Neutron-activated, plasmonically excitable Fe-Pt-Yb2O3 nanoparticles delivering anti-cancer radiation against human glioblastoma cells.

Magnetic nanoparticles can be functionalized in many ways for biomedical applications. Here, we combine four advantageous features in a novel Fe-Pt-Yb2O3 core-shell nanoparticle. (a) The nanoparticles have a size of 10 nm allowing them to diffuse through neuronal tissue. (b) The particles are superparamagnetic after synthesis and ferromagnetic after annealing, enabling directional control by magnetic fields, enhance NMRI contrast, and hyperthermia treatment. (c) After neutron-activation of the shell, they carry low-energetic, short half-life ß-radiation from 175Yb, 177Yb, and 177Lu. (d) Additionally, the particles can be optically visualized by plasmonic excitation and luminescence. To demonstrate the potential of the particles for cancer treatment, we exposed cultured human glioblastoma cells (LN-18) to non-activated and activated particles to confirm that the particles are internalized, and that the ß-radiation of the radioisotopes incorporated in the neutron-activated shell of the nanoparticles kills more than 98% of the LN-18 cancer cells, promising for future anti-cancer applications.

Radiation-induced alterations in multi-layered, in-vitro skin models detected by optical coherence tomography and histological methods.

BACKGROUND: Inflammatory skin reactions and skin alterations are still a potential side effect in radiation therapy (RT), which also need attention for patients' health care. METHOD: In a pre-clinical study we consider alterations in irradiated in-vitro skin models of epidermal and dermal layers. Typical dose regimes in radiation therapy are applied for irradiation. For non-invasive imaging and characterization optical coherence tomography (OCT) is used. Histological staining method is additionally applied for comparison and discussion. RESULTS: Structural features, such as keratinization, modifications in epidermal cell layer thickness and disorder in the layering-as indications for reactions to ionizing radiation and aging-could be observed by means of OCT and confirmed by histology. We were able to recognize known RT induced changes such as hyper-keratosis, acantholysis, and epidermal hyperplasia as well as disruption and/or demarcation of the dermo-epidermal junction. CONCLUSION: The results may pave the way for OCT to be considered as a possible adjunctive tool to detect and monitor early skin inflammation and side effects of radiotherapy, thus supporting patient healthcare in the future.

Author Correction: DNA damage interactions on both nanometer and micrometer scale determine overall cellular damage.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Proton pencil minibeam irradiation of an in-vivo mouse ear model spares healthy tissue dependent on beam size.

Proton radiotherapy using minibeams of sub-millimeter dimensions reduces side effects in comparison to conventional proton therapy due to spatial fractionation. Since the proton minibeams widen with depth, the homogeneous irradiation of a tumor can be ensured by adjusting the beam distances to tumor size and depth to maintain tumor control as in conventional proton therapy. The inherent advantages of protons in comparison to photons like a limited range that prevents a dosage of distal tissues are maintained by proton minibeams and can even be exploited for interlacing from different beam directions. A first animal study was conducted to systematically investigate and quantify the tissue-sparing effects of proton pencil minibeams as a function of beam size and dose distributions, using beam widths between σ = 95, 199, 306, 411, 561 and 883 µm (standard deviation) at a defined center-to-center beam distance (ctc) of 1.8 mm. The average dose of 60 Gy was distributed in 4x4 minibeams using 20 MeV protons (LET ~ 2.7 keV/µm). The induced radiation toxicities were measured by visible skin reactions and ear swelling for 90 days after irradiation. The largest applied beam size to ctc ratio (σ/ctc = 0.49) is similar to a homogeneous irradiation and leads to a significant 3-fold ear thickness increase compared to the control group. Erythema and desquamation was also increased significantly 3-4 weeks after irradiation. With decreasing beam sizes and thus decreasing σ/ctc, the maximum skin reactions are strongly reduced until no ear swelling or other visible skin reactions should occur for σ/ctc < 0.032 (extrapolated from data). These results demonstrate that proton pencil minibeam radiotherapy has better tissue-sparing for smaller σ/ctc, corresponding to larger peak-to-valley dose ratios PVDR, with the best effect for σ/ctc < 0.032. However, even quite large σ/ctc (e.g. σ/ctc = 0.23 or 0.31, i.e. PVDR = 10 or 2.7) show less acute side effects than a homogeneous dose distribution. This suggests that proton minibeam therapy spares healthy tissue not only in the skin but even for dose distributions appearing in deeper layers close to the tumor enhancing its benefits for clinical proton therapy.

Beam size limit for pencil minibeam radiotherapy determined from side effects in an in-vivo mouse ear model.

