Impact of superparamagnetic iron oxide nanoparticles on in vitro and in vivo radiosensitisation of cancer cells.

PURPOSE: The recent implementation of MR-Linacs has highlighted theranostic opportunities of contrast agents in both imaging and radiotherapy. There is a lack of data exploring the potential of superparamagnetic iron oxide nanoparticles (SPIONs) as radiosensitisers. Through preclinical 225 kVp exposures, this study aimed to characterise the uptake and radiobiological effects of SPIONs in tumour cell models in vitro and to provide proof-of-principle application in a xenograft tumour model. METHODS: SPIONs were also characterised to determine their hydrodynamic radius using dynamic light scattering and uptake was measured using ICP-MS in 6 cancer cell lines; H460, MiaPaCa2, DU145, MCF7, U87 and HEPG2. The impact of SPIONs on radiobiological response was determined by measuring DNA damage using 53BP1 immunofluorescence and cell survival. Sensitisation Enhancement Ratios (SERs) were compared with the predicted Dose Enhancement Ratios (DEFs) based on physical absorption estimations. In vivo efficacy was demonstrated using a subcutaneous H460 xenograft tumour model in SCID mice by following intra-tumoural injection of SPIONs. RESULTS: The hydrodynamic radius was found to be between 110 and 130 nm, with evidence of being monodisperse in nature. SPIONs significantly increased DNA damage in all cell lines with the exception of U87 cells at a dose of 1 Gy, 1 h post-irradiation. Levels of DNA damage correlated with the cell survival, in which all cell lines except U87 cells showed an increased sensitivity (P < 0.05) in the linear quadratic curve fit for 1 h exposure to 23.5 µg/ml SPIONs. There was also a 30.1% increase in the number of DNA damage foci found for HEPG2 cells at 2 Gy. No strong correlation was found between SPION uptake and DNA damage at any dose, yet the biological consequences of SPIONs on radiosensitisation were found to be much greater, with SERs up to 1.28 ± 0.03, compared with predicted physical dose enhancement levels of 1.0001. In vivo, intra-tumoural injection of SPIONs combined with radiation showed significant tumour growth delay compared to animals treated with radiation or SPIONs alone (P < 0.05). CONCLUSIONS: SPIONs showed radiosensitising effects in 5 out of 6 cancer cell lines. No correlation was found between the cell-specific uptake of SPIONs into the cells and DNA damage levels. The in vivo study found a significant decrease in the tumour growth rate.

Effects of Gadolinium MRI Contrast Agents on DNA Damage and Cell Survival when Used in Combination with Radiation.

Gadolinium is a commonly used contrast agent for magnetic resonance imaging (MRI). The goal of this work was to determine how MRI contrast agents affect radiosensitivity for tumour cells. Using a 225kVp X-ray cabinet source, immunofluorescence and clonogenic assays were performed on six cancer cell lines: lung (H460), pancreas (MiaPaCa2), prostate (DU145), breast (MCF7), brain (U87) and liver (HEPG2). Dotarem® contrast agent, at concentrations of 0.2, 2 and 20 mM, was used to determine its effect on DNA damage and cell survival. Measurements were performed using inductively coupled plasma mass spectrometry (ICP-MS) to determine the amount of gadolinium taken up by each cell line for each concentration. A statistically significant increase in DNA damage was seen for all cell lines at a dose of 1 Gy for concentrations of 2 and 20 mM, at 1 h postirradiation. At 24 h postirradiation, most of the DNA damage had been repaired, with approximately 90% repair for almost all doses of radiation and concentrations of Dotarem. Clonogenic results showed no statistically significant decrease in cell survival for any cell line or concentration. Uptake measurements showed cell line-specific variations in uptake, with MCF7 and HEPG2 cells having a high percentage uptake compared to other cell lines, with 151.4 ± 0.3 × 10-15 g and 194.8 ± 0.4 × 10-15 g per cell, respectively, at 2 mM Dotarem concentration. In this work, a variability in gadolinium uptake was observed between cell lines. A significant increase was seen in initial levels of DNA damage after 1 Gy irradiation for all six cancer cell lines; however, no significant decrease in cell survival was seen with the clonogenic assay. The observation of high levels of repair suggest that while initial levels of DNA damage are increased, this damage is almost entirely repaired within 24 h, and does not affect the ability of cells to survive and produce colonies.

Radiobiological Implications of Nanoparticles Following Radiation Treatment.

Materials with a high atomic number (Z) are shown to cause an increase in the level of cell kill by ionizing radiation when introduced into tumor cells. This study uses in vitro experiments to investigate the differences in radiosensitization between two cell lines (MCF-7 and U87) and three commercially available nanoparticles (gold, gadolinium, and iron oxide) irradiated by 6 MV X-rays. To assess cell survival, clonogenic assays are carried out for all variables considered, with a concentration of 0.5 mg mL-1 for each nanoparticle material used. This study demonstrates differences in cell survival between nanoparticles and cell line. U87 shows the greatest enhancement with gadolinium nanoparticles (2.02 ± 0.36), whereas MCF-7 cells have higher enhancement with gold nanoparticles (1.74 ± 0.08). Mass spectrometry, however, shows highest elemental uptake with iron oxide and U87 cells with 4.95 ± 0.82 pg of iron oxide per cell. A complex relationship between cellular elemental uptake is demonstrated, highlighting an inverse correlation with the enhancement, but a positive relation with DNA damage when comparing the same nanoparticle between the two cell lines.

Lysozyme encapsulated gold nanoclusters for probing the early stage of lysozyme aggregation under acidic conditions.

