Nikolitsa Nomikou

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.

Studies on neutral, cationic and biotinylated cationic microbubbles in enhancing ultrasound-mediated gene delivery in vitro and in vivo

Ultrasound-mediated gene transfer is emerging as a practical means of facilitating targeted gene expression and is significantly enhanced in the presence of exogenously added microbubbles. This study explores the influence of microbubble surface modifications on their interaction with plasmid DNA and target cells, and the functional consequences of those interactions in terms of ultrasound-mediated gene transfer. Polyethylene glycol-stabilized, lipid-shelled microbubbles with neutral (SDM201), cationic (SDM202) and biotinylated cationic (SDM302) surfaces were compared in terms of their abilities to interact with a luciferase-encoding reporter plasmid DNA and with target cells in vitro. The results demonstrate that the biotinylated cationic microbubble>cationic microbubble>neutral microbubble, in terms of their abilities to interact with target cells and to enhance ultrasound-mediated gene transfer, particularly at low microbubble concentration. The presence of a net positive charge on both cationic microbubbles promoted the formation of microbubble-nucleic acid complexes, although preformation of the complexes prior to addition to target cells inhibited the interaction between the microbubbles and target cells in vitro. The impact of these findings on potential in vitro or ex vivo therapeutic applications of microbubble-enhanced ultrasound-mediated gene transfer is discussed. All three microbubble preparations could be used to facilitate gene transfer in vivo and the potential advantages associated with the use of the cationic microbubbles for targeted gene delivery are discussed.

Treating cancer with sonodynamic therapy: A review

Abstract Sonodynamic therapy (SDT) has emerged as a promising option for the minimally invasive treatment of solid cancerous tumours. SDT requires the combination of three distinct components: a sensitising drug, ultrasound, and molecular oxygen. Individually, these components are non-toxic but when combined together generate cytotoxic reactive oxygen species (ROS). The major advantage of SDT over its close relative photodynamic therapy (PDT), is the increased penetration of ultrasound through mammalian tissue compared to light. As a result, SDT can be used to treat a wider array of deeper and less accessible tumours than PDT. In this article, we critically review the current literature on SDT and discuss strategies that have been developed in combination with SDT to enhance the therapeutic outcome.

Water soluble quantum dots as hydrophilic carriers and two-photon excited energy donors in photodynamic therapy†

In search of strategies to develop deeply penetrating agents for use in Photodynamic Therapy (PDT), we have devised a Quantum Dot-Rose Bengal conjugate that is effective at producing singlet oxygen upon two-photon irradiation. The CdSe/ZnS Quantum Dot, with its high two photon absorption cross section, serves as a two-photon absorbing antenna and transfers its excited state energy to the attached photosensitiser which engages with molecular oxygen to produce cytotoxic singlet oxygen. Thus, we were able to excite the photosensitiser indirectly, which has an absorption maximum of 565 nm, with two-photon irradiation at 800 nm. Given the tissue penetration depth of 800 nm light is at least four times greater than 565 nm light, this offers the opportunity to access much deeper-seated tumours than is currently possible with pharmaceutically approved photosensitisers. Furthermore, the attachment of the photosensitiser to the hydrophilic quantum dot improved the aqueous solubility of the photosensitiser by 48 fold, thus overcoming another limitation of currently used photosensitisers, that of poor aqueous solubility.

The Effects of Ultrasound and Light on Indocyanine‐Green‐Treated Tumour Cells and Tissues

Photodynamic therapy (PDT) is emerging as a treatment modality for the management of neoplastic disease. Despite considerable clinical success, its application for the treatment of deep‐seated lesions is constrained by the inability of visible light to penetrate deeply into tissues. An emerging alternative approach exploits the fact that many photosensitisers respond to ultrasound, eliciting cytotoxic effects on target cells and tissues; this has become known as sonodynamic therapy (SDT). The objectives of this study were 1) to determine whether the IR‐absorbing dye, indocyanine green (ICG), can be employed as a sonosensitiser and 2) to determine whether ultrasound can be used to enhance ICG‐mediated PDT. Exposing ICG‐treated mouse fibrosarcoma cells to ultrasound at an energy density of 30 J cm−2 decreased cell viability by 65 %. Prior exposure of ICG‐treated cells to light (λ 830 nm) and subsequent treatment with ultrasound led to a 90 % decrease in cell viability. In combination treatments a synergistic effect was observed at lower doses of ultrasound. Microscopic examination of cell populations treated with light or ultrasound demonstrated the production of intracellular reactive oxygen species (ROS). Using a mouse tumour model, treatment with light, ultrasound, or a combination thereof led to respective decreases in tumour growth of 42, 67, and 98 % at day 27 post‐treatment. These results could provide a means of circumventing light‐penetration issues that currently challenge the widespread use of PDT in the treatment of 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.

