Evaluating Nanoparticles Penetration by a New Microfluidic Hydrogel-Based Approach.

Reliable predictions for the route and accumulation of nanotherapeutics in vivo are limited by the huge gap between the 2D in vitro assays used for drug screening and the 3D physiological in vivo environment. While developing a standard 3D in vitro model for screening nanotherapeutics remains challenging, multi-cellular tumor spheroids (MCTS) are a promising in vitro model for such screening. Here, we present a straightforward and flexible 3D-model microsystem made out of agarose-based micro-wells, which enables the formation of hundreds of reproducible spheroids in a single pipetting. Immunostaining and fluorescent imaging, including live high-resolution optical microscopy, can be done in situ without manipulating spheroids.

Enhancing radioprotection: A chitosan-based chelating polymer is a versatile radioprotective agent for prophylactic and therapeutic interventions against radionuclide contamination.

To mitigate the risk of radioactive isotope dissemination, the development of preventative and curative measures is of particular interest. For mass treatment, the developed solution must be easily administered, preferably orally, with effective, nontoxic decorporating properties against a wide range of radioactive isotopes. Currently, most orally administered chelation therapy products are quickly absorbed into the blood circulation, where chelation of the radioactive isotope is a race against time due to the short circulation half-life of the therapeutic. This report presents an alternative therapeutic approach by using a functionalized chitosan (chitosan@DOTAGA) with chelating properties that remains within the gastrointestinal tract and is eliminated in feces, that can protect against ingested radioactive isotopes. The polymer shows important in vitro chelation properties towards different metallic cations of importance, including (Cs(I), Ir(III), Th(IV), Tl(I), Sr(II), U(VI) and Co(II)), at different pH (from 1 to 7) representing the different environments in the gastrointestinal tract. An in vivo proof of concept is presented on a rodent model of uranium contamination following an oral administration of Chitosan@DOTAGA. The polymer partially prevents the accumulation of uranium within the kidneys (providing a protective effect) and completely prevents its uptake by the spleen.

Supramolecular Heterodimer Peptides Assembly for Nanoparticles Functionalization.

Nanoparticle (NP) surface functionalization with proteins, including monoclonal antibodies (mAbs), mAb fragments, and various peptides, has emerged as a promising strategy to enhance tumor targeting specificity and immune cell interaction. However, these methods often rely on complex chemistry and suffer from batch-dependent outcomes, primarily due to limited control over the protein orientation and quantity on NP surfaces. To address these challenges, a novel approach based on the supramolecular assembly of two peptides is presented to create a heterotetramer displaying VH Hs on NP surfaces. This approach effectively targets both tumor-associated antigens (TAAs) and immune cell-associated antigens. In vitro experiments showcase its versatility, as various NP types are biofunctionalized, including liposomes, PLGA NPs, and ultrasmall silica-based NPs, and the VH Hs targeting of known TAAs (HER2 for breast cancer, CD38 for multiple myeloma), and an immune cell antigen (NKG2D for natural killer (NK) cells) is evaluated. In in vivo studies using a HER2+ breast cancer mouse model, the approach demonstrates enhanced tumor uptake, retention, and penetration compared to the behavior of nontargeted analogs, affirming its potential for diverse applications.

AGuIX nanoparticle-nanobody bioconjugates to target immune checkpoint receptors.

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.

Subcutaneous Administration of a Zwitterionic Chitosan-Based Hydrogel for Controlled Spatiotemporal Release of Monoclonal Antibodies.

Subcutaneous (SC) administration of monoclonal antibodies (mAbs) is a proven strategy for improving therapeutic outcomes and patient compliance. The current FDA-/EMA-approved enzymatic approach, utilizing recombinant human hyaluronidase (rHuPH20) to enhance mAbs SC delivery, involves degrading the extracellular matrix's hyaluronate to increase tissue permeability. However, this method lacks tunable release properties, requiring individual optimization for each mAb. Seeking alternatives, physical polysaccharide hydrogels emerge as promising candidates due to their tunable physicochemical and biodegradability features. Unfortunately, none have demonstrated simultaneous biocompatibility, biodegradability, and controlled release properties for large proteins (≥150 kDa) after SC delivery in clinical settings. Here, a novel two-component hydrogel comprising chitosan and chitosan@DOTAGA is introduced that can be seamlessly mixed with sterile mAbs formulations initially designed for intravenous (IV) administration, repurposing them as novel tunable SC formulations. Validated in mice and nonhuman primates (NHPs) with various mAbs, including trastuzumab and rituximab, the hydrogel exhibited biodegradability and biocompatibility features. Pharmacokinetic studies in both species demonstrated tunable controlled release, surpassing the capabilities of rHuPH20, with comparable parameters to the rHuPH20+mAbs formulation. These findings signify the potential for rapid translation to human applications, opening avenues for the clinical development of this novel SC biosimilar formulation.

