Ultrasound targeted microbubble destruction using docetaxel and Rose Bengal loaded Microbubbles for targeted Chemo-Sonodynamic therapy treatment of prostate cancer.

Docetaxel (DTX) chemotherapy is commonly used in the treatment of patients with advanced prostate cancer demonstrating modest improvements in survival. As these patients are often elderly and the chemotherapy treatment is not targeted, it is often poorly tolerated. More targeted approaches that increase therapeutic efficacy yet reduce the amount of toxic chemotherapy administered are needed. In this manuscript, we investigate the potential of ultrasound targeted microbubble destruction (UTMD) to deliver a combination of docetaxel chemotherapy and Rose Bengal mediated sonodynamic therapy (SDT) in pre-clinical prostate cancer models. A Rose Bengal modified phospholipid was synthesized and used as a component lipid to prepare a microbubble (MB) formulation that was also loaded with DTX. The DTX-MB-RB formulation was used in the UTMD mediated treatment of androgen sensitive and androgen resistant 3D spheroid and murine models of prostate cancer. Results from the 3D spheroid experiments showed UTMD mediated DTX-MB-RB chemo-sonodynamic therapy to be significantly more effective at reducing cell viability than UTMD mediated DTX or SDT treatment alone. In an androgen sensitive murine model of prostate cancer, UTMD mediated DTX-MB-RB chemo-sonodynamic therapy was as effective as androgen deprivation therapy (ADT) at controlling tumour growth. However, when both treatments were combined, a significant improvement in tumour growth delay was observed. In an androgen resistant murine model, UTMD mediated DTX-MB-RB chemo-sonodynamic therapy was significantly more effective than standard DTX monotherapy. Indeed, the DTX dose administered using the DTX-MB-RB formulation was 91% less than standard DTX monotherapy. As a result, UTMD mediated DTX-MB-RB treatment was well tolerated while animals treated with DTX monotherapy displayed significant weight loss which was attributed to acute toxic effects. These results highlight the potential of UTMD mediated DTX-MB-RB chemo-sonodynamic therapy as a targeted, well tolerated alternative treatment for advanced prostate cancer.

Non-Invasive, Targeted Nanoparticle-Mediated Drug Delivery across a Novel Human BBB Model.

The blood-brain barrier (BBB) is a highly sophisticated system with the ability to regulate compounds transporting through the barrier and reaching the central nervous system (CNS). The BBB protects the CNS from toxins and pathogens but can cause major issues when developing novel therapeutics to treat neurological disorders. PLGA nanoparticles have been developed to successfully encapsulate large hydrophilic compounds for drug delivery. Within this paper, we discuss the encapsulation of a model compound Fitc-dextran, a large molecular weight (70 kDa), hydrophilic compound, with over 60% encapsulation efficiency (EE) within a PLGA nanoparticle (NP). The NP surface was chemically modified with DAS peptide, a ligand that we designed which has an affinity for nicotinic receptors, specifically alpha 7 nicotinic receptors, found on the surface of brain endothelial cells. The attachment of DAS transports the NP across the BBB by receptor-mediated transcytosis (RMT). Assessment of the delivery efficacy of the DAS-conjugated Fitc-dextran-loaded PLGA NP was studied in vitro using our optimal triculture in vitro BBB model, which successfully replicates the in vivo BBB environment, producing high TEER (≥230 ) and high expression of ZO1 protein. Utilising our optimal BBB model, we successfully transported fourteen times the concentration of DAS-Fitc-dextran-PLGA NP compared to non-conjugated Fitc-dextran-PLGA NP. Our novel in vitro model is a viable method of high-throughput screening of potential therapeutic delivery systems to the CNS, such as our receptor-targeted DAS ligand-conjugated NP, whereby only lead therapeutic compounds will progress to in vivo studies.

A single microbubble formulation carrying 5-fluorouridine, Irinotecan and oxaliplatin to enable FOLFIRINOX treatment of pancreatic and colon cancer using ultrasound targeted microbubble destruction.

