Design and Validation of Experimental Setup for Cell Spheroid Radiofrequency-Induced Heating.

While hyperthermia has been shown to induce a variety of cytotoxic and sensitizing effects on cancer tissues, the thermal dose-effect relationship is still not well quantified, and it is still unclear how it can be optimally combined with other treatment modalities. Additionally, it is speculated that different methods of applying hyperthermia, such as water bath heating or electromagnetic energy, may have an effect on the resulting biological mechanisms involved in cell death or in sensitizing tumor cells to other oncological treatments. In order to further quantify and characterize hyperthermia treatments on a cellular level, in vitro experiments shifted towards the use of 3D cell spheroids. These are in fact considered a more representative model of the cell environment when compared to 2D cell cultures. In order to perform radiofrequency (RF)-induced heating in vitro, we have recently developed a dedicated electromagnetic field applicator. In this study, using this applicator, we designed and validated an experimental setup which can heat 3D cell spheroids in a conical polypropylene vial, thus providing a reliable instrument for investigating hyperthermia effects at the cellular scale.

Design and Characterization of an RF Applicator for In Vitro Tests of Electromagnetic Hyperthermia.

The evaluation of the biological effects of therapeutic hyperthermia in oncology and the precise quantification of thermal dose, when heating is coupled with radiotherapy or chemotherapy, are active fields of research. The reliable measurement of hyperthermia effects on cells and tissues requires a strong control of the delivered power and of the induced temperature rise. To this aim, we have developed a radiofrequency (RF) electromagnetic applicator operating at 434 MHz, specifically engineered for in vitro tests on 3D cell cultures. The applicator has been designed with the aid of an extensive modelling analysis, which combines electromagnetic and thermal simulations. The heating performance of the built prototype has been validated by means of temperature measurements carried out on tissue-mimicking phantoms and aimed at monitoring both spatial and temporal temperature variations. The experimental results demonstrate the capability of the RF applicator to produce a well-focused heating, with the possibility of modulating the duration of the heating transient and controlling the temperature rise in a specific target region, by simply tuning the effectively supplied power.

Selenium-doped hydroxyapatite nanoparticles for potential application in bone tumor therapy.

In the present study we have studied the incorporation and release of selenite ions (SeO32-) in hydroxyapatite nanoparticles for the treatment of bone tumors. Two types of selenium-doped hydroxyapatite (HASe) nanoparticles (NPs) with a nominal Se/(P + Se) molar ratio ranging from 0.01 up to 0.40 have been synthesized by a new and mild wet method. The two series of samples were thoroughly characterized and resulted to be slightly different in chemical composition, but they had similar properties in terms of morphology and degree of crystallinity. Selenium release from HASe was investigated under neutral and acidic conditions to simulate both healthy tissues and the low-pH environment surrounding a tumor mass, respectively. The comparison of the release profiles at two pH values clearly showed the possibility of modulating the Se release by simply changing the amount of Se in the HASe particles. The correlation between the physicochemical properties of HASe and their dissolution as a function of pH has been also investigated to facilitate future application of the NPs as chemotherapeutic adjuvant agents. Finally, the cytotoxic activity of HASe was evaluated using prostate (PC3) and breast (MDA-MB-231) cancer cells as well as healthy human bone marrow stem cells (hBMSc). HASe NPs exerted a good cytocompatibility at low concentration of Se but, with high Se doping concentration, they displayed strong cytotoxicity.

Preclinical evaluation of platinum-loaded hydroxyapatite nanoparticles in an embryonic zebrafish xenograft model.

