Treatment protocols of cancer combining radiotherapy and nanoparticles are rapidly evolving. To evaluate their efficacy, spheroids provide a 3D in vitro model that better reflects tumor architecture than traditional 2D cell cultures. This article presents a workflow to prepare and characterize spheroids, optimize the protocols for irradiation with medical photon and ion beams and for exposure to nanoparticles, and, finally, to evaluate the effects of radiation, nanoparticles, and their combination on spheroids. Illustrations with HeLa, U-87 MG, and BxPC-3 tumor cell lines and HDFn are reported. Using this workflow, we observed that spheroids exhibit variable cell organization and interstitial spaces, which affect nutrient and oxygen diffusion and, consequently, cell proliferation. These structural differences also affect enzyme diffusion, limiting the applicability of the clonogenic assay in densely packed spheroids, as the assay requires enzymatic disaggregation of the spheroids. The clonogenic assay remains essential for quantitatively comparing spheroid irradiation results with 2D cell culture experiments. It has long been used as the reference method in radiobiology because it assesses mitotic death and long-term proliferative capacity. Protocols were adapted to ensure the feasibility of the clonogenic assay when possible, depending on cell line characteristics. Interstitial spaces also influenced nanoparticle internalization, which was more efficient in spheroids with larger interstitial spaces. This workflow and associated techniques verified the characteristic effect of carbon ion irradiation with a relative biological effectiveness of 3 and demonstrated a 30

This study investigates the effects of platinum nanoparticles (Pt NPs) on breast cancer cell lines (MCF7 and MDA-MB-231) under proton beam irradiation, with a focus on nanoparticle accumulation behavior, cytotoxicity and radiosensitization. Platinum NPs used in this research exhibited a uniform, flower-like morphology with an average size of 26.2 ± 3.9 nm and showed no agglomeration. Cytotoxicity assays revealed that Pt NPs induced time- and concentration-dependent decrease in cell viability, with significant toxic effects observed at concentrations ≥ 0.375 mM. Moreover, holotomographic imaging demonstrated dynamic nanoparticle accumulation in both cell lines, initially localizing at the membrane and progressively internalizing over time. Importantly, no specific accumulation site preference was detected. Quantitative analysis showed that Pt NP uptake by MCF7 cells was approximately twice that of MDA-MB-231 cells, with simulation models aligning closely with experimental data (86 % and 90 % fit, respectively). Proton irradiation studies indicated a marked radiosensitizing effect of Pt NPs, with cell viability reduced by up to 85 % at higher NP concentrations and radiation doses (10 Gy). These findings underscore the potential of flower-like Pt NPs as effective radiosensitizers in proton therapy, owing to their high cellular uptake, surface reactivity and cytotoxicity.

Combination of nanoagents with radiations has opened up new perspectives in cancer treatment, improving both tumor diagnosis and therapeutic index. This work presents the first investigation of an innovative strategy that combines porous metal-organic frameworks (nanoMOFs) loaded with the anti-cancer drug Gemcitabine monophosphate (GemMP) and particle therapy-a globally emerging technique that offers more precise radiation targeting and enhanced biological efficacy compared to conventional radiotherapy. This radiochemotherapy has been confronted with two major obstacles limiting the efficacy of therapeutics when tested in vivo: (i) the presence of hypoxia, one of the most important causes for radiotherapy failure and (ii) the presence of a microenvironment, main biological barrier to the direct penetration of nanoparticles into cancer cells. On the one hand, this study explore the effects of hypoxia on drug delivery systems in combination with radiation, demonstrating that GemMP-loaded nanoMOFs significantly enhance the anticancer efficacy of particle therapy under both normoxic (pO2 = 20 %) and hypoxic (pO2 = 0.5 %) conditions. Notably, the presence of GemMP-loaded nanoMOFs allows the irradiation dose to be reduced by 1.4-fold in normoxia and at least 1.6-fold in hypoxia, achieving the same cytotoxic effect (SF=10 %) as carbon or helium ions alone. Synergistic effects between GemMP-loaded nanoMOFs and radiations have been observed and quantified. On the other hand, we also highlighted the ability of the nanoMOFs to diffuse through an extracellular matrix and accumulate in cells. An higher effect of the encapsulated GemMP than the free drug was observed, confirming the key role of the nanoMOFs in transporting the active substance to the cancer cells as a Trojan horse. This paves the way to the design of "all-in-one" nanodrugs where each component plays a role in the optimization of cancer therapy to maximize cytotoxic effects on hypoxic tumor cells while minimizing toxicity on healthy tissue.