FOLFIRINOX and FOLFOXIRI are combination chemotherapy treatments that incorporate the same drug cocktail (folinic acid, 5-fluorouracil, oxaliplatin and irinotecan) but exploit an altered dosing regimen when used in the management of pancreatic and colorectal cancer, respectively. Both have proven effective in extending life when used to treat patients with metastatic disease but are accompanied by significant adverse effects. To facilitate improved tumour-targeting of this drug combination, an ultrasound responsive microbubble formulation loaded with 5-fluorouridine, irinotecan and oxaliplatin (FIRINOX MB) was developed and its efficacy tested, together with the non-toxic folinic acid, in preclinical murine models of pancreatic and colorectal cancer. A significant improvement in tumour growth delay was observed in both models following ultrasound targeted microbubble destruction (UTMD) mediated FIRINOX treatment with pancreatic tumours 189% and colorectal tumours 82% smaller at the conclusion of the study when compared to animals treated with a standard dose of FOLFIRINOX. Survival prospects were also improved for animals in the UTMD mediated FIRINOX treatment group with an average survival of 22.17 ± 12.19 days (pancreatic) and 44.40 ± 3.85 days (colorectal) compared to standard FOLFIRINOX treatment (15.83 ± 4.17 days(pancreatic) and 37.50 ± 7.72 days (colon)). Notably, this improved efficacy was achieved using FIRINOX MB that contained 5-fluorouricil, irinotecan and oxaliplatin loadings that were 13.44-fold, 9.19-fold and 1.53-fold lower than used for the standard FOLFIRINOX treatment. These results suggest that UTMD enhances delivery of FIRINOX chemotherapy, making it significantly more effective at a substantially lower dose. In addition, the reduced systemic levels of 5-fluorouracil, irinotecan and oxaliplatin should also make the treatment more tolerable and reduce the adverse effects often associated with this treatment.

Sonodynamic therapy complements PD-L1 immune checkpoint inhibition in a murine model of pancreatic cancer.

The emergence of immune checkpoint inhibitors (ICI's) in the past decade has proven transformative in the area of immuno-oncology. The PD-1/PD-L1 axis has been particularly well studied and monoclonal antibodies developed to block either the receptor (anti PD-1) or its associated ligand (anti PD-L1) can generate potent anti-tumour immunity in certain tumour models. However, many "immune cold" tumours remain unresponsive to ICI's and strategies to stimulate the adaptive immune system and make these tumours more susceptible to ICI treatment are currently under investigation. Sonodynamic therapy (SDT) is a targeted anti-cancer treatment that uses ultrasound to activate a sensitiser with the resulting generation of reactive oxygen species (ROS) causing direct cell death by apoptosis and necrosis. SDT has also been shown to stimulate the adaptive immune system in a pre-clinical model of colorectal cancer. In this manuscript, we investigate the ability of microbubble mediated SDT to control tumour growth in a bilateral tumour mouse model of pancreatic cancer by treating the target tumour with SDT and observing the effects at the off-target untreated tumour. The results demonstrated a significant 287% decrease in tumour volume when compared to untreated animals 11 days following the initial treatment with SDT, which reduced further to 369% when SDT was combined with anti-PD-L1 ICI treatment. Analysis of residual tumour tissues remaining after treatment revealed increased levels of infiltrating CD4+ and CD8+ T-lymphocytes (respectively 4.65 and 3.16-fold more) in the off-target tumours of animals where the target tumour was treated with SDT and anti-PD-L1, when compared to untreated tumours. These results suggest that SDT treatment elicits an adaptive immune response that is potentiated by the anti-PD-L1 ICI in this particular model of pancreatic cancer.

Synthesis of a gemcitabine-modified phospholipid and its subsequent incorporation into a single microbubble formulation loaded with paclitaxel for the treatment of pancreatic cancer using ultrasound-targeted microbubble destruction.

