutations are estimated to account for 10% of familial and between 1 % and 3 % of sporadic PD. LRRK2 proximate single nucleotide polymorphisms have also been significantly associated with idiopathic/sporadic PD by genome-wide association studies. LRRK2 is a multidomain-containing protein and belongs to the protein kinase super-family. We constructed two inducible dopaminergic cell lines expressing either human-LRRK2-wild-type or human-LRRK2-mutant (G2019S). Phenotypes of these LRRK2 cell lines were examined with respect to cell viability, morphology, and protein function with or without induction of LRRK2 gene expression. The overexpression of G2019S gene promoted (1) low cellular metabolic activity without affecting cell viability, (2) blunted neurite extension, and (3) increased phosphorylation at S910 and S935. Our observations are consistent with reported general phenotypes in LRRK2 cell lines by other investigators. We used these cell lines to interrogate the biological function of LRRK2, to evaluate their potential as a drug-screening tool, and to investigate screening for small hairpin RNA-mediated LRRK2 G2019S gene knockdown as a potential therapeutic strategy. A proposed LRRK2 kinase inhibitor (i.e., IN-1) decreased LRRK2 S910 and S935 phosphorylation in our MN9DLRRK2 cell lines in a dose-dependent manner. Lentivirus-mediated transfer of LRRK2 G2019S allele-specific small hairpin RNA reversed the blunting of neurite extension caused by LRRK2 G2019S overexpression. Taken together, these inducible LRRK2 cell lines are suitable reagents for LRRK2 functional studies, and the screening of potential LRRK2 therapeutics.