Noble metal materials, especially platinum nanoparticles (Pt NPs), have immense potential in nanomedicine as therapeutic agents on account of their high electron density and their high surface area. Intravenous injection is proposed as the best mode to deliver the product to patients. However, our understanding of the reaction of nanoparticles with blood components, especially proteins, is far behind the explosive development of these agents. Using synchrotron radiation circular dichroism (SRCD), we investigated the structural and stability changes of human serum albumin (HSA) upon interaction with PEG-OH coated Pt NPs at nanomolar concentrations, conditions potentially encountered for intravenous injection. There is no strong complexation found between HSA and Pt NPs. However, for the highest molar ratio of NP:HSA of 1:1, an increase of 18 °C in the thermal unfolding of HSA was observed, which is attributed to increased thermal stability of HSA generated by preferential hydration. This work proposes a new and fast method to probe the potential toxicity of nanoparticles intended for clinical use with intravenous injection.

Radiation therapy is an effective and localised method for cancer treatment and its efficacy is considerably improved by using radioenhancing agents. Nanoparticles containing high atomic number (Z) elements have emerged as an appealing class of enhancers. This work reports the first example of radioenhancers based on ultrasmall platinum (Pt)-polycyanometallate nanoparticles. Importantly, both novel nano-enhancers (Pt[Pt(CN)4] and Pt[Fe(CN)6]) are prepared in fairly large quantities using a water-based, surfactant-free, green route. The post-coating of the inorganic cores by different biocompatible polymers is straightforward and allows probing of the influence of the coating on the radiotherapeutic efficiency of the nanosystems. The structural/chemical integrity is maintained after high doses of gamma-radiation and high colloidal stability is obtained in biological media. At 250 mu M total metal concentration, these nanoparticles are non-toxic to cells, but when coupled with gamma-radiation, they amplify apoptosis of HeLa cells by 20% at 4.5 Gray, which rises to 60% at larger doses. Furthermore, irradiation studies conducted on a model DNA probe evidence that the amount of lethal Double Strand Breaks (DSB) is significantly enhanced in the presence of these novel Pt-based radioenhancers. The radiotherapeutic efficiency of these versatile coordination nanoparticles obtained using a cheap, fast and green process opens interesting possibilities in the field of nanomedicine. Two novel nanoparticles based on Pt-containing coordination networks, produced by a green method, reveal important radioenhancement activity on cancerous cells.

Clinical trials incorporating metallic nanoparticles (NPs) have recently begun. Radiotherapy planning does not take into account NPs concentrations observed in the patients' target volumes. In the framework of the NANOCOL clinical trial including patients treated for locally advanced cervical cancers, this study proposes a complete method to evaluate the radiation-induced biological effects of NPs. For this, calibration phantom was developed and MRI sequences with variable flip angles were acquired. This process allowed the quantification of NPs in the tumor of 4 patients, which was compared to the results of mass spectrometry obtained from 3 patient biopsies. The concentration of the NPs was reproduced in 3D cell models. Based on clonogenic assays, the radio-enhancement effects were quantified for radiotherapy and brachytherapy, and the impact in terms of local control was evaluated. T1 signal change in GTVs revealed NPs accumulation ∼12.4 μmol/L, in agreement with mass spectrometry. Radio-enhancement effects of about 15 % at 2 Gy were found for both modalities, with a positive impact on local tumor control. Even if further follow-up of patients in this and subsequent clinical trials will be necessary to assess the reliability of this proof of concept, this study opens the way to the integration of a dose modulation factor to better take into account the impact of NPs in radiotherapy treatment.

Personalized medicine approach in radiotherapy requires the delivery of precise dose to the tumor. The concept is to increase the effectiveness of radiotherapy while sparing the surrounding heathy tissue. This can be achieved by the use of high-Z metal-based nanoparticles (NPs) as radio-enhancers and PET imaging for mapping NPs distribution to guide the irradiation. In the present study, radio-enhancing platinum NPs were radiolabeled and imaged to assess their pharmacokinetics over time. PET imaging of these NPs revealed high enhanced permeation and retention effect. The maximal tumor accumulation (4.8 ± 0.8 %ID/cc) was observed at 24 h post-injection along with persistent accumulation of the NPs, especially at the tumor ring, even after several days. These properties positively suggest the potential clinical use of these NPs.

Correction for 'Quantifying nanotherapeutic penetration using a hydrogel-based microsystem as a new 3D in vitro platform' by Saba Goodarzi et al., Lab Chip, 2021, 21, 2495-2510, DOI: 10.1039/D1LC00192B.