Gemcitabine and nab-paclitaxel (Abraxane®) is a standard of care chemotherapy combination used in the treatment of patients with advanced pancreatic cancer. While the combination has shown a survival benefit when compared to gemcitabine monotherapy, it is associated with significant off-target toxicity. Ultrasound targeted microbubble destruction (UTMD) has emerged as an effective strategy for the site-specific deposition of drug-payloads. However, loading a single microbubble formulation with two drug payloads can be challenging and often involves several manipulations post-microbubble preparation that can be cumbersome and generally results in low / inconsistent drug loadings. In this manuscript, we report the one-pot synthesis of a gemcitabine functionalised phospholipid and use it to successfully generate stable microbubble formulations loaded with gemcitabine (Lipid-Gem MB) or a combination of gemcitabine and paclitaxel (Lipid-Gem-PTX MB). Efficacy of the Lipid-Gem MB and Lipid-Gem-PTX MB formulations, following ultrasound (US) stimulation, was evaluated in a three-dimensional (3D) PANC-1 spheroid model of pancreatic cancer and a mouse model bearing ectopic BxPC-3 tumours. The results demonstrated a significant reduction in the cell viability in spheroids for both formulations reducing from 90 ± 10% to 62 ± 5% for Lipid-Gem MB and 84 ± 10% to 30 ± 6% Lipid-Gem-PTX MB following US irradiation. When compared with a clinically relevant dose of free gemcitabine and paclitaxel (i.e. non-particle bound) in a BxPC-3 murine pancreatic tumour model, both formulations also improved tumour growth delay with tumours 40 ± 20% and 40 ± 30% smaller than the respective free drug formulation when treated with Lipid-Gem MB and Lipid-Gem-PTX MB respectively, at the conclusion of the experiment. These results highlight the potential of UTMD mediated Gem / PTX as a treatment for pancreatic cancer and the facile preparation of Lipid-Gem-PTX MBs using a gemcitabine functionalised lipid should expedite clinical translation of this technology.

Exploiting a Rose Bengal-bearing, oxygen-producing nanoparticle for SDT and associated immune-mediated therapeutic effects in the treatment of pancreatic cancer.

Sonodynamic therapy (SDT) is an emerging stimulus-responsive approach for the targeted treatment of solid tumours. However, its ability to generate stimulus-responsive cytotoxic reactive oxygen species (ROS), is compromised by tumour hypoxia. Here we describe a robust means of preparing a pH-sensitive polymethacrylate-coated CaO2 nanoparticle that is capable of transiently alleviating tumour hypoxia. Systemic administration of particles to animals bearing human xenograft BxPC3 pancreatic tumours increases oxygen partial pressures (PO2) to 20-50 mmHg for over 40 min. RT-qPCR analysis of expression of selected tumour marker genes in treated animals suggests that the transient production of oxygen is sufficient to elicit effects at a molecular genetic level. Using particles labelled with the near infra-red (nIR) fluorescent dye, indocyanine green, selective uptake of particles by tumours was observed. Systemic administration of particles containing Rose Bengal (RB) at concentrations of 0.1 mg/mg of particles are capable of eliciting nanoparticle-induced, SDT-mediated antitumour effects using the BxPC3 human pancreatic tumour model in immuno-compromised mice. Additionally, a potent abscopal effect was observed in off-target tumours in a syngeneic murine bilateral tumour model for pancreatic cancer and an increase in tumour cytotoxic T cells (CD8+) and a decrease in immunosuppressive tumour regulatory T cells [Treg (CD4+, FoxP3+)] was observed in both target and off-target tumours in SDT treated animals. We suggest that this approach offers significant potential in the treatment of both focal and disseminated (metastatic) pancreatic cancer.

An ultrasound responsive microbubble-liposome conjugate for targeted irinotecan-oxaliplatin treatment of pancreatic cancer.

