Cytotoxicity of PEG-Coated Gold and Gold-Iron Alloy Nanoparticles: ROS or Ferroptosis?

Nanomedicine relies on the exploitation of nanoscale constructs for therapeutic and diagnostic functions. Gold and gold-iron alloy nanoparticles (NPs) are two examples of nanomaterials with favorable features for use in nanomedicine. While gold NPs have been studied extensively in the last decades, they are not biodegradable. Nonetheless, biodegradation was recently observed in gold alloys with iron obtained using laser ablation in liquid (LAL). Hence, there is a significant interest in the study of the biological effects of gold and gold-iron alloy nanoparticles, starting from their tolerability and cytotoxicity. In this study, these two classes of NPs, obtained via LAL and coated with biocompatible polymers such as polyethylene glycol, were investigated in terms of their cytotoxicity in fibroblasts, prostate cancer cells (PC3) and embryonic kidney cells (HEK). We also explored the effects of different synthetic procedures, stabilizing additives, and the possible mechanisms behind cell mortality such as the formation of reactive oxygen species (ROS) or ferroptosis. NPs larger than 200 nm were associated with lower cell tolerability. The most tolerable formulations were pure PEG-Au NPs, followed by PEG-Au-Fe NPs with a hydrodynamic size < 50 nm, which displayed a toxicity of only 20% in fibroblasts after 72 h of incubation. In addition, tumor cells and highly proliferating HEK cells are more sensitive to the NPs than fibroblasts. However, a protective effect of catalase was found for cells incubated with PEG-Au-Fe NPs, indicating an important role of hydrogen peroxide in alloy NP interactions with cells. These results are crucial for directing future synthetic efforts for the realization of biocompatible Au NPs and biodegradable and cytocompatible Au-Fe alloy NPs. Moreover, the correlation of the cytocompatibility of NPs with ROS and ferroptosis in cells is of general interest and applicability to other types of nanomaterials.

Radioluminescence imaging feasibility for robotic radiosurgery field size quality assurance.

PURPOSE: To investigate the feasibility of radioluminescence imaging (RLI) as a novel 2D quality assurance (QA) dosimetry system for CyberKnife®. METHODS: We developed a field size measurement system based on a commercial complementary metal oxide semiconductor (CMOS) camera facing a radioluminescence screen located at the isocenter normal to the beam axis. The radioluminescence light collected by a lens was used to measure 2D dose distributions. An image transformation procedure, based on two reference phantoms, was developed to correct for projective distortion due to the angle (15°) between the optical and beam axis. Dose profiles were measured for field sizes ranging from 5 mm to 60 mm using fixed circular and iris collimators and compared against gafchromic (GC) film. The corresponding full width at half maximum (FWHM) was measured using RLI and benchmarked against GC film. A small shift in the source-to-surface distance (SSD) of the measurement plane was intentionally introduced to test the sensitivity of the RLI system to field size variations. To assess reproducibility, the entire RLI procedure was tested by acquiring the 60 mm circle field three times on two consecutive days. RESULTS: The implemented procedure for perspective image distortion correction showed improvements of up to 1 mm using the star phantom against the square phantom. The FWHM measurements using the RLI system indicated a strong agreement with GC film with maximum absolute difference equal to 0.131 mm for fixed collimators and 0.056 mm for the iris. A 2D analysis of RLI with respect to GC film showed that the differences in the central region are negligible, while small discrepancies are in the penumbra region. Changes in field sizes of 0.2 mm were detectable by RLI. Repeatability measurements of the beam FWHM have shown a standard deviation equal to 0.11 mm. CONCLUSIONS: The first application of a RLI approach for CyberKnife® field size measurement was presented and tested. Results are in agreement with GC film measurements. Spatial resolution and immediate availability of the data indicate that RLI is a feasible technique for robotic radiosurgery QA.

Photon emission and changes in fluorescent properties of bone after laser irradiation.

Laser scalpels used in medical surgery concentrate light energy, heating the tissues. Recently, we reported thermoluminescence emission from laser-treated soft tissues. Here we investigated the thermo-optical effects caused by a laser operating at 808 nm on animal bones (beef ribs) through luminescence and fluorescence imaging, thermal imaging and scanning electron microscopy. Laser-induced artificial lesions emitted luminescence peaking around 650 nm, with a half-life of almost 1 hour. As concerns fluorescence, 24 hours after laser treatment we observed an increase of the emission and a shift from 500 (untreated) to 580 nm (treated). Recrystallization observed by SEM indicates that the temperature in the artificial lesions is over 600°C. We can conclude that laser treatment induces specific luminescent and fluorescent emissions due to heating of the bone and modification of its components. Monitoring these emissions could help prevent tissue overheating and its potential damages during laser-assisted medical procedures.