The huge gap between 2D in vitro assays used for drug screening and the in vivo 3D physiological environment hampered reliable predictions for the route and accumulation of nanotherapeutics in vivo. For such nanotherapeutics, multi-cellular tumour spheroids (MCTS) are emerging as a good alternative in vitro model. However, the classical approaches to produce MCTS suffer from low yield, slow process, difficulties in MCTS manipulation and compatibility with high-magnification fluorescence optical microscopy. On the other hand, spheroid-on-chip set-ups developed so far require a practical knowledge of microfluidics difficult to transfer to a cell biology laboratory. We present here a simple yet highly flexible 3D model microsystem consisting of agarose-based microwells. Fully compatible with the multi-well plate format conventionally used in cell biology, our simple process enables the formation of hundreds of reproducible spheroids in a single pipetting. Immunostaining and fluorescence imaging including live high-resolution optical microscopy can be performed in situ, with no manipulation of spheroids. As a proof of principle of the relevance of such an in vitro platform for nanotherapeutic evaluation, this study investigates the kinetics and localisation of nanoparticles within colorectal cancer MCTS cells (HCT-116). The nanoparticles chosen are sub-5 nm ultrasmall nanoparticles made of polysiloxane and gadolinium chelates that can be visualized in MRI (AGuIX®, currently implicated in clinical trials as effective radiosensitizers for radiotherapy) and confocal microscopy after addition of Cy5.5. We show that the amount of AGuIX® nanoparticles within cells is largely different in 2D and 3D. Using our flexible agarose-based microsystems, we are able to resolve spatially and temporally the penetration and distribution of AGuIX® nanoparticles within MCTS. The nanoparticles are first found in both extracellular and intracellular space of MCTS. While the extracellular part is washed away after a few days, we evidenced intracellular localisation of AGuIX®, mainly within the lysosomal compartment, but also occasionally within mitochondria. Hence, our agarose-based microsystem appears as a promising 3D in vitro user-friendly platform for investigation of nanotherapeutic transport, ahead of in vivo studies.

2D cell cultures are commonly used to rapidly evaluate the therapeutic potential of various treatments on living cells. However, the effects of the extracellular matrix (ECM) including the 3D arrangement of cells and the complex physiology of native environment are missing, which makes these models far from in vivo conditions. 3D cell models have emerged in preclinical studies to simulate the impact of the ECM and partially bridge the gap between monolayer cultures and in vivo tissues. To date, the difficulty to handle the existing 3D models, the cost of their production and their poor reproducibility have hindered their use. Here, we present a reproducible and commercially available "3D cell collagen-based model" (3D-CCM) that allows to study the influence of the matrix on nanoagent uptake and radiation effects. The cell density in these samples is homogeneous. The oxygen concentration in the 3D-CCM is tunable, which opens the opportunity to investigate hypoxic effects. In addition, thanks to the intrinsic properties of the collagen, the second harmonic imaging microscopy may be used to probe the whole volume and visualize living cells in real-time. Thus, the architecture and composition of 3D-CCMs as well as the impact of various therapeutic strategies on cells embedded in the ECM is observed directly. Moreover, the disaggregation of the collagen matrix allows recovering of cells without damaging them. It is a major advantage that makes possible single cell analysis and quantification of treatment effects using clonogenic assay. In this work, 3D-CCMs were used to evaluate the correlative efficacies of nanodrug exposure and medical radiation on cells contained in a tumor like sample. A comparison with monolayer cell cultures was performed showing the advantageous outcome and the higher potential of 3D-CCMs. This cheap and easy to handle approach is more ethical than in vivo experiments, thus, giving a fast evaluation of cellular responses to various treatments.