Side effects caused by radiation are a limiting factor to the amount of dose that can be applied to a tumor volume. A novel method to reduce side effects in radiotherapy is the use of spatial fractionation, in which a pattern of sub-millimeter beams (minibeams) is applied to spare healthy tissue. In order to determine the skin reactions in dependence of single beam sizes, which are relevant for spatially fractionated radiotherapy approaches, single pencil beams of submillimeter to 6 millimeter size were applied in BALB/c mice ears at a Small Animal Radiation Research Platform (SARRP) with a plateau dose of 60 Gy. Radiation toxicities in the ears were observed for 25 days after irradiation. Severe radiation responses were found for beams ≥ 3 mm diameter. The larger the beam diameter the stronger the observed reactions. No ear swelling and barely reddening or desquamation were found for the smallest beam sizes (0.5 and 1 mm). The findings were confirmed by histological sections. Submillimeter beams are preferred in minibeam therapy to obtain optimized tissue sparing. The gradual increase of radiation toxicity with beam size shows that also larger beams are capable of healthy tissue sparing in spatial fractionation.

DNA damage interactions on both nanometer and micrometer scale determine overall cellular damage.

DNA double strand breaks (DSB) play a pivotal role for cellular damage, which is a hazard encountered in toxicology and radiation protection, but also exploited e.g. in eradicating tumors in radiation therapy. It is still debated whether and in how far clustering of such DNA lesions leads to an enhanced severity of induced damage. Here we investigate - using focused spots of ionizing radiation as damaging agent - the spatial extension of DNA lesion patterns causing cell inactivation. We find that clustering of DNA damage on both the nm and µm scale leads to enhanced inactivation compared to more homogeneous lesion distributions. A biophysical model interprets these observations in terms of enhanced DSB production and DSB interaction, respectively. We decompose the overall effects quantitatively into contributions from these lesion formation processes, concluding that both processes coexist and need to be considered for determining the resulting damage on the cellular level.

Increased cell survival and cytogenetic integrity by spatial dose redistribution at a compact synchrotron X-ray source.

X-ray microbeam radiotherapy can potentially widen the therapeutic window due to a geometrical redistribution of the dose. However, high requirements on photon flux, beam collimation, and system stability restrict its application mainly to large-scale, cost-intensive synchrotron facilities. With a unique laser-based Compact Light Source using inverse Compton scattering, we investigated the translation of this promising radiotherapy technique to a machine of future clinical relevance. We performed in vitro colony-forming assays and chromosome aberration tests in normal tissue cells after microbeam irradiation compared to homogeneous irradiation at the same mean dose using 25 keV X-rays. The microplanar pattern was achieved with a tungsten slit array of 50 µm slit size and a spacing of 350 µm. Applying microbeams significantly increased cell survival for a mean dose above 2 Gy, which indicates fewer normal tissue complications. The observation of significantly less chromosome aberrations suggests a lower risk of second cancer development. Our findings provide valuable insight into the mechanisms of microbeam radiotherapy and prove its applicability at a compact synchrotron, which contributes to its future clinical translation.

Live cell imaging of mitochondria following targeted irradiation in situ reveals rapid and highly localized loss of membrane potential.

The reliance of all cell types on the mitochondrial function for survival makes mitochondria an interesting target when trying to understand their role in the cellular response to ionizing radiation. By harnessing highly focused carbon ions and protons using microbeams, we have performed in situ live cell imaging of the targeted irradiation of individual mitochondria stained with Tetramethyl rhodamine ethyl ester (TMRE), a cationic fluorophore which accumulates electrophoretically in polarized mitochondria. Targeted irradiation with both carbon ions and protons down to beam spots of <1 µm induced a near instant loss of mitochondrial TMRE fluorescence signal in the targeted area. The loss of TMRE after targeted irradiation represents a radiation induced change in mitochondrial membrane potential. This is the first time such mitochondrial responses have been documented in situ after targeted microbeam irradiation. The methods developed and the results obtained have the ability to shed new light on not just mitochondria's response to radiation but to further elucidate a putative mechanism of radiation induced depolarization and mitochondrial response.

Proton Minibeam Radiation Therapy Reduces Side Effects in an In Vivo Mouse Ear Model.

PURPOSE: Proton minibeam radiation therapy is a novel approach to minimize normal tissue damage in the entrance channel by spatial fractionation while keeping tumor control through a homogeneous tumor dose using beam widening with an increasing track length. In the present study, the dose distributions for homogeneous broad beam and minibeam irradiation sessions were simulated. Also, in an animal study, acute normal tissue side effects of proton minibeam irradiation were compared with homogeneous irradiation in a tumor-free mouse ear model to account for the complex effects on the immune system and vasculature in an in vivo normal tissue model. METHODS AND MATERIALS: At the ion microprobe SNAKE, 20-MeV protons were administered to the central part (7.2 × 7.2 mm(2)) of the ear of BALB/c mice, using either a homogeneous field with a dose of 60 Gy or 16 minibeams with a nominal 6000 Gy (4 × 4 minibeams, size 0.18 × 0.18 mm(2), with a distance of 1.8 mm). The same average dose was used over the irradiated area. RESULTS: No ear swelling or other skin reactions were observed at any point after minibeam irradiation. In contrast, significant ear swelling (up to fourfold), erythema, and desquamation developed in homogeneously irradiated ears 3 to 4 weeks after irradiation. Hair loss and the disappearance of sebaceous glands were only detected in the homogeneously irradiated fields. CONCLUSIONS: These results show that proton minibeam radiation therapy results in reduced adverse effects compared with conventional homogeneous broad-beam irradiation and, therefore, might have the potential to decrease the incidence of side effects resulting from clinical proton and/or heavy ion therapy.