Pre-clinical Research on Bladder Toxicity After Radiotherapy for Pelvic Cancers: State-of-the Art and Challenges.

Despite the dramatic advancements in pelvic radiotherapy, urinary toxicity remains a significant side-effect. The assessment of clinico-dosimetric predictors of radiation cystitis (RC) based on clinical data has improved substantially over the last decade; however, a thorough understanding of the physiopathogenetic mechanisms underlying the onset of RC, with its variegated acute and late urinary symptoms, is still largely lacking, and data from pre-clinical research is still limited. The aim of this review is to provide an overview of the main open issues and, ideally, to help investigators in orienting future research. First, anatomy and physiology of bladder, as well as the current knowledge of dose and dose-volume effects in humans, are briefly summarized. Subsequently, pre-clinical radiobiology aspects of RC are discussed. The findings suggest that pre-clinical research on RC in animal models is a lively field of research with growing interest in the development of new radioprotective agents. The availability of new high precision micro-irradiators and the rapid advances in small animal imaging might lead to big improvement into this field. In particular, studies focusing on the definition of dose and fractionation are warranted, especially considering the growing interest in hypo-fractionation and ablative therapies for prostate cancer treatment. Moreover, improvement in radiotherapy plans optimization by selectively reducing radiation dose to more radiosensitive substructures close to the bladder would be of paramount importance. Finally, thanks to new pre-clinical imaging platforms, reliable and reproducible methods to assess the severity of RC in animal models are expected to be developed.

A non-invasive ultrasound imaging method to measure acute radiation-induced bladder wall thickening in rats.

BACKGROUND: Methods for the non-invasive quantification of changes in bladder wall thickness as potential predictors of radiation cystitis in pre-clinical research would be desirable. The use of ultrasound for this aim seems promising, but is still relatively unexplored. A method using ultrasound for bladder wall thickness quantification in rats was developed and applied to measure early radiation-induced bladder wall thickness changes. METHODS: Two groups (n = 9 each) of female Fischer rats were treated with a single radiation dose of 25-30 and 35-40 Gy respectively, using an image-guided micro-irradiator; six untreated rats were monitored as a control group. Empty, half-filled and fully-filled bladder volumes were determined for four non-irradiated rats by measuring axes from ultrasound 3D-images and applying the ellipsoid formula. Mean bladder wall thickness was estimated for both ventral and dorsal bladder sides through the measurement of the bladder wall area along a segment of 4 mm in the central sagittal scan, in order to minimize operator-dependence on the measurement position. Ultrasound acquisitions of all fully-filled rat bladders were also acquired immediately before, and 4 and 28 days after irradiation. Mean bladder wall thickness normalized to the baseline value and corrected for filling were then used to evaluate acute bladder wall thickening and to quantify the dose-effect. RESULTS: The relationship between mean bladder wall thickness and volume in unirradiated rats showed that for a bladder volume > 1.5 mL the bladder wall thickness is almost constant and equal to 0.30 mm with variations within ± 15%. The average ratios between post and pre irradiation showed a dose-effect relationship. Bladder wall thickening was observed for the 25-30 Gy and 35-40 Gy groups in 2/9 (22%) and 5/9 (56%) cases at day 4 and in 4/9 (44%) and 8/9 (89%) cases at day 28, respectively. The two groups showed significantly different bladder wall thickness both relative to the control group (p < 0.0001) and between them (p = 0.022). The bladder wall thickness increment was on average 1.32 ± 0.41, and was 1.30 ± 0.21 after 25-30 Gy and 1.47 ± 0.29 and 1.90 ± 0.83 after 35-40 Gy at days 4 and 28 respectively. CONCLUSIONS: The feasibility of using ultrasound on a preclinical rat model to detect bladder wall thickness changes after bladder irradiation was demonstrated, and a clear dose-effect relationship was quantified. Although preliminary, these results are promising in addressing the potential role of this non-invasive approach in quantifying radiation cystitis.

4D Multimodal Nanomedicines Made of Nonequilibrium Au-Fe Alloy Nanoparticles.