Survival rates in pancreatic cancer have remained largely unchanged over the past four decades with less than 5% of patients surviving five years following initial diagnosis. FOLFIRINOX chemotherapy, a combination of folinic acid, 5-fluoruracil, irinotecan and oxaliplatin, has shown the greatest survival benefit for patients with advanced disease but is only indicated for those with good physical performance status due to its extreme off-target toxicity. Ultrasound targeted microbubble destruction (UTMD) has emerged as an effective strategy for the targeted delivery of drug payloads to solid tumours and involves using low intensity ultrasound to disrupt (burst) MBs in the tumour vasculature, releasing encapsulated or attached drugs in a targeted manner. In this manuscript, we describe the preparation of a microbubble-liposome complex (IRMB-OxLipo) carrying two of the three cytotoxic drugs present in the FOLFIRINOX combination, namely irinotecan and oxaliplatin. Efficacy of the IRMB-OxLipo complex following UTMD was determined in Panc-01 3D spheroid and BxPC-3 human xenograft murine models of pancreatic cancer. The results revealed that tumours treated with the IRMB-OxLipo complex and ultrasound were 136% smaller than tumours treated with the same concentration of irinotecan/oxaliplatin but delivered in a conventional manner, i.e. as a non-complexed mixture. This suggests that UTMD facilitates a more effective delivery of irinotecan/oxaliplatin improving the overall effectiveness of this drug combination and to the best of our knowledge, is the first reported example of a microbubble-liposome complex used to deliver these two chemotherapies.

Liposome mimicking polymersomes; A comparative study of the merits of polymersomes in terms of formulation and stability.

The past few decades have seen a significant rise in research into alternative polymer based nanosized unilamellar drug delivery systems, termed polymersomes. The reported benefits of polymersomes over the more traditional liposomes include increased stability, higher encapsulation efficacies, better adaptability and reduced water permeation due to an increased bilayer thickness. Together, these advantages render them suitable for a plethora of therapies. The work presented in this manuscript creates and compares four such drug delivery systems, two based on the traditional liposome and two prepared from amphiphilic polymers. From there we assess these systems in terms of size, stability, encapsulation efficiency, drug release, cellular toxicity and cellular uptake. We can confirm from this comprehensive investigation that the multi-functional synthetic polymersomes are undoubtedly a future contender in this expanding field of nanomedicines. Their ability to encapsulate a cocktail of different compounds, high stability as well as their ease of adaptability will ensure that they feature prominently in the future of advanced drug delivery systems.

Rose Bengal-Amphiphilic Peptide Conjugate for Enhanced Photodynamic Therapy of Malignant Melanoma.

Malignant melanoma is an aggressive skin cancer with poor survival outcomes for patients diagnosed at an advanced stage. While targeted serine/threonine-protein kinase B-Raf (BRAF) and immune checkpoint inhibitors have improved survival outcomes for a proportion of these patients, response rates remain variable. There is a need, therefore, for more effective treatments to bolster the options available for melanoma patients. In this manuscript, we covalently attached Rose Bengal (RB) to the amphipathic peptide (AMP) C(KLAKLAK)2 and determined the effectiveness of the resulting RB-C(KLAKLAK)2 conjugate as a photodynamic therapy (PDT) sensitizer. RB-C(KLAKLAK)2-mediated PDT treatment of subcutaneous B16-F10-Luc2 tumors in C57 mice resulted in lesions that were 479% smaller at the end of the study than animals treated with RB-mediated PDT. The synergistic effect between RB and C(KLAKLAK)2 has been attributed to the AMP sensitizing cells to reactive oxygen species (ROS), making them more susceptible to ROS-induced oxidative stress.

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.

Electroneutral polymersomes for combined cancer chemotherapy.

