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This article presents bioconjugates combining nanoparticles (AGuIX) with nanobodies (VHH) targeting Programmed Death-Ligand 1 (PD-L1, A12 VHH) and Cluster of Differentiation 47 (CD47, A4 VHH) for active tumor targeting. AGuIX nanoparticles offer theranostic capabilities and an efficient biodistribution/pharmacokinetic profile (BD/PK), while VHH's reduced size (15 kDa) allows efficient tumor penetration. Site-selective sortagging and click chemistry were compared for bioconjugation. While both methods yielded bioconjugates with similar functionality, click chemistry demonstrated higher yield and could be used for the conjugation of various VHH. The specific targeting of AGuIX@VHH has been demonstrated in both in vitro and ex vivo settings, paving the way for combined targeted immunotherapies, radiotherapy, and cancer imaging.

In the frame of radiotherapy treatment of cancer, radioresistance remains a major issue that still needs solutions to be overcome. To effectively improve the radiosensitivity of tumors and reduce the damage of radiation to neighboring normal tissues, radiosensitizers have been given increasing attention in recent years. As nanoparticles based on the metal element gadolinium, AGuIX nanoparticles have been shown to increase the radiosensitivity of cancers. Although it is a rare nanomaterial that has entered preclinical trials, the unclear biological mechanism hinders its further clinical application. In this study, we demonstrated the effectiveness of AGuIX nanoparticles in the radiosensitization of triple-negative breast cancer. We found that AGuIX nanoparticles increased the level of DNA damage by compromising the homologous recombination repair pathway instead of the non-homologous end joining pathway. Moreover, the results showed that AGuIX nanoparticles induced apoptosis, but the degree of apoptosis ability was very low, which cannot fully explain their strong radiosensitizing effect. Ferroptosis, the other mode of cell death, was also discovered to play a significant role in radiation sensitization, and AGuIX nanoparticles may regulate the anti-ferroptosis system by inhibiting the NRF2-GSH-GPX4 signaling pathway., (© 2022. The Author(s).)

Radiotherapy suffers from its high-dose radiation-induced systemic toxicity and radioresistance caused by the immunosuppressive tumor microenvironment. Immunotherapy using checkpoint blocking in solid tumors shows limited anticancer efficacy due to insufficient T-cell infiltration and inadequate systemic immune responses. Activation and guiding of irradiation by X-ray (AGuIX) nanoparticles with sizes below 5 nm have entered a phase III clinical trial as efficient radiosensitizers. This study aimed to develop a unique synergistic strategy based on AGuIX-mediated radiotherapy and immune checkpoint blockade to further improve the efficiency for B16 tumor therapy. AGuIX exacerbated radiation-induced DNA damage, cell cycle arrest, and apoptosis on B16 cells. More importantly, it could efficiently induce the immunogenic cell death of irradiated B16 tumor cells, and consequently trigger the maturation of dendritic cells and activation of systemic T-cell responses. Combining AGuIX-mediated radiotherapy with programmed cell death protein 1 blockade demonstrated excellent synergistic therapeutic effects in both bilateral and metastatic B16 tumor models, as indicated by a significant increase in the infiltration of effector CD8 + T cells and effective alleviation of the immunosuppressive tumor microenvironment. Our findings indicate that the synergy between radiosensitization and immunomodulation provides a new and powerful therapy regimen to achieve durable antitumor T-cell responses, which is promising for cancer treatment.

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.

Interest of tumor targeting through EPR effect is still controversial due to intrinsic low targeting efficacy and rare translation to human cancers. Moreover, due to different reasons, it has generally been described for relatively large nanoparticles (NPs) (hydrodynamic diameter > 10 nm). In this review EPR effect will be discussed for ultrasmall NPs using the example of the AGuIX® NP (Activation and Guiding of Irradiation by X-ray) recently translated in clinic. AGuIX® NP is a 4 ± 2 nm hydrodynamic diameter polysiloxane based NP. Since AGuIX® NP biodistribution is monitored by magnetic resonance imaging (MRI) and its activation is triggered by irradiation upon X-rays, this NP is well adapted for a theranostic approach of radiotherapy cancer treatment. Here we show that AGuIX® NP is particularly well suited to benefit from EPR-mediated tumor targeting thanks to an ultrasmall size and efficacy under irradiation at small dose. Indeed, intravenously-injected AGuIX® NP into rodent cancer models passively reached the tumor and revealed no toxicity, favoured by renal clearance. Moreover, translation of AGuIX® NP accumulation and retention into humans carrying brain metastases was validated during a first-in-man phase Ib trial taking advantage of easy biodistribution monitoring by MRI., Competing Interests: Competing Interests: F.L. and O.T. have one patent (WO2011135101) protecting the AGuIX® NP, are employed by NH TherAguix owner of AGuIX® NP, and possess shares of this company. S.D. is employed by NH TherAguix and possesses shares of this company. Y.C. is employed by NH TherAguix., (© The author(s).)