Several examples of nanosized therapeutic and imaging agents have been proposed to date, yet for most of them there is a low chance of clinical translation due to long-term in vivo retention and toxicity risks. The realization of nanoagents that can be removed from the body after use remains thus a great challenge. Here, we demonstrate that nonequilibrium gold-iron alloys behave as shape-morphing nanocrystals with the properties of self-degradable multifunctional nanomedicines. DFT calculations combined with mixing enthalpy-weighted alloying simulations predict that Au-Fe solid solutions can exhibit self-degradation in an aqueous environment if the Fe content exceeds a threshold that depends upon element topology in the nanocrystals. Exploiting a laser-assisted synthesis route, we experimentally confirm that nonequilibrium Au-Fe nanoalloys have a 4D behavior, that is, the ability to change shape, size, and structure over time, becoming ultrasmall Au-rich nanocrystals. In vivo tests show the potential of these transformable Au-Fe nanoalloys as efficient multimodal contrast agents for magnetic resonance imaging and computed X-ray absorption tomography and further demonstrate their self-degradation over time, with a significant reduction of long-term accumulation in the body, when compared to benchmark gold or iron oxide contrast agents. Hence, Au-Fe alloy nanoparticles exhibiting 4D behavior can respond to the need for safe and degradable inorganic multifunctional nanomedicines required in clinical translation.

Small animal irradiator dose distribution verification using radioluminescence imaging.

The main objective of this work was the development of a novel 2D dosimetry approach for small animal external radiotherapy using radioluminescence imaging (RLI) with a commercial complementary metal oxide semiconductor detector. Measurements of RLI were performed on the small animal image-guided platform SmART, RLI data were corrected for perspective distortion using Matlab. Four irradiation fields were tested and the planar 2D dose distributions and dose profiles were compared against dose calculations performed with a Monte Carlo based treatment planning system and gafchromic film. System linearity and RLI image noise against dose were also measured. The maximum difference between beam size measured with RLI and nominal beam size was less than 8% for all the tested beams. The image correction procedure was able to reduce perspective distortion. A novel RLI approach for quality assurance of a small animal irradiator was presented and tested. Results are in agreement with MC dose calculations and gafchromic film measurements.

Severe Heterotopic Ossification in the Skeletal Muscle and Endothelial Cells Recruitment to Chondrogenesis Are Enhanced by Monocyte/Macrophage Depletion.

Altered macrophage infiltration upon tissue damage results in inadequate healing due to inappropriate remodeling and stem cell recruitment and differentiation. We investigated in vivo whether cells of endothelial origin phenotypically change upon heterotopic ossification induction and whether infiltration of innate immunity cells influences their commitment and alters the ectopic bone formation. Liposome-encapsulated clodronate was used to assess macrophage impact on endothelial cells in the skeletal muscle upon acute damage in the ECs specific lineage-tracing Cdh5CreERT2:R26REYFP/dtTomato transgenic mice. Macrophage depletion in the injured skeletal muscle partially shifts the fate of ECs toward endochondral differentiation. Upon ectopic stimulation of BMP signaling, monocyte depletion leads to an enhanced contribution of ECs chondrogenesis and to ectopic bone formation, with increased bone volume and density, that is reversed by ACVR1/SMAD pathway inhibitor dipyridamole. This suggests that macrophages contribute to preserve endothelial fate and to limit the bone lesion in a BMP/injury-induced mouse model of heterotopic ossification. Therefore, alterations of the macrophage-endothelial axis may represent a novel target for molecular intervention in heterotopic ossification.

Weak biophoton emission after laser surgery application in soft tissues: Analysis of the optical features.

Nowadays, laser scalpels are commonly used in surgery, replacing the traditional surgical scalpels for several applications involving cutting or ablating living biological tissue. Laser scalpels are generally used to concentrate light energy in a very small-sized area; light energy is then converted in heat by the tissues. In other cases, the fiber glass tip of the laser scalpel is heated to high temperature and used to cut the tissues. Depending on the temperature reached in the irradiated area, different effects are visible in the tissues. In this study, we report the discovery and characterization of the light emitted by soft mammalian biological tissues from seconds to hours after laser surgery application. A laser diode (with hot fiber glass tip) working at 808 nm and commercially available for medical and dentistry applications was used. The irradiated tissues (red meat, chicken breast and fat) showed light emission in the visible range, well detectable with a commercial charge coupled device (CCD) camera. The time decay of the light emission, the laser power effects and the spectral features in the range 500 to 840 nm in the different tissues are here reported.

