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.

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.

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.

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.

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.

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.

Supramolecular nanoreactors for intracellular singlet-oxygen sensitization.

An amphiphilic polymer with multiple decyl and oligo(ethylene glycol) chains attached to a common poly(methacrylate) backbone assembles into nanoscaled particles in aqueous environments. Hydrophobic anthracene and borondipyrromethene (BODIPY) chromophores can be co-encapsulated within the self-assembling nanoparticles and transported across hydrophilic media. The reversible character of the noncovalent bonds, holding the supramolecular containers together, permits the exchange of their components with fast kinetics in aqueous solution. Incubation of cervical cancer (HeLA) cells with a mixture of two sets of nanoparticles, pre-loaded independently with anthracene or BODIPY chromophores, results in guest scrambling first and then transport of co-entrapped species to the intracellular space. Alternatively, incubation of cells with the two sets of nanocarriers in consecutive steps permits the sequential transport of the anthracene and BODIPY chromophores across the plasma membrane and only then allows their co-encapsulation within the same supramolecular containers. Both mechanisms position the two sets of chromophores with complementary spectral overlap in close proximity to enable the efficient transfer of energy intracellularly from the anthracene donors to the BODIPY acceptors. In the presence of iodine substituents on the BODIPY platform, intersystem crossing follows energy transfer. The resulting triplet state can transfer energy further to molecular oxygen with the concomitant production of singlet oxygen to induce cell mortality. Furthermore, the donor can be excited with two near-infrared photons simultaneously to permit the photoinduced generation of singlet oxygen intracellularly under illumination conditions compatible with applications in vivo. Thus, these supramolecular strategies to control the excitation dynamics of multichromophoric assemblies in the intracellular environment can evolve into valuable protocols for photodynamic therapy.

Oxygen carrying microbubbles for enhanced sonodynamic therapy of hypoxic tumours.

Tumour hypoxia represents a major challenge in the effective treatment of solid cancerous tumours using conventional approaches. As oxygen is a key substrate for Photo-/Sono-dynamic Therapy (PDT/SDT), hypoxia is also problematic for the treatment of solid tumours using these techniques. The ability to deliver oxygen to the vicinity of the tumour increases its local partial pressure improving the possibility of ROS generation in PDT/SDT. In this manuscript, we investigate the use of oxygen-loaded, lipid-stabilised microbubbles (MBs), decorated with a Rose Bengal sensitiser, for SDT-based treatment of a pancreatic cancer model (BxPc-3) in vitro and in vivo. We directly compare the effectiveness of the oxygen-loaded MBs with sulphur hexafluoride (SF6)-loaded MBs and reveal a significant improvement in therapeutic efficacy. The combination of oxygen-carrying, ultrasound-responsive MBs, with an ultrasound-responsive therapeutic sensitiser, offers the possibility of delivering and activating the MB-sensitiser conjugate at the tumour site in a non-invasive manner, providing enhanced sonodynamic activation at that site.

Carbon quantum dot-NO photoreleaser nanohybrids for two-photon phototherapy of hypoxic tumors.

We report a conjugate between carbon quantum dots and a NO photoreleaser able to photogenerate the anticancer NO radical via an energy transfer mechanism. This nanohybrid proved toxic to cancer cells in vitro and significantly reduced tumor volume in mice bearing human xenograft BxPC-3 pancreatic tumors upon two-photon excitation with the highly biocompatible 800 nm light.

Microbubble-sonosensitiser conjugates as therapeutics in sonodynamic therapy.

A Rose Bengal sonosensitiser has been covalently attached to a lipid microbubble and the resulting conjugate shown to produce higher levels of singlet oxygen, enhanced cytotoxicity in a cancer cell line and a greater reduction in tumour growth than the sonosensitiser alone.

Enhanced ROS production and cell death through combined photo- and sono-activation of conventional photosensitising drugs.

A combination of light and ultrasound activation of two conventional photosensitising drugs, methylene blue and rose bengal, was shown to generate higher levels of reactive oxygen species (ROS) and lower LD50 values than either light or ultrasound activation alone.