Combination cancer chemotherapy provides an important treatment tool, both as an adjuvant and neoadjuvant treatment, this shift in focus from mono to combination therapies has led to increased interest in drug delivery systems (DDS). DDSs, such as polymersomes, are capable of encapsulating large amounts of multiple drugs with both hydrophilic and hydrophobic properties simultaneously, as well as offering a mechanism to combat multi drug resistant cancers and poor patient tolerance of the cytotoxic compounds utilised. In this article, we report the formulation and evaluation of a novel electroneutral polymersome capable of high encapsulation efficacies for multiple drugs (Doxorubicin, 5-Fluorouracil and leucovorin). The in-vivo biodistribution of the polymersome were established and they were found to accumulate largely in tumour tissue. Polymersome encapsulating the three chemotherapeutic drugs were assessed both in-vitro (BxPC-3 cell line) and in-vivo (following intratumoral and intravenous administration) and compared with the same concentration of the three drugs in solution. We report better efficacy and higher maximum tolerated dose for our combination drug loaded polymersomes in all experiments. Furthermore, intratumorally injected combination drug loaded polymersomes exhibited a 62% reduction in tumour volume after 13 days when compared with the free combination solutions. A smaller differential of 13% was observed for when treatment was administered intravenously however, importantly less cardiotoxicity was displayed from the polymersomal DDS. In this study, expression of a number of survival-relevant genes in tumours treated with the free chemotherapy combination was compared with expression of those genes in tumours treated with the polymersomes harbouring those drugs and the significance of findings is discussed. STATEMENT OF SIGNIFICANCE: The shift in focus from mono to combination chemotherapies has led to an increased interest in the role of drug delivery systems (DDS). Liposomes, although commercialized for mono therapy, have lower loading capacities and stability than their polymeric counterpart, polymersomes. Polymersomes are growing in prevalence as their advantageous properties are better understood and exploited. Here we present a novel polymersome for the encapsulation of three anticancer compounds. This is the first time this particular polymersome has been used to encapsulate these three compounds with both an in-vitro and in-vivo evaluation carried out. This work will be of interest to those in the field of combination therapy, drug delivery, drug toxicity, multidrug resistance, liposomes, DDS and polymersomes.

Rapid paper based colorimetric detection of glucose using a hollow microneedle device.

The monitoring of blood glucose is a key aspect of diabetes care in limiting the negative effects of hyperglycaemia to both the microvasculature and macrovasculature. Self-monitoring of blood glucose (SMBG) gives an indication of blood glucose at a specific point in time and is recommended to be carried out four times daily. However, due to the inconvenience and associated pain of blood withdrawal, SMBG is often carried out less frequently than recommended or not at all. Extraction and subsequent determination of glucose in interstitial fluid (ISF) using microneedles (MNs) is an emerging area of research due to their minimally invasive nature and lack of associated pain. In this manuscript, a novel method for the fabrication of a hollow microneedle device is reported. The microneedle produced had a sharp bevelled edge and was 400 µm in length. Additionally, a paper backplate embedded with a colorimetric system for the rapid visual determination of glucose in simulated ISF was developed and paired with the hollow MN. This device rapidly extracted simulated ISF within five seconds and its ability to produce a glucose concentration dependent colour change within 30 s was demonstrated. Using this approach, it was possible to discriminate between glucose concentrations in normal glycaemia (4-7 mM) and hyperglycaemia (>7 mM) ranges using the naked eye. While further development is required, the results herein highlight the potential of this device to be used as a blood-free minimally invasive approach to glucose monitoring.

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.

A descriptive systematic review of salivary therapeutic drug monitoring in neonates and infants.

AIMS: Saliva, as a matrix, offers many benefits over blood in therapeutic drug monitoring (TDM), in particular for infantile TDM. However, the accuracy of salivary TDM in infants remains an area of debate. This review explored the accuracy, applicability and advantages of using saliva TDM in infants and neonates. METHODS: Databases were searched up to and including September 2016. Studies were included based on PICO as follows: P: infants and neonates being treated with any medication, I: salivary TDM vs. C: traditional methods and O: accuracy, advantages/disadvantages and applicability to practice. Compounds were assessed by their physicochemical and pharmacokinetic properties, as well as published quantitative saliva monitoring data. RESULTS: Twenty-four studies and their respective 13 compounds were investigated. Four neutral and two acidic compounds, oxcarbazepine, primidone, fluconazole, busulfan, theophylline and phenytoin displayed excellent/very good correlation between blood plasma and saliva. Lamotrigine was the only basic compound to show excellent correlation with morphine exhibiting no correlation between saliva and blood plasma. Any compound with an acid dissociation constant (pKa) within physiological range (pH 6-8) gave a more varied response. CONCLUSION: There is significant potential for infantile saliva testing and in particular for neutral and weakly acidic compounds. Of the properties investigated, pKa was the most influential with both logP and protein binding having little effect on this correlation. To conclude, any compound with a pKa within physiological range (pH 6-8) should be considered with extra care, with the extraction and analysis method examined and optimized on a case-by-case basis.

Oxygen generating nanoparticles for improved photodynamic therapy of hypoxic tumours.