Hydroxyapatite (HA) nanoparticles are commonly used as building blocks in the design of bone-substituting biomaterials. Recently, these nanoparticles have been considered for the treatment of metastasis disease, since their pH-dependent dissolution behavior allows for precise tuning of release kinetics of loaded cargo. Herein we show that the capacity of drug-loaded nanoparticles stabilized with citrate ions reduce cancer cell survival in an embryonic zebrafish xenograft model. In particular, in vitro studies demonstrate that PtPP-loaded HA nanoparticles exhibit anti-proliferative activity against breast cancer cells at reduced pH. In vivo studies using an embryonic zebrafish xenograft model reveal that PtPP co-delivered with human breast cancer cells strongly reduce cancer cell survival. Similarly, co-injection of breast cancer cells with citrate-functionalized and PtPP-loaded HA nanoparticles into zebrafish significantly reduces survival of cancer cells due to release of chemotherapeutically active kiteplatin species. These results demonstrate the preclinical efficacy of drug-loaded nanoparticles against human breast cancer cells in a xenogenic embryonic in vivo model.

Targeting of radioactive platinum-bisphosphonate anticancer drugs to bone of high metabolic activity.

Platinum-based chemotherapeutics exhibit excellent antitumor properties. However, these drugs cause severe side effects including toxicity, drug resistance, and lack of tumor selectivity. Tumor-targeted drug delivery has demonstrated great potential to overcome these drawbacks. Herein, we aimed to design radioactive bisphosphonate-functionalized platinum (195mPt-BP) complexes to confirm preferential accumulation of these Pt-based drugs in metabolically active bone. In vitro NMR studies revealed that release of Pt from Pt BP complexes increased with decreasing pH. Upon systemic administration to mice, Pt-BP exhibited a 4.5-fold higher affinity to bone compared to platinum complexes lacking the bone-seeking bisphosphonate moiety. These Pt-BP complexes formed less Pt-DNA adducts compared to bisphosphonate-free platinum complexes, indicating that in vivo release of Pt from Pt-BP complexes proceeded relatively slow. Subsequently, radioactive 195mPt-BP complexes were synthesized using 195mPt(NO3)2(en) as precursor and injected intravenously into mice. Specific accumulation of 195mPt-BP was observed at skeletal sites with high metabolic activity using micro-SPECT/CT imaging. Furthermore, laser ablation-ICP-MS imaging of proximal tibia sections confirmed that 195mPt BP co-localized with calcium in the trabeculae of mice tibia.

Platinum-loaded, selenium-doped hydroxyapatite nanoparticles selectively reduce proliferation of prostate and breast cancer cells co-cultured in the presence of stem cells.

Chemotherapeutic treatment of patients with bone tumors or bone metastases often leads to severe side effects such as high drug toxicity, lack of tumor specificity and induced drug resistance. A novel strategy to treat early stages of bone metastases involves local co-delivery of multiple chemotherapeutic agents to synergistically improve the curative effect and overcome shortcomings of traditional chemotherapy. Herein we show that selenite-doped hydroxyapatite nanoparticles loaded with a hydroxyapatite-binding anti-tumor platinum complex (PtPP-HASe) selectively reduce proliferation of cancer cells without reducing proliferation of bone marrow stem cells. These PtPP-HASe particles were nanocrystalline with selenium (Se) and platinum (Pt) contents ranging between 0-10 and 1.5-3 wt%, respectively. Release kinetics of Se and Pt from PtPP-HASe nanoparticles resulted in a cumulative release of ∼10 and ∼66 wt% after 7 days, respectively. At a Pt/Se ratio of 8, released Pt and Se species selectively reduced cell number of human prostate (PC3) and human breast cancer cells (MDA-MB-231) by a factor of >10 with limited effects on co-cultured human bone marrow stem cells (hBMSc). These novel nanoparticles demonstrate high anti-cancer selectivity, which offers ample opportunities for the design of novel biomaterials with potent and selective chemotherapeutic efficacy against cancer cells.

Thermoresponsive Brushes Facilitate Effective Reinforcement of Calcium Phosphate Cements.