T-cell tracking using Cerenkov and radioluminescence imaging.

Cancer immunotherapy is a promising strategy based on the ability of the immune system to kill selected cells. In the development of an effective T-cell therapy, the noninvasive cell tracking methods play a crucial role. Here, we investigate the potentialities of T-cell marked with radionuclides in order to detect their localization with imaging techniques in small animal rodents. A protocol to label T-cells with 32 P-ATP was tested and evaluated. The homing of 32 P-ATP labeled T lymphocytes was investigated by Cerenkov luminescence imaging (CLI) and radioluminescence imaging (RLI). The first approach relies on the acquisition of Cerenkov photons produced by the beta particles emitted by the 32 P internalized by lymphocytes; the second one on the detection of photons coming from the conversion of radioactive energy in light done by scintillator crystals layered on the animals. The results show that T-cell biodistribution can be optically observed by both CLI and RLI in small animal rodents in in vivo and ex vivo acquisitions. T-cell localization in the tumor mass was definitively confirmed by flow cytometry.

Weak light emission of soft tissues induced by heating.

The main goal of this work is to show that soft tissue interaction with high-intensity focused ultrasound (HIFU) or direct heating leads to a weak light emission detectable using a small animal optical imaging system. Our results show that the luminescence signal is detectable after 30 min of heating, resembling the time scale of delayed luminescence. The imaging of a soft tissue after heating it using an HIFU field shows that the luminescence pattern closely matches the shape of the cone typical of the HIFU beam. We conclude that heating a soft tissue using two different sources leads to the emission of a weak luminescence signal from the heated region with a decay half-life of a few minutes (4 to 6 min). The origin of such light emission needs to be further investigated.

Optical emission of 223 Radium: in vitro and in vivo preclinical applications.

223 Radium (223 Ra) is widely used in nuclear medicine to treat patients with osseous metastatic prostate cancer. In clinical practice 223 Ra cannot be imaged directly; however, gamma photons produced by its short-lived daughter nuclides can be captured by conventional gamma cameras. In this work, we show that 223 Ra and its short-lived daughter nuclides can be detected with optical imaging techniques. The light emission of 223 Ra was investigated in vitro using different setups in order to clarify the mechanism of light production. The results demonstrate that the luminescence of the 223 Ra chloride solution, usually employed in clinical treatments, is compatible with Cerenkov luminescence having an emission spectrum that is almost indistinguishable from CR one. This study proves that luminescence imaging can be successfully employed to detect 223 Ra in vivo in mice by imaging whole body 223 Ra biodistribution and more precisely its uptake in bones.

Theranostic Role of 32P-ATP as Radiopharmaceutical for the Induction of Massive Cell Death within Avascular Tumor Core.

Drug inaccessibility to vast areas of the tumor parenchyma is amongst the major hurdles for conventional therapies. Treatment efficacy rapidly decreases with distance from vessels and most of the tumor cells survive therapy. Also, between subsequent cycles of treatment, spared cancer cells replace those killed near the vessels, improving their access to nutrients, boosting their proliferation rate, and thus enabling tumor repopulation. Because of their property of "acting at a distance," radioisotopes are believed to overcome the physical barrier of vascular inaccessibility. Methods A novel molecular imaging tool called Cerenkov Luminescence Imaging (CLI) was employed for the detection of Cerenkov radiation emitted by beta particles, allowing in vivo tracking of beta-emitters. More precisely we investigated using a xenograft model of colon carcinoma the potential use of 32P-ATP as a novel theranostic radiopharmaceutical for tracing tumor lesions while simultaneously hampering their growth. Results Our analyses demonstrated that 32P-ATP injected into tumor-bearing mice reaches tumor lesions and persists for days and weeks within the tumor parenchyma. Also, the high-penetrating beta particles of 32P-ATP exert a "cross-fire" effect that induces massive cell death throughout the entire tumor parenchyma including core regions. Conclusion Our findings suggest 32P-ATP treatment as a potential approach to complement conventional therapies that fail to reach the tumor core and to prevent tumor repopulation.

Radioluminescence from Tc-99m in glass predicts local dose.

The widely-used gamma-emitter Tc-99m has been shown to lead to optical emissions in mice and glass. We investigated the possibility that these emissions are due to the Cerenkov effect and whether the light emitted is proportional to local dose. By using a Geant4 Monte Carlo model matched to an experimental measurement, we show that the light detected by a small animal optical imaging system provides a 2D map of the dose throughout a glass sample. We conclude that radioluminescence from Tc-99m can be used to quantitatively measure dose in transparent materials, which could have applications in dosimetry and quality assurance.