Radiation therapy is a major treatment regimen for more than 50% of cancer patients. The collateral damage induced on healthy tissues during radiation and the minimal therapeutic effect on the organ-of-interest (target) is a major clinical concern. Ultra-small, renal clearable, silica based gadolinium chelated nanoparticles (SiGdNP) provide simultaneous MR contrast and radiation dose enhancement. The high atomic number of gadolinium provides a large photoelectric cross-section for increased photon interaction, even for high-energy clinical radiation beams. Imaging and therapy functionality of SiGdNP were tested in cynomolgus monkeys and pancreatic tumor-bearing mice models, respectively. A significant improvement in tumor cell damage (double strand DNA breaks), growth suppression, and overall survival under clinical radiation therapy conditions were observed in a human pancreatic xenograft model. For the first time, safe systemic administration and systematic renal clearance was demonstrated in both tested species. These findings strongly support the translational potential of SiGdNP for MR-guided radiation therapy in cancer treatment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Since twenty years, many nanoparticles based on high atomic number elements have been developed as radiosensitizers. The design of these nanoparticles is limited by the classical rules associated with the development of nanoparticles for oncology and by the specific ones associated to radiosensitizers, which aim to increase the effect of the dose in the tumor area and to spare the health tissues. For this application, systemic administration of nanodrugs is possible. This paper will discuss the development of AGuIX nanoparticles and will emphasize on this example the critical points for the development of a nanodrug for this application. AGuIX nanoparticles display hydrodynamic diameters of a few nanometers and are composed of polysiloxane and gadolinium chelates. This particle has been used in many preclinical studies and is evaluated for a further phase I clinical trial. Finally, in addition to its high radiosensitizing potential, AGuIX display MRI functionality and can be used as theranostic nanodrug for personalized medicine., (Copyright © 2015 Société française de radiothérapie oncologique (SFRO). Published by Elsevier SAS. All rights reserved.)

A rapid development of gadolinium-based nanoparticles is observed due to their attractive properties as MRI-positive contrast agents. Indeed, they display high relaxivity, adapted biodistribution and passive uptake in the tumor thanks to enhanced permeability and retention effect. In addition to these imaging properties, it has been recently shown that they can act as effective radiosensitizers under different types of irradiation (radiotherapy, neutron therapy or hadron therapy). These new therapeutic modalities pave the way to therapy guided by imaging and to personalized medicine.

A new efficient type of gadolinium-based theranostic agent (AGuIX®) has recently been developed for MRI-guided radiotherapy (RT). These new particles consist of a polysiloxane network surrounded by a number of gadolinium chelates, usually 10. Owing to their small size (<5 nm), AGuIX typically exhibit biodistributions that are almost ideal for diagnostic and therapeutic purposes. For example, although a significant proportion of these particles accumulate in tumours, the remainder is rapidly eliminated by the renal route. In addition, in the absence of irradiation, the nanoparticles are well tolerated even at very high dose (10 times more than the dose used for mouse treatment). AGuIX particles have been proven to act as efficient radiosensitizers in a large variety of experimental in vitro scenarios, including different radioresistant cell lines, irradiation energies and radiation sources (sensitizing enhancement ratio ranging from 1.1 to 2.5). Pre-clinical studies have also demonstrated the impact of these particles on different heterotopic and orthotopic tumours, with both intratumoural or intravenous injection routes. A significant therapeutical effect has been observed in all contexts. Furthermore, MRI monitoring was proven to efficiently aid in determining a RT protocol and assessing tumour evolution following treatment. The usual theoretical models, based on energy attenuation and macroscopic dose enhancement, cannot account for all the results that have been obtained. Only theoretical models, which take into account the Auger electron cascades that occur between the different atoms constituting the particle and the related high radical concentrations in the vicinity of the particle, provide an explanation for the complex cell damage and death observed.