Iron as an emerging therapeutic target in critically ill patients.

The multiple roles of iron in the body have been known for decades, particularly its involvement in iron overload diseases such as hemochromatosis. More recently, compelling evidence has emerged regarding the critical role of non-transferrin bound iron (NTBI), also known as catalytic iron, in the care of critically ill patients in intensive care units (ICUs). These trace amounts of iron constitute a small percentage of the serum iron, yet they are heavily implicated in the exacerbation of diseases, primarily by catalyzing the formation of reactive oxygen species, which promote oxidative stress. Additionally, catalytic iron activates macrophages and facilitates the growth of pathogens. This review aims to shed light on this underappreciated phenomenon and explore the various common sources of NTBI in ICU patients, which lead to transient iron dysregulation during acute phases of disease. Iron serves as the linchpin of a vicious cycle in many ICU pathologies that are often multifactorial. The clinical evidence showing its detrimental impact on patient outcomes will be outlined in the major ICU pathologies. Finally, different therapeutic strategies will be reviewed, including the targeting of proteins involved in iron metabolism, conventional chelation therapy, and the combination of renal replacement therapy with chelation therapy.

Gadolinium-Based Nanoparticles Sensitize Ovarian Peritoneal Carcinomatosis to Targeted Radionuclide Therapy.

Ovarian cancer (OC) is the most lethal gynecologic malignancy (5-y overall survival rate, 46%). OC is generally detected when it has already spread to the peritoneal cavity (peritoneal carcinomatosis). This study investigated whether gadolinium-based nanoparticles (Gd-NPs) increase the efficacy of targeted radionuclide therapy using [177Lu]Lu-DOTA-trastuzumab (an antibody against human epidermal growth factor receptor 2). Gd-NPs have radiosensitizing effects in conventional external-beam radiotherapy and have been tested in clinical phase II trials. Methods: First, the optimal activity of [177Lu]Lu-DOTA-trastuzumab (10, 5, or 2.5 MBq) combined or not with 10 mg of Gd-NPs (single injection) was investigated in athymic mice bearing intraperitoneal OC cell (human epidermal growth factor receptor 2-positive) tumor xenografts. Next, the therapeutic efficacy and toxicity of 5 MBq of [177Lu]Lu-DOTA-trastuzumab with Gd-NPs (3 administration regimens) were evaluated. NaCl, trastuzumab plus Gd-NPs, and [177Lu]Lu-DOTA-trastuzumab alone were used as controls. Biodistribution and dosimetry were determined, and Monte Carlo simulation of energy deposits was performed. Lastly, Gd-NPs' subcellular localization and uptake, and the cytotoxic effects of the combination, were investigated in 3 cancer cell lines to obtain insights into the involved mechanisms. Results: The optimal [177Lu]Lu-DOTA-trastuzumab activity when combined with Gd-NPs was 5 MBq. Moreover, compared with [177Lu]Lu-DOTA-trastuzumab alone, the strongest therapeutic efficacy (tumor mass reduction) was obtained with 2 injections of 5 mg of Gd-NPs/d (separated by 6 h) at 24 and 72 h after injection of 5 MBq of [177Lu]Lu-DOTA-trastuzumab. In vitro experiments showed that Gd-NPs colocalized with lysosomes and that their radiosensitizing effect was mediated by oxidative stress and inhibited by deferiprone, an iron chelator. Exposure of Gd-NPs to 177Lu increased the Auger electron yield but not the absorbed dose. Conclusion: Targeted radionuclide therapy can be combined with Gd-NPs to increase the therapeutic effect and reduce the injected activities. As Gd-NPs are already used in the clinic, this combination could be a new therapeutic approach for patients with ovarian peritoneal carcinomatosis.