High-Z metallic nanoparticles (NPs) are new players in the therapeutic arsenal against cancer, especially radioresistant cells. Indeed, the presence of these NPs inside malignant cells is believed to enhance the effect of ionizing radiation by locally increasing the dose deposition. In this context, the potential of platinum nanoparticles (PtNPs) as radiosensitizers was investigated in two breast cancer cell lines, T47D and MDA-MB-231, showing a different radiation sensitivity. PtNPs were internalized in the two cell lines and localized in lysosomes and multivesicular bodies. Analyses of cell responses in terms of clonogenicity, survival, mortality, cell-cycle distribution, oxidative stress, and DNA double-strand breaks did not reveal any significant enhancement effect when cells were pre-exposed to PtNPs before being irradiated, as compared to radiation alone. This result is different from that reported in a previous study performed, under the same conditions, on cervical cancer HeLa cells. This shows that the efficacy of radio-enhancement is strongly cell-type-dependent. Simulation of the early stage ionization processes, taking into account the irradiation characteristics and realistic physical parameters in the biological sample, indicated that PtNPs could weakly increase the dose deposition (by 3%) in the immediate vicinity of the nanoparticles. Some features that are potentially responsible for the biological effect could not be taken into account in the simulation. Thus, chemical and biological effects could explain this discrepancy. For instance, we showed that, in these breast cancer cell lines, PtNPs exhibited ambivalent redox properties, with an antioxidant potential which could counteract the radio-enhancement effect. This work shows that the efficacy of PtNPs for enhancing radiation effects is strongly cell-dependent and that no effect is observed in the case of the breast cancer cell lines T47D and MDA-MB-231. Thus, more extensive experiments using other relevant biological models are needed in order to evaluate such combined strategies, since several clinical trials have already demonstrated the success of combining nanoagents with radiotherapy in the treatment of a range of tumor types.

Nanomedicine has stepped into the spotlight of radiation therapy over the last two decades. Nanoparticles (NPs), especially metallic NPs, can potentiate radiotherapy by specific accumulation into tumors, thus enhancing the efficacy while alleviating the toxicity of radiotherapy. Water radiolysis is a simple, fast and environmentally-friendly method to prepare highly controllable metallic nanoparticles in large scale. In this study, we used this method to prepare biocompatible PEGylated (with Poly(Ethylene Glycol) diamine) platinum nanoflowers (Pt NFs). These nanoagents provide unique surface chemistry, which allows functionalization with various molecules such as fluorescent markers, drugs or radionuclides. The Pt NFs were produced with a controlled aggregation of small Pt subunits through a combination of grafted polymers and radiation-induced polymer cross-linking. Confocal microscopy and fluorescence lifetime imaging microscopy revealed that Pt NFs were localized in the cytoplasm of cervical cancer cells (HeLa) but not in the nucleus. Clonogenic assays revealed that Pt NFs amplify the gamma rays induced killing of HeLa cells with a sensitizing enhancement ratio (SER) of 23%, thus making them promising candidates for future cancer radiation therapy. Furthermore, the efficiency of Pt NFs to induce nanoscopic biomolecular damage by interacting with gamma rays, was evaluated using plasmids as molecular probe. These findings show that the Pt NFs are efficient nano-radio-enhancers. Finally, these NFs could be used to improve not only the performances of radiation therapy treatments but also drug delivery and/or diagnosis when functionalized with various molecules.

Purpose Metal-based nanoparticles (M-NPs) have attracted great attention in nanomedicine due to their capacity to amplify and improve the tumor targeting of medical beams. However, their simple, efficient, high-yield and reproducible production remains a challenge. Currently, M-NPs are mainly synthesized by chemical methods or radiolysis using toxic reactants. The waste of time, loss of material and potential environmental hazards are major limitations. Materials and Methods This work proposes a simple, fast and green strategy to synthesize small, non-toxic and stable NPs in water with a 100% production rate. Ionizing radiation is used to simultaneously synthesize and sterilize the containing NPs solutions. The synthesis of platinum nanoparticles (Pt NPs) coated with biocompatible poly(ethylene glycol) ligands (PEG) is presented as proof of concept. The physicochemical properties of NPs were studied by complementary specialized techniques. Their toxicity and radio-enhancing properties were evaluated in a cancerous in vitro model. Using plasmid nanoprobes, we investigated the elementary mechanisms underpinning radio-enhancement. Results and Discussion Pt NPs showed nearly spherical-like shapes and an average hydrodynamic diameter of 9 nm. NPs are zero-valent platinum successfully coated with PEG. They were found non-toxic and have the singular property of amplifying cell killing induced by γ-rays (14%) and even more, the effects of carbon ions (44%) used in particle therapy. They induce nanosized-molecular damage, which is a major finding to potentially implement this protocol in treatment planning simulations. Conclusion This new eco-friendly, fast and simple proposed method opens a new era of engineering water-soluble biocompatible NPs and boosts the development of NP-aided radiation therapies.

This roadmap outlines the potential roles of metallic nanoparticles (MNPs) in the field of radiation therapy. MNPs made up of a wide range of materials (from Titanium, Z = 22, to Bismuth, Z = 83) and a similarly wide spectrum of potential clinical applications, including diagnostic, therapeutic (radiation dose enhancers, hyperthermia inducers, drug delivery vehicles, vaccine adjuvants, photosensitizers, enhancers of immunotherapy) and theranostic (combining both diagnostic and therapeutic), are being fabricated and evaluated. This roadmap covers contributions from experts in these topics summarizing their view of the current status and challenges, as well as expected advancements in technology to address these challenges.