Gadolinium based Small Rigid Plaforms (SRPs) have previously demonstrated their efficiency for multimodal imaging and radiosensitization. Since the RGD sequence is well-known to be highly selective for αvβ3 integrins, a cyclic pentapeptide containing the RGD motif (cRGDfK) has been grafted onto the SRP surface. An appropriate protocol led to the grafting of two targeting ligands per nano-object. The resulting nanoparticles have demonstrated a strong association with αvβ3 integrins in comparison with cRADfK grafted SRPs as negative control. Flow cytometry and fluorescence microscopy have also been used to highlight the ability of the nanoparticles to target efficiently HEK293(β3) and U87MG cells. Finally the grafted radiosensitizing nanoparticles were intravenously injected into Nude mice bearing subcutaneous U87MG tumors and the signal observed by optical imaging was twice as high for SRP-cRGDfK compared to their negative analogue.

Owing to their tunable optical properties and their high absorption cross-section of X- and γ-ray, gold nanostructures appear as promising agents for remotely controlled therapy. Since the efficiency of cancer therapy is not limited to the eradication of the tumour but rests also on the sparing of healthy tissue, a biodistribution study is required in order to determine whether the behaviour of the nanoparticles after intravenous injection is safe (no accumulation in healthy tissue, no uptake by phagocytic cell-rich organs (liver, spleen) and renal clearance). The biodistribution of Au@DTDTPA nanoparticles which are composed of a gold core and a DTDTPA (dithiolated polyaminocarboxylate) shell can be established by X-ray imaging (owing to the X-ray absorption of the gold core) and by magnetic resonance imaging (MRI) since the DTDTPA shell was designed for the immobilization of paramagnetic gadolinium ions. However scintigraphy appears better suited for a biodistribution study owing to a great sensitivity. The successful immobilization of radioelements ((99m)Tc, (111)In) in the DTDTPA shell, instead of gadolinium ions, renders possible the follow up of Au@DTDTPA by scintigraphy which showed that Au@DTDTPA nanoparticles exhibit a safe behaviour after intravenous injection to healthy rats.

Over the last few decades, nanoparticles have been studied in theranostic field with the objective of exhibiting a long circulation time through the body coupled to major accumulation in tumor tissues, rapid elimination, therapeutic potential and contrast properties. In this context, we developed sub-5 nm gadolinium-based nanoparticles that possess in vitro efficient radiosensitizing effects at moderate concentration when incubated with head and neck squamous cell carcinoma cells (SQ20B). Two main cellular internalization mechanisms were evidenced and quantified: passive diffusion and macropinocytosis. Whereas the amount of particles internalized by passive diffusion is not sufficient to induce in vitro a significant radiosensitizing effect, the cellular uptake by macropinocytosis leads to a successful radiotherapy in a limited range of particles incubation concentration. Macropinocytosis processes in two steps: formation of agglomerates at vicinity of the cell followed by their collect via the lamellipodia (i.e. the "arms") of the cell. The first step is strongly dependent on the physicochemical characteristics of the particles, especially their zeta potential that determines the size of the agglomerates and their distance from the cell. These results should permit to control the quantity of particles internalized in the cell cytoplasm, promising ambitious opportunities towards a particle-assisted radiotherapy using lower radiation doses., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Early and accurate detection of tumors, like the development of targeted treatments, is a major field of research in oncology. The generation of specific vectors, capable of transporting a drug or a contrast agent to the primary tumor site as well as to the remote (micro-) metastasis would be an asset for early diagnosis and cancer therapy. Our goal was to develop new treatments based on the use of tumor-targeted delivery of large biomolecules (DNA, siRNA, peptides, or nanoparticles), able to induce apoptosis while dodging the specific mechanisms developed by tumor cells to resist this programmed cell death. Nonetheless, the insufficient effectiveness of the vectorization systems is still a crucial issue. In this context, we generated new targeting vectors for drug and biomolecules delivery and developed several optical imaging systems for the follow-up and evaluation of these vectorization systems in live mice. Based on our recent work, we present a brief overview of how noninvasive optical imaging in small animals can accelerate the development of targeted therapeutics in oncology.