Clonogenic assay and computational modeling using real cell images to study physical enhancement and cellular sensitization induced by metal nanoparticles under MV and kV X-ray irradiation.

This study was initiated due to the physically unexplainable tumor controls resulting from metal nanoparticle (MNP) experiments even under MV X-ray irradiation. A more accurate explanation of the mechanism of radiosensitization induced by MNP is warranted, considering both its physical dose enhancement and biological sensitization, as related research is lacking. Thus, we aimed to examine the intricate dynamics involved in MNP-induced radiosensitization. We conducted specifically designed clonogenic assays for the A549 lung cancer cell line with MNP irradiated by 6 MV and 300 kVp X-rays. Two types of MNP were employed: one based on iron oxide, promoting ferroptosis, and the other on gold nanoparticles known for inducing a significant dose enhancement, particularly at low-energy X-rays. We introduced the lethality enhancement factor (LEF) as the fraction in the cell killing attributed to biological sensitization. Subsequently, Monte Carlo simulations were conducted to evaluate the radial dose profiles for each MNP, corresponding to the physical enhancement. Finally, the local effect model was applied to the clonogenic assay results on real cell images. The LEF and the dose enhancement in the cytoplasm were incorporated to increase the accuracy in the average lethal events and, consequently, in the survival fraction. The results reveal an increased cell killing for both of the MNP under MV and kV X-ray irradiation. In both types of MNP, the LEF reveals a biological sensitization evident. The sensitizer enhancement ratio, derived from the calculations, exhibited only 3% and 1% relative differences compared to the conventional linear-quadratic model for gold and ferroptosis inducer nanoparticles, respectively. These findings indicate that MNPs sensitize cells via radiation through mechanisms akin to ferroptosis inducers, not exclusively relying on a physical dose enhancement. Their own contributions to survival fractions were successfully integrated into computational modeling.

Synergistic Effect of Ferroptosis-Inducing Nanoparticles and X-Ray Irradiation Combination Therapy.

Ferroptosis, characterized by the induction of cell death via lipid peroxidation, has been actively studied over the last few years and has shown the potential to improve the efficacy of cancer nanomedicine in an iron-dependent manner. Radiation therapy, a common treatment method, has limitations as a stand-alone treatment due to radiation resistance and safety as it affects even normal tissues. Although ferroptosis-inducing drugs help alleviate radiation resistance, there are no safe ferroptosis-inducing drugs that can be considered for clinical application and are still in the research stage. Here, the effectiveness of combined treatment with radiotherapy with Fe and hyaluronic acid-based nanoparticles (FHA-NPs) to directly induce ferroptosis, considering the clinical applications is reported. Through the induction of ferroptosis by FHA-NPs and apoptosis by X-ray irradiation, the therapeutic efficiency of cancer is greatly improved both in vitro and in vivo. In addition, Monte Carlo simulations are performed to assess the physical interactions of the X-rays with the iron-oxide nanoparticle. The study provides a deeper understanding of the synergistic effect of ferroptosis and X-ray irradiation combination therapy. Furthermore, the study can serve as a valuable reference for elucidating the role and mechanisms of ferroptosis in radiation therapy.

Radiosensitization by Au-nanofilm at micrometer level using confocal Raman spectroscopy.