Tuning ultrasmall theranostic nanoparticles for MRI contrast and radiation dose amplification.

Background: The introduction of magnetic resonance (MR)-guided radiation treatment planning has opened a new space for theranostic nanoparticles to reduce acute toxicity while improving local control. In this work, second-generation AGuIX® nanoparticles (AGuIX-Bi) are synthesized and validated. AGuIX-Bi are shown to maintain MR positive contrast while further amplifying the radiation dose by the replacement of some Gd3+ cations with higher Z Bi3+. These next-generation nanoparticles are based on the AGuIX® platform, which is currently being evaluated in multiple Phase II clinical trials in combination with radiotherapy. Methods: In this clinically scalable methodology, AGuIX® is used as an initial chelation platform to exchange Gd3+ for Bi3+. AGuIX-Bi nanoparticles are synthesized with three ratios of Gd/Bi, each maintaining MR contrast while further amplifying radiation dose relative to Bi3+. Safety, efficacy, and theranostic potential of the nanoparticles were evaluated in vitro and in vivo in a human non-small cell lung cancer model. Results: We demonstrated that increasing Bi3+ in the nanoparticles is associated with more DNA damage and improves in vivo efficacy with a statistically significant delay in tumor growth and 33% complete regression for the largest Bi/Gd ratio tested. The addition of Bi3+ by our synthetic method leads to nanoparticles that present slightly altered pharmacokinetics and lengthening of the period of high tumor accumulation with no observed evidence of toxicity. Conclusions: We confirmed the safety and enhanced efficacy of AGuIX-Bi with radiation therapy at the selected ratio of 30Gd/70Bi. These results provide crucial evidence towards patient translation.

Added Value of Scintillating Element in Cerenkov-Induced Photodynamic Therapy.

Cerenkov-induced photodynamic therapy (CR-PDT) with the use of Gallium-68 (68Ga) as an unsealed radioactive source has been proposed as an alternative strategy to X-ray-induced photodynamic therapy (X-PDT). This new strategy still aims to produce a photodynamic effect with the use of nanoparticles, namely, AGuIX. Recently, we replaced Gd from the AGuIX@ platform with Terbium (Tb) as a nanoscintillator and added 5-(4-carboxyphenyl succinimide ester)-10,15,20-triphenylporphyrin (P1) as a photosensitizer (referred to as AGuIX@Tb-P1). Although Cerenkov luminescence from 68Ga positrons is involved in nanoscintillator and photosensitizer activation, the cytotoxic effect obtained by PDT remains controversial. Herein, we tested whether free 68Ga could substitute X-rays of X-PDT to obtain a cytotoxic phototherapeutic effect. Results were compared with those obtained with AGuIX@Gd-P1 nanoparticles. We showed, by Monte Carlo simulations, the contribution of Tb scintillation in P1 activation by an energy transfer between Tb and P1 after Cerenkov radiation, compared to the Gd-based nanoparticles. We confirmed the involvement of the type II PDT reaction during 68Ga-mediated Cerenkov luminescence, id est, the transfer of photon to AGuIX@Tb-P1 which, in turn, generated P1-mediated singlet oxygen. The effect of 68Ga on cell survival was studied by clonogenic assays using human glioblastoma U-251 MG cells. Exposure of pre-treated cells with AGuIX@Tb-P1 to 68Ga resulted in the decrease in cell clone formation, unlike AGuIX@Gd-P1. We conclude that CR-PDT could be an alternative of X-PDT.

Design of a water-soluble chitosan-based polymer with antioxidant and chelating properties for labile iron extraction.

Loosely bound iron, due to its contribution to oxidative stress and inflammation, has become an important therapeutic target for many diseases. A water-soluble chitosan-based polymer exhibiting both antioxidant and chelating properties due to the dual functionalization with DOTAGA and DFO has been developed to extract this iron therefore preventing its catalytic production of reactive oxygen species. This functionalized chitosan was shown to have stronger antioxidant properties compared to conventional chitosan, improved iron chelating properties compared to the clinical therapy, deferiprone, and provided promising results for its application and improved metal extraction within a conventional 4 h hemodialysis session with bovine plasma.