The gadolinium-based nanoagent named AGuIX® is a unique radiosensitizer and contrast agent which improves the performance of radiotherapy and medical imaging. Currently tested in clinical trials, AGuIX® is administrated to patients via intravenous injection. The presence of nanoparticles in the blood stream may induce harmful effects due to undesired interactions with blood components. Thus, there is an emerging need to understand the impact of these nanoagents when meeting blood proteins. In this work, the influence of nanoagents on the structure and stability of the most abundant blood protein, human serum albumin, is presented. Synchrotron radiation circular dichroism showed that AGuIX® does not bind to the protein, even at the high ratio of 45 nanoparticles per protein at 3 mg/L. However, it increases the stability of the albumin. Isothermal thermodynamic calorimetry and fluorescence emission spectroscopy demonstrated that the effect is due to preferential hydration processes. Thus, this study confirms that intravenous injection of AGuIX® presents limited risks of perturbing the blood stream. In a wider view, the methodology developed in this work may be applied to rapidly evaluate the impact and risk of other nano-products that could come into contact with the bloodstream.

Ultrasmall polyaminocarboxylate-coated gold nanoparticles (NPs), Au@DTDTPA and Au@TADOTAGA, that have been recently developed exhibit a promising potential for image-guided radiotherapy. In order to render the radiosensitizing effect of these gold nanoparticles even more efficient, the study of their localization in cells is required to better understand the relation between the radiosensitizing properties of the agents and their localization in cells and in tumors. To achieve this goal, post-functionalization of Au@DTDTPA nanoparticles by near-infrared (NIF) organic dyes (aminated derivative of cyanine 5, Cy5-NH2) was performed. The immobilization of organic Cy5-NH2 dyes onto the gold nanoparticles confers to these radiosensitizers fluorescence properties which can be exploited for monitoring their internalization in cancerous cells, for determining their localization in cells by fluorescence microscopy (a common and powerful imaging tool in biology), and for following up on their accumulation in tumors after intravenous injection.

The use of nanoparticles, in combination with ionizing radiation, is considered a promising method to improve the performance of radiation therapies. In this work, we engineered mono- and bimetallic core-shell gold-platinum nanoparticles (NPs) grafted with poly (ethylene glycol) (PEG). Their radio-enhancing properties were investigated using plasmids as bio-nanomolecular probes and gamma radiation. We found that the presence of bimetallic Au:Pt-PEG NPs increased by 90% the induction of double-strand breaks, the signature of nanosize biodamage, and the most difficult cell lesion to repair. The radio-enhancement of Au:Pt-PEG NPs were found three times higher than that of Au-PEG NPs. This effect was scavenged by 80% in the presence of dimethyl sulfoxide, demonstrating the major role of hydroxyl radicals in the damage induction. Geant4-DNA Monte Carlo simulations were used to elucidate the physical processes involved in the radio-enhancement. We predicted enhancement factors of 40% and 45% for the induction of nanosize damage, respectively, for mono- and bimetallic nanoparticles, which is attributed to secondary electron impact processes. This work contributed to a better understanding of the interplay between energy deposition and the induction of nanosize biomolecular damage, being Monte Carlo simulations a simple method to guide the synthesis of new radio-enhancing agents.

Nanomedicine recently emerged as a novel strategy to improve the performance of radiotherapy. Herein we report the first application of radioenhancers made of nanoscale metal-organic frameworks (nanoMOFs), loaded with gemcitabine monophosphate (Gem-MP), a radiosensitizing anticancer drug. Iron trimesate nanoMOFs possess a regular porous structure with oxocentered Fe trimers separated by around 5 Å (trimesate linkers). This porosity is favorable to diffuse the electrons emitted from nanoMOFs due to activation by γ radiation, leading to water radiolysis and generation of hydroxyl radicals which create nanoscale damages in cancer cells. Moreover, nanoMOFs act as "Trojan horses", carrying their Gem-MP cargo inside cancer cells to interfere with DNA repair. By displaying different mechanisms of action, both nanoMOFs and incorporated Gem-MP contribute to improve radiation efficacy. The radiation enhancement factor of Gem-MP loaded nanoMOFs reaches 1.8, one of the highest values ever reported. These results pave the way toward the design of engineered nanoparticles in which each component plays a role in cancer treatment by radiotherapy.