PURPOSE: To measure the radiosensitization by an Au-nanofilm (GNF) at a micrometer level on a radiochromic film (RCF) using confocal Raman spectroscopy (CRS). METHODS: Unlaminated radiochromic films were irradiated by 200 kVp x-ray from 0.3 to 50 Gy to obtain a calibration curve. Raman spectra of these films were measured by positioning the postirradiated RCF perpendicular to the CRS monochromatic beam and reading a depth profile of the film along the lateral axis. The Raman peak corresponding to the C ≡ C peak was obtained from a region of interest of 100 × 5 µm2 . To investigate the radiosensitization by GNF, two sets of RCF, one attached to a 100-nm thick GNF and the other without GNF were irradiated at 0.5 Gy by 50 and 120 kVp X-rays. The spatial resolution of the CRS on the RCF was quantified by the modulation transfer function method (MTF). Thus, in the spatial resolution determined by MTF, the doses deposited on the films were evaluated. The dose enhancement factor (DEF) was obtained in the measurable micro-size by comparing doses deposited on the RCFs with and without GNF. To verify the experimental results, Monte Carlo simulations following the experimental set up were performed using Geant4. In addition, analytical calculations for the radiosensitization by GNF were carried out. RESULTS: The confocal Raman spectroscopy on the RCF achieved a spatial resolution of ~6 µm. An experimental DEF within the first 6 µm depth from the surface of RCF was found to be 17.9 for 50 kVp and 14.7 for 120 kVp. The DEF for the same depth obtained by MC and analytical calculations was 13.53 and 9.75 for 50 kVp, and 10.63 and 6.67 for 120 kVp, respectively. CONCLUSIONS: The experimental DEF as a function of the distance from GNF was consistent with data from previous studies and the MC simulations, supporting that CRS in conjunction with the RCF is a feasible micrometer-resolution dosimeter.

Characterization of radiochromic films as a micrometer-resolution dosimeter by confocal Raman spectroscopy.

PURPOSE: Micrometer spatial resolution dosimetry has become inevitable for advanced radiotherapy techniques. A new approach using radiochromic films was developed to measure a radiation dose at a micrometer spatial resolution by confocal Raman spectroscopy. METHODS: The commercial radiochromic films (RCF), EBT3 and EBT-XD, were irradiated with known doses using 50, 100, 200, and 300 kVp, and 6-MV x rays. The dose levels ranged from 0.3 to 50 Gy. The Raman mapping technique developed in our early study was used to readout an area of 100 × 100 µm2 on RCF with improved lateral and depth resolutions with confocal Raman spectrometry. The variation in Raman spectra of C-C-C deformation and C≡C stretching modes of diacetylene polymers around 676 and 2060 cm-1 , respectively, as a function of therapeutic x-ray doses, was measured. The single peak (SP) of C≡C and the peak ratio (PR) of C≡C band height to C-C-C band height with a spatial resolution of 10 µm on both types of RCF were evaluated, averaged, and plotted as a function of dose. An achievable spatial resolution, clinically useful dose range, dosimetric sensitivity, dose uniformity, and postirradiation stability as well as the orientation, energy, and dose rate dependence, of both types of RCFs, were characterized by the technique developed in this study. RESULTS: A spatial resolution on RCF achieved by SP and PR methods was ~4.5 and ~2.9 µm, respectively. Raman spectroscopy data showed dose nonuniformity of ~11% in SP method and <3% in PR method. The SP method provided dose ranges of up to ~10 and ~20 Gy for EBT3 and EBT-XD films, respectively while the PR method up to ~30 and ~50 Gy. The PR method diminished the orientation effect. The percent difference between landscape and portrait orientations for the EBT3 and the EBT-XD films at 4 Gy had an acceptable level of 1.2% and 2.4%, respectively. With both SP and PR methods, the EBT3 and the EBT-XD films showed weak energy (within ~10% and ~3% for SP and PR methods, respectively) and dose rate dependence (within ~5% and ~3% for SP and PR methods, respectively) and had a stable response after 24-h postirradiation. CONCLUSIONS: A technique for micrometer-resolution dosimetry was successfully developed by detecting radiation-induced Raman shift on EBT3 and EBT-XD. Both types of RCFs were suitable for micrometer-resolution dosimetry using CRS. With CRS both lateral and depth resolutions on RCF were improved. The PR method provided superior characteristics in dose uniformity, dose ranges, orientation dependence, and laser effect for both types of RCFs. The overall dosimetric characteristics of the RCFs determined by this technique were similar to those known by optical density scanning. The CRS with the PR method is advantageous over other the traditional scanning systems as a spatial resolution of <10 µm on RCF can be achieved with less deviations.