Clinical transfer of AGuIX®-based radiation treatments for locally advanced cervical cancer: MR quantification and in vitro insights in the NANOCOL clinical trial framework.

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.

Targeting Glioblastoma-Associated Macrophages for Photodynamic Therapy Using AGuIX®-Design Nanoparticles.

Glioblastoma (GBM) is the most difficult brain cancer to treat, and photodynamic therapy (PDT) is emerging as a complementary approach to improve tumor eradication. Neuropilin-1 (NRP-1) protein expression plays a critical role in GBM progression and immune response. Moreover, various clinical databases highlight a relationship between NRP-1 and M2 macrophage infiltration. In order to induce a photodynamic effect, multifunctional AGuIX®-design nanoparticles were used in combination with a magnetic resonance imaging (MRI) contrast agent, as well as a porphyrin as the photosensitizer molecule and KDKPPR peptide ligand for targeting the NRP-1 receptor. The main objective of this study was to characterize the impact of macrophage NRP-1 protein expression on the uptake of functionalized AGuIX®-design nanoparticles in vitro and to describe the influence of GBM cell secretome post-PDT on the polarization of macrophages into M1 or M2 phenotypes. By using THP-1 human monocytes, successful polarization into the macrophage phenotypes was argued via specific morphological traits, discriminant nucleocytoplasmic ratio values, and different adhesion abilities based on real-time cell impedance measurements. In addition, macrophage polarization was confirmed via the transcript-level expression of TNFα, CXCL10, CD-80, CD-163, CD-206, and CCL22 markers. In relation to NRP-1 protein over-expression, we demonstrated a three-fold increase in functionalized nanoparticle uptake for the M2 macrophages compared to the M1 phenotype. The secretome of the post-PDT GBM cells led to nearly a three-fold increase in the over-expression of TNFα transcripts, confirming the polarization to the M1 phenotype. The in vivo relationship between post-PDT efficiency and the inflammatory effects points to the extensive involvement of macrophages in the tumor zone.

Combating lead and cadmium exposure with an orally administered chitosan-based chelating polymer.

Heavy metals present a threat to human health, even at minimal concentrations within the body. One source of exposure is due to the consumption of low-level contaminated foodstuff and water. Lead and cadmium have been shown to be absorbed by and accumulate within organs like the kidneys and liver, and they have also been associated to many diseases including cardiovascular disease and kidney dysfunction as well as developmental disorders and neurodegenerative diseases. Since this contamination of lead and cadmium is found worldwide, limiting the exposure is complicated and novel strategies are required to prevent the absorption and accumulation of these metals by forcing their elimination. In this study, a DOTAGA-functionalized chitosan polymer is evaluated for this preventative strategy. It shows promising results when orally administered in mice to force the elimination and negate the toxic effects of lead and cadmium found within foodstuff.

Proof of Concept of the Radiosensitizing Effect of Gadolinium Oxide Nanoparticles in Cell Spheroids and a Tumor-Implanted Murine Model of Chondrosarcoma.

Purpose: Chondrosarcomas (CHSs), which represent 20% of primary bone tumors in adults, are mostly resistant to radio- and chemotherapy. It is therefore essential that new therapeutic approaches, targeted to the tumour, be developed to improve the prognosis of patients. The effectiveness, as a radiosensitizing agent, of gadolinium oxide nanoparticles (GdoNP, AGuIX®) nanoparticles in CHS was evaluated in vitro, in spheroid CHS models allowing to reproduce cell-cell extracellular matrix interactions, and, in vivo, in a nude mouse model with heterotopic tumour xenograft. Methods: Spheroids from SW1353 and HEMC-SS cells were characterized by confocal microscopy with or without GdoNP treatment. Real-time microscopy enabled quantification of cell viability, cell migration and invasion. In vivo, the efficacy of the association of GdoNP combined with a single (4Gy) or fractionated (4x1Gy) irradiation was evaluated in HEMC-SS tumor-bearing mice by monitoring tumor growth, mouse survival and gene expression profile. Results: The expression of proteoglycans in the extra-cellular matrix (ECM) of spheroids demonstrated the relevance of the 3-D model. The combination of GdoNP with single or fractionated irradiation increased the lethal effects of irradiation on 2-D- and 3-D-cultured cells. In vivo, a single or a fractionated dose of 4 Gy associated with IT or IV injection of GdoNP decreased tumor growth significantly. Only IT injection increased mice survival. Unexpectedly, the radiosensitizing effect of GdoNP was associated, in vitro, with a significant decrease in invasion-migration capacities and, in vivo, with the decreased expression of PTX3, a protein involved in the epithelial-to-mesenchymal transition process, suggesting a potential impact of GdoNP on metastasis formation. Conclusion: These results provide the first proof of concept of the radiosensitizing effect of GdoNP in CHSs and opened the way for a multicentre, randomized Phase 2 trial evaluating the association of GdoNP with radiotherapy for the therapeutic management of patients with symptomatic inoperable musculoskeletal tumor lesions.