Monte Carlo simulations support non-Cerenkov radioluminescence production in tissue.

There is experimental evidence for the production of non-Cerenkov radioluminescence in a variety of materials, including tissue. We constructed a Geant4 Monte Carlo simulation of the radiation from P32 and Tc99m interacting in chicken breast and used experimental imaging data to model a scintillation-like emission. The same radioluminescence spectrum is visible from both isotopes and cannot otherwise be explained through fluorescence or filter miscalibration. We conclude that chicken breast has a near-infrared scintillation-like response with a light yield three orders of magnitude smaller than BGO.

High-throughput screening for modulators of ACVR1 transcription: discovery of potential therapeutics for fibrodysplasia ossificans progressiva.

The ACVR1 gene encodes a type I receptor of bone morphogenetic proteins (BMPs). Activating mutations in ACVR1 are responsible for fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by congenital toe malformation and progressive heterotopic endochondral ossification leading to severe and cumulative disability. Until now, no therapy has been available to prevent soft-tissue swelling (flare-ups) that trigger the ossification process. With the aim of finding a new therapeutic strategy for FOP, we developed a high-throughput screening (HTS) assay to identify inhibitors of ACVR1 gene expression among drugs already approved for the therapy of other diseases. The screening, based on an ACVR1 promoter assay, was followed by an in vitro and in vivo test to validate and characterize candidate molecules. Among compounds that modulate the ACVR1 promoter activity, we selected the one showing the highest inhibitory effect, dipyridamole, a drug that is currently used as a platelet anti-aggregant. The inhibitory effect was detectable on ACVR1 gene expression, on the whole Smad-dependent BMP signaling pathway, and on chondrogenic and osteogenic differentiation processes by in vitro cellular assays. Moreover, dipyridamole reduced the process of heterotopic bone formation in vivo Our drug repositioning strategy has led to the identification of dipyridamole as a possible therapeutic tool for the treatment of FOP. Furthermore, our study has also defined a pipeline of assays that will be useful for the evaluation of other pharmacological inhibitors of heterotopic ossification.

Bremsstrahlung radiation detection for small animal imaging using a CCD detector.

The use of optical methods for the detection of radionuclides is becoming an established tool for preclinical molecular imaging experiments. In this paper we present a set of proof of principle experiments showing that planar bremsstrahlung radiation images can be detected with an intensifying screen using a small animal optical imager based on charge coupled device detector. We develop a bremsstrahlung source using a (32)P-ATP vial placed in a Plexiglas box, the source with an intensifying screen on top was placed inside a small animal optical imaging system. Bremsstrahlung radiation images were produced with the (32)P-ATP source only and also with a pair of pliers placed between the source and the screen. We found that the pair of pliers absorption image matches the shape of the object. Spatial resolution measurements were not performed however, the bremsstrahlung image of the pliers show that the resolution is relatively poor due to a large penumbra effect. We conclude that it is possible to produce planar bremsstrahlung images using optical imaging devices.

Human malignant mesothelioma is recapitulated in immunocompetent BALB/c mice injected with murine AB cells.

Malignant Mesothelioma is a highly aggressive cancer, which is difficult to diagnose and treat. Here we describe the molecular, cellular and morphological characterization of a syngeneic system consisting of murine AB1, AB12 and AB22 mesothelioma cells injected in immunocompetent BALB/c mice, which allows the study of the interplay of tumor cells with the immune system. Murine mesothelioma cells, like human ones, respond to exogenous High Mobility Group Box 1 protein, a Damage-Associated Molecular Pattern that acts as a chemoattractant for leukocytes and as a proinflammatory mediator. The tumors derived from AB cells are morphologically and histologically similar to human MM tumors, and respond to treatments used for MM patients. Our system largely recapitulates human mesothelioma, and we advocate its use for the study of MM development and treatment.

Unified approach for bioluminescence, Cerenkov, ß, X and γ rays imaging.

The goal of this work is to demonstrate that a CCD-based system can be used as a unified device which allows visible, ß, X and γ rays imaging. A system composed of a CCD coupled with lens mounted on a black light-tight box and a high resolution intensifying screen for the radiations conversion were used. In order to investigate the detection of different type of radiations in vitro and in vivo experiments were performed. The comparison of the results obtained with our prototype and those obtained with dedicated commercial devices showed a good agreement.