Protein aggregation can lead to several incurable amyloidosis diseases. The full aggregation pathway is not fully understood, creating the need for new methods of studying this important biological phenomenon. Lysozyme is an amyloidogenic protein which is often used as a model protein for studying amyloidosis. This work explores the potential of employing Lysozyme encapsulated gold nanoclusters (Ly-AuNCs) to study the protein's aggregation. The fluorescence emission properties of Ly-AuNCs were studied in the presence of increasing concentrations of native lysozyme and as a function of pH, of relevance in macromolecular crowding and inflammation-triggered aggregation. AuNC fluorescence was observed to both redshift and increase in intensity as pH is increased or when native lysozyme is added to a solution of Ly-AuNCs at pH 3. The long (µs) fluorescence lifetime component of AuNC emission was observed to decrease under both conditions. Interestingly it was found via Time-Resolved Emission Spectra (TRES) that both AuNC fluorescence components increase in intensity and redshift with increasing pH while only the long lifetime component of AuNC was observed to change when adding native lysozyme to solution; indicating that the underlying mechanisms for the changes observed are fundamentally different for each case. It is possible that the sensitivity of Ly-AuNCs to native lysozyme concentration could be utilized to study early-stage aggregation.

Chemical Purification of Terbium-155 from Pseudo-Isobaric Impurities in a Mass Separated Source Produced at CERN.

Four terbium radioisotopes (149, 152, 155, 161Tb) constitute a potential theranostic quartet for cancer treatment but require any derived radiopharmaceutical to be essentially free of impurities. Terbium-155 prepared by proton irradiation and on-line mass separation at the CERN-ISOLDE and CERN-MEDICIS facilities contains radioactive 139Ce16O and also zinc or gold, depending on the catcher foil used. A method using ion-exchange and extraction chromatography resins in two column separation steps has been developed to isolate 155Tb with a chemical yield of ≥95% and radionuclidic purity ≥99.9%. Conversion of terbium into a form suitable for chelation to targeting molecules in diagnostic nuclear medicine is presented. The resulting 155Tb preparations are suitable for the determination of absolute activity, SPECT phantom imaging studies and pre-clinical trials.

Detecting lysozyme unfolding via the fluorescence of lysozyme encapsulated gold nanoclusters.

Protein misfolding plays a critical role in the manifestation of amyloidosis type diseases. Therefore, understanding protein unfolding and the ability to track protein unfolding in a dynamic manner are of considerable interest. Fluorescence-based techniques are powerful tools for gaining real-time information about the local environmental conditions of a probe on the nanoscale. Fluorescent gold nanoclusters (AuNCs) are a new type of fluorescent probes which are <2 nm in diameter, incredibly robust and offer highly sensitive, wavelength tuneable emission. Their small size minimises intrusion and makes AuNCs ideal for studying protein dynamics. Lysozyme has previously been used to encapsulate AuNCs. The unfolding dynamics of lysozyme under different environmental conditions have been well-studied and being an amyloid type protein makes lysozyme an ideal candidate for encapsulating AuNCs in order to test their sensitivity to protein unfolding. In this study, we tracked the fluorescence characteristics of AuNCs encapsulated in lysozyme while inducing protein unfolding using urea, sodium dodecyl sulphate (SDS) and elevated temperature and compared them to complimentary circular dichroism spectra. It is found that AuNC fluorescence emission is quenched upon induced protein unfolding either due to a decrease in Forster Resonance Energy Transfer (FRET) efficiency between tryptophan and AuNCs or solvent exposure of the AuNC. Fluorescence lifetime measurements confirmed quenching to be collisional via oxygen dissolved in a solution which increases as the AuNC was exposed to the solvent during unfolding. Moreover, the longer decay component τ1 was observed to decrease as the protein unfolded, due to the increased collisional quenching. It is suggested that AuNC sensitivity to solvent exposure might be utilised in the future as a new approach to studying and possibly even detecting amyloidosis type diseases.

Comprehensive proteome profiling of glioblastoma-derived extracellular vesicles identifies markers for more aggressive disease.

Extracellular vesicles (EVs) play key roles in glioblastoma (GBM) biology and represent novel sources of biomarkers that are detectable in the peripheral circulation. Despite this notionally non-invasive approach to assess GBM tumours in situ, a comprehensive GBM EV protein signature has not been described. Here, EVs secreted by six GBM cell lines were isolated and analysed by quantitative high-resolution mass spectrometry. Overall, 844 proteins were identified in the GBM EV proteome, of which 145 proteins were common to EVs secreted by all cell lines examined; included in the curated EV compendium (Vesiclepedia_559; http://microvesicles.org ). Levels of 14 EV proteins significantly correlated with cell invasion (invadopodia production; r2 > 0.5, p < 0.05), including several proteins that interact with molecules responsible for regulating invadopodia formation. Invadopodia, actin-rich membrane protrusions with proteolytic activity, are associated with more aggressive disease and are sites of EV release. Gene levels corresponding to invasion-related EV proteins showed that five genes (annexin A1, actin-related protein 3, integrin-ß1, insulin-like growth factor 2 receptor and programmed cell death 6-interacting protein) were significantly higher in GBM tumours compared to normal brain in silico, with common functions relating to actin polymerisation and endosomal sorting. We also show that Cavitron Ultrasonic Surgical Aspirator (CUSA) washings are a novel source of brain tumour-derived EVs, demonstrated by particle tracking analysis, TEM and proteome profiling. Quantitative proteomics corroborated the high levels of proposed invasion-related proteins in EVs enriched from a GBM compared to low-grade astrocytoma tumour. Large-scale clinical follow-up of putative biomarkers, particularly the proposed survival marker annexin A1, is warranted.