Calcium phosphate ceramics are frequently applied to stimulate regeneration of bone in view of their excellent biological compatibility with bone tissue. Unfortunately, these bioceramics are also highly brittle. To improve their toughness, fibers can be incorporated as the reinforcing component for the calcium phosphate cements. Herein, we functionalize the surface of poly(vinyl alcohol) fibers with thermoresponsive poly(N-isopropylacrylamide) brushes of tunable thickness to improve simultaneously fiber dispersion and fiber-matrix affinity. These brushes shift from hydrophilic to hydrophobic behavior at temperatures above their lower critical solution temperature of 32 °C. This dual thermoresponsive shift favors fiber dispersion throughout the hydrophilic calcium phosphate cements (at 21 °C) and toughens these cements when reaching their hydrophobic state (at 37 °C). The reinforcement efficacy of these surface-modified fibers was almost double at 37 versus 21 °C, which confirms the strong potential of thermoresponsive fibers for reinforcement of calcium phosphate cements.

Quantitative imaging of platinum-based antitumor complexes in bone tissue samples using LA-ICP-MS.

There is a need for effective medication against bone metastases because todays drugs are not able to penetrate the bone and reach the affected areas. To analyze if current or future platinum-containing drugs are able to achieve this, a quantitative imaging method is urgently needed. In this study, the platinum distribution in thin sections of mice tibia was determined using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) in a spatially resolved manner. The hard bone tissue visible in microscopic images and signals found for calcium and phosphorous recorded via LA-ICP-MS and micro X-ray fluorescence spectroscopy (µXRF) correlate well. Furthermore, the platinum concentration was quantified using polymer-based matrix-matched standards. A limit of detection of 6 µg/g and a linearity of almost three decades could be achieved. Concentrations surpassing 300 µg/g could be found in the tibia samples. The method presented herein is a powerful approach for the visualization and quantification of platinum. As such, this method is a valuable tool to unravel the mechanism of delivery and optimize the therapeutic potency of platinum-containing drugs targeting bone diseases like bone metastases.

Bisphosphonate-Functionalized Imaging Agents, Anti-Tumor Agents and Nanocarriers for Treatment of Bone Cancer.

Bone metastases result from the invasion of primary tumors to bone. Current treatment modalities include local treatments such as surgery and radiotherapy, while systemic treatments include chemotherapy and (palliative) treatment of skeletal metastases. Nevertheless, once bone metastases have been established they remain incurable leading to morbidity and mortality. Bisphosphonates are a well-established class of drugs, which are increasingly applied in the treatment of bone cancers owing to their effective inhibition of tumor cells and suppression of bone metastases. The increased understanding of the mechanism of action of bisphosphonates on bone and tumor cells has prompted the development of novel bisphosphonate-functionalized imaging and therapeutic agents. This review provides an update on the preclinical efficacy of bisphosphonate-functionalized fluorophore, anti-tumor agents and nanocarriers for the treatment of bone metastases. After an overview of the general characteristics of bisphosphonates and their mechanisms of action, an outline is provided on the various conjugation strategies that have become available to functionalize imaging agents, anti-tumor agents and nanocarriers with bisphosphonates. Finally, the efficacy of these bisphosphonate-modified agents and carriers in preclinical studies is evaluated by reviewing their potential to target tumors and inhibit tumor growth in clinically relevant animal models for the treatment of bone cancer.

Radiolabelled Polymeric Materials for Imaging and Treatment of Cancer: Quo Vadis?

Owing to their tunable blood circulation time and suitable plasma stability, polymer-based nanomaterials hold a great potential for designing and utilising multifunctional nanocarriers for efficient imaging and effective treatment of cancer. When tagged with appropriate radionuclides, they may allow for specific detection (diagnosis) as well as the destruction of tumours (therapy) or even customization of materials, aiming to both diagnosis and therapy (theranostic approach). This review provides an overview of recent developments of radiolabelled polymeric nanomaterials (natural and synthetic polymers) for molecular imaging of cancer, specifically, applying nuclear techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT). Different approaches to radiolabel polymers are evaluated from the methodical radiochemical point of view. This includes new bifunctional chelating agents (BFCAs) for radiometals as well as novel labelling methods. Special emphasis is given to eligible strategies employed to evade the mononuclear phagocytic system (MPS) in view of efficient targeting. The discussion encompasses promising strategies currently employed as well as emerging possibilities in radionuclide-based cancer therapy. Key issues involved in the clinical translation of radiolabelled polymers and future scopes of this intriguing research field are also discussed.