Role of membrane Hsp70 in radiation sensitivity of tumor cells.

BACKGROUND: The major stress-inducible heat shock protein 70 (Hsp70) is frequently overexpressed in the cytosol and integrated in the plasma membrane of tumor cells via lipid anchorage. Following stress such as non-lethal irradiation Hsp70 synthesis is up-regulated. Intracellular located Hsp70 is known to exert cytoprotective properties, however, less is known about membrane (m)Hsp70. Herein, we investigate the role of mHsp70 in the sensitivity towards irradiation in tumor sublines that differ in their cytosolic and/or mHsp70 levels. METHODS: The isogenic human colon carcinoma sublines CX(+) with stable high and CX(-) with stable low expression of mHsp70 were generated by fluorescence activated cell sorting, the mouse mammary carcinoma sublines 4 T1 (4 T1 ctrl) and Hsp70 knock-down (4 T1 Hsp70 KD) were produced using the CRISPR/Cas9 system, and the Hsp70 down-regulation in human lung carcinoma sublines H1339 ctrl/H1339 HSF-1 KD and EPLC-272H ctrl/EPLC-272H HSF-1 KD was achieved by small interfering (si)RNA against Heat shock factor 1 (HSF-1). Cytosolic and mHsp70 was quantified by Western blot analysis/ELISA and flow cytometry; double strand breaks (DSBs) and apoptosis were measured by flow cytometry using antibodies against γH2AX and real-time PCR (RT-PCR) using primers and antibodies directed against apoptosis related genes; and radiation sensitivity was determined using clonogenic cell surviving assays. RESULTS: CX(+)/CX(-) tumor cells exhibited similar cytosolic but differed significantly in their mHsp70 levels, 4 T1 ctrl/4 T1 Hsp70 KD cells showed significant differences in their cytosolic and mHsp70 levels and H1339 ctrl/H1339 HSF-1 KD and EPLC-272H ctrl/EPLC-272H HSF-1 KD lung carcinoma cell sublines had similar mHsp70 but significantly different cytosolic Hsp70 levels. γH2AX was significantly up-regulated in irradiated CX(-) and 4 T1 Hsp70 KD with low basal mHsp70 levels, but not in their mHsp70 high expressing counterparts, irrespectively of their cytosolic Hsp70 content. After irradiation γH2AX, Caspase 3/7 and Annexin V were up-regulated in the lung carcinoma sublines, but no significant differences were observed in H1339 ctrl/H1339 HSF-1 KD, and EPLC-272H ctrl/EPLC-272H HSF-1 KD that exhibit identical mHsp70 but different cytosolic Hsp70 levels. Clonogenic cell survival was significantly lower in CX(-) and 4 T1 Hsp70 KD cells with low mHsp70 expression, than in CX+ and 4 T1 ctrl cells, whereas no difference in clonogenic cell survival was observed in H1339 ctrl/H1339 HSF-1 KD and EPLC-272H ctrl/ EPLC-272H HSF-1 KD sublines with identical mHsp70 but different cytosolic Hsp70 levels. CONCLUSION: In summary, our results indicate that mHsp70 has an impact on radiation resistance.

The relative biological effectiveness for carbon and oxygen ion beams using the raster-scanning technique in hepatocellular carcinoma cell lines.

BACKGROUND: Aim of this study was to evaluate the relative biological effectiveness (RBE) of carbon (12C) and oxygen ion (16O)-irradiation applied in the raster-scanning technique at the Heidelberg Ion beam Therapy center (HIT) based on clonogenic survival in hepatocellular carcinoma cell lines compared to photon irradiation. METHODS: Four human HCC lines Hep3B, PLC, HepG2 and HUH7 were irradiated with photons, 12C and 16O using a customized experimental setting at HIT for in-vitro trials. Cells were irradiated with increasing physical photon single doses of 0, 2, 4 and 6 Gy and heavy ion-single doses of 0, 0.125, 0.5, 1, 2, 3 Gy (12C and 16O). SOBP-penetration depth and extension was 35 mm +/-4 mm and 36 mm +/-5 mm for carbon ions and oxygen ions respectively. Mean energy level and mean linear energy transfer (LET) were 130 MeV/u and 112 keV/um for 12C, and 154 MeV/u and 146 keV/um for 16O. Clonogenic survival was computated and relative biological effectiveness (RBE) values were defined. RESULTS: For all cell lines and both particle modalities α- and ß-values were determined. As expected, α-values were significantly higher for 12C and 16O than for photons, reflecting a steeper decline of the initial slope of the survival curves for high-LET beams. RBE-values were in the range of 2.1-3.3 and 1.9-3.1 for 12C and 16O, respectively. CONCLUSION: Both irradiation with 12C and 16O using the raster-scanning technique leads to an enhanced RBE in HCC cell lines. No relevant differences between achieved RBE-values for 12C and 16O were found. Results of this work will further influence biological-adapted treatment planning for HCC patients that will undergo particle therapy with 12C or 16O.