Sonodynamic Therapy: Concept, Mechanism and Application to Cancer Treatment

Sonodynamic therapy (SDT) represents an emerging approach that offers the possibility of non-invasively eradicating solid tumors in a site-directed manner. It involves the sensitization of target tissues with a non-toxic sensitizing chemical agent and subsequent exposure of the sensitized tissues to relatively low-intensity ultrasound. Essentially, both aspects (the sensitization and ultrasound exposure) are harmless, and cytotoxic events occur when both are combined. Due to the significant depth that ultrasound penetrates tissue, the approach provides an advantage over similar alternative approaches, such as photodynamic therapy (PDT), in which less penetrating light is employed to provide the cytotoxic effect in sensitized tissues. This suggests that sonodynamic therapy may find wider clinical application, particularly for the non-invasive treatment of less accessible lesions. Early SDT-based approaches employed many of the sensitizers used in PDT, although the manner in which ultrasound activates the sensitizer differs from activation events in PDT. Here we will review the currently accepted mechanisms by which ultrasound activates sensitizers to elicit cytotoxic effects. In addition, we will explore the breath of evidence from in-vitro and in-vivo SDT-based studies, providing the reader with an insight into the therapeutic potential offered by SDT in the treatment of cancer.

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.

Polymeric Microbubbles as Delivery Vehicles for Sensitizers in Sonodynamic Therapy

Microbubbles (MBs) have recently emerged as promising delivery vehicles for sensitizer drugs in sonodynamic therapy (SDT). The ability to selectively destroy the MB and activate the sensitizer using an external ultrasound trigger could provide a minimally invasive and highly targeted therapy. While lipid MBs have been approved for use as contrast agents in diagnostic ultrasound, the attachment of sensitizer drugs to their surface results in a significant reduction in particle stability. In this Article, we prepare both lipid and polymer (PLGA) MBs with rose bengal attached to their surface and demonstrate that PLGA MB conjugates are significantly more stable than their lipid counterparts. In addition, the improved stability offered by the PLGA shell does not hinder their selective destruction using therapeutically acceptable ultrasound intensities. Furthermore, we demonstrate that treatment of ectopic human tumors (BxPC-3) in mice with the PLGA MB-rose bengal conjugate and ultrasound reduced tumor volume by 34% 4 days after treatment while tumors treated with the conjugate alone increased in volume by 48% over the same time period. Therefore, PLGA MBs may offer a more stable alternative to lipid MBs for the site specific delivery of sensitizers in SDT.

Sonoporation increases therapeutic efficacy of inducible and constitutive BMP2/7 in vivo gene delivery.

An ideal novel treatment for bone defects should provide regeneration without autologous or allogenous grafting, exogenous cells, growth factors, or biomaterials while ensuring spatial and temporal control as well as safety. Therefore, a novel osteoinductive nonviral in vivo gene therapy approach using sonoporation was investigated in ectopic and orthotopic models. Constitutive or regulated, doxycycline-inducible, bone morphogenetic protein 2 and 7 coexpression plasmids were repeatedly applied for 5 days. Ectopic and orthotopic gene transfer efficacy was monitored by coapplication of a luciferase plasmid and bioluminescence imaging. Orthotopic plasmid DNA distribution was investigated using a novel plasmid-labeling method. Luciferase imaging demonstrated an increased trend (61% vs. 100%) of gene transfer efficacy, and micro-computed tomography evaluation showed significantly enhanced frequency of ectopic bone formation for sonoporation compared with passive gene delivery (46% vs. 100%) dependent on applied ultrasound power. Bone formation by the inducible system (83%) was stringently controlled by doxycycline in vivo, and no ectopic bone formation was observed without induction or with passive gene transfer without sonoporation. Orthotopic evaluation in a rat femur segmental defect model demonstrated an increased trend of gene transfer efficacy using sonoporation. Investigation of DNA distribution demonstrated extensive binding of plasmid DNA to bone tissue. Sonoporated animals displayed a potentially increased union rate (33%) without extensive callus formation or heterotopic ossification. We conclude that sonoporation of BMP2/7 coexpression plasmids is a feasible, minimally invasive method for osteoinduction and that improvement of bone regeneration by sonoporative gene delivery is superior to passive gene delivery.