Photodynamic therapy (PDT) is a clinically approved anti-cancer treatment that involves the activation of an otherwise inactive sensitiser drug with light, which in the presence of molecular oxygen, generates cytotoxic reactive oxygen species (ROS). As oxygen is a key requirement for the generation of ROS in PDT and given the fact that hypoxia is a characteristic of most solid cancerous tumours, treating hypoxic tumours using PDT can be a challenge. In this manuscript, we have prepared a CaO2 nanoparticle (NP) formulation coated with a pH-sensitive polymer to enable the controlled generation of molecular oxygen as a function of pH. The polymer coat was designed to protect the particles from decomposition while in circulation but enable their activation at lower pH values in hypoxic regions of solid tumours. The oxygen generating capability of the polymer coated NPs was demonstrated in aqueous solution with minimal oxygen produced at pH7.4, whereas it increased significantly when the pH was reduced to 6.2. The polymer coated CaO2 NPs were also observed to significantly increase tumour pO2 levels (p<0.05) in mice bearing ectopic human xenograft MIA PaCa-2 pancreatic tumours with an average increase in tumour pO2 of 6.5mmHg in the period 10-30min following administration. A statistically significant improvement in PDT mediated efficacy (p<0.001) was also observed when the particles were administered to mice bearing the same tumours 20min prior to PDT treatment. These results suggest that the polymer coated CaO2 NP formulation offers significant potential as an in situ method for oxygen generation to enhance the efficacy of treatments that depend on the presence of oxygen to elicit a cytotoxic effect.

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.

The integration of triggered drug delivery with real time quantification using FRET; creating a super 'smart' drug delivery system.

The ability to control drug release at a specific physiological target enables the possibility of an enhanced therapeutic effect with reduced off-target toxic side effects. The discipline of controlled drug release has grown to include most areas of medicine with examples in the literature of targeted drug delivery to the majority of organs within the human body. In addition, a variety of external stimuli used to meditate the drug release process have also been investigated. Nonetheless, the concurrent real time monitoring of drug release has not been widely studied. In this manuscript, we present a novel micellar drug delivery system that is not only capable of releasing its cargo when stimulated by light but also provides a real time analysis of the amount of cargo remaining. Controlled drug release from the delivery system was mediated by physicochemical changes of a spiropyran-merocyanine photochromic dyad, while drug quantification was enabled using a Förster Resonance Energy Transfer (FRET) relationship between the photochrome and a co-encapsulated BODIPY fluorophore. The percentage of drug released from the delivery system was significantly greater (24%) when exposed to light irradiation compared to an analogous control maintained in the dark (5%). Furthermore, the fluorescence read-out capability also enabled the drug-release process to be followed in living cells with a significantly reduced fluorescence emission observed for those cells incubated with the delivery system and exposed to light irradiation compared to control cells maintained in the dark. Combined, these results highlight the utility of this approach to theranostic drug delivery with the potential of light-triggered released together with a fluorescence read-out to enable quantification of the drug release process.

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.

Cholesteryl to improve the cellular uptake of polymersomes within HeLa cells.

The need to develop a greater understanding of drug delivery systems has arisen through the development of alternative biological based therapeutics. Drug delivery systems need to adapt and respond to this increasing demand for cellular transportation of highly charged species. Polymersomal drug delivery systems have displayed great potential and versatility for such a task. In this manuscript we present the synthesis, characterisation and biological evaluation of six amphiphilic random co polymers with varying amounts of cholesteryl (0-39%wt) before the subsequent formation into polymersomes. The polymersomes were then analysed for size, zeta potential, encapsulation efficiency, release kinetics and cellular uptake. Results confirmed that the polymersome containing 12%wt cholesteryl polymer displayed a ten-fold increase in cellular uptake of Fitc-CM-dextran when compared to un-encapsulated drug, crossing the cellular membrane via endocytosis. The size of these vehicles ranged between 100 and 500nm, zeta potential was shown to be neutral at -0.82mV ±0.2 with encapsulation efficiencies in the region of 60%. The ease of adaptability and preparation of such systems renders them a viable alternative to liposomal drug delivery systems.

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.