AGuIX nanoparticles enhance ionizing radiation-induced ferroptosis on tumor cells by targeting the NRF2-GPX4 signaling pathway.

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.

A New Generation of Ultrasmall Nanoparticles Inducing Sensitization to Irradiation and Copper Depletion to Overcome Radioresistant and Invasive Cancers.

An emerging target to overcome cancer resistance to treatments is copper, which is upregulated in a wide variety of tumors and may be associated with cancer progression and metastases. The aim of this study was to develop a multimodal ultrasmall nanoparticle, CuPRiX, based on the clinical AGuIX nanoparticle made of the polysiloxane matrix on which gadolinium chelates are grafted. Such hybrid nanoparticles allow: (i) a localized depletion of copper in tumors to prevent tumor cell dissemination and metastasis formation and (ii) an increased sensitivity of the tumor to radiotherapy (RT) due to the presence of high Z gadolinium (Gd) atoms. CuPRiX nanoparticles are obtained by controlled acidification of AGuIX nanoparticles. They were evaluated in vitro on two cancer cell lines (lung and head and neck) using the scratch-wound assay and clonogenic cell survival assay. They were able to reduce cell migration and invasion and displayed radiosensitizing properties.

Identification of Molecular Fragments in Equilibrium with Polysiloxane Ultrasmall Nanoparticles.

During recent decades, ultrasmall inorganic nanoparticles have attracted considerable interest due to their favorable biodistribution, pharmacokinetics and theranostic properties. In particular, AGuIX nanoparticles made of polysiloxane and gadolinium chelates were successfully translated to the clinics. In an aqueous medium, these nanoparticles are in dynamic equilibrium with polysiloxane fragments due to the hydrolysis of Si-O-Si bonds. Thanks to high-performance liquid chromatography coupled with electrospray ionization mass spectrometry, all these fragments were separated and identified.

Anti-BCMA Immuno-NanoPET Radiotracers for Improved Detection of Multiple Myeloma.

Current clinical imaging modalities for the sensitive and specific detection of multiple myeloma (MM) rely on nonspecific imaging contrast agents based on gadolinium chelates for magnetic resonance imaging (MRI) or for 18 F-FDG-directed and combined positron emission tomography (PET) and computed tomography (CT) scans. These tracers are not, however, able to detect minute plasma cell populations in the tumor niche, leading to false negative results. Here, a novel PET-based anti-BCMA nanoplatform labeled with 64 Cu is developed to improve the monitoring of these cells in both the spine and femur and to compare its sensitivity and specificity to more conventional immunoPET (64 Cu labeled anti-BCMA antibody) and passively targeted PET radiotracers (64 CuCl2 and 18 F-FDG). This proof-of-concept preclinical study confirmed that by conjugating up to four times more radioisotopes per antibody with the immuno-nanoPET platform, an improvement in the sensitivity and in the specificity of PET to detect tumor cells in an orthotopic model of MM is observed when compared to the traditional immunoPET approach. It is anticipated that when combined with tumor biopsy, this immuno-nanoPET platform may improve the management of patients with MM.

Correction: Quantifying nanotherapeutic penetration using a hydrogel-based microsystem as a new 3D in vitro platform.

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.

Feasibility study and direct extraction of endogenous free metallic cations combining hemodialysis and chelating polymer.

In this article, we report the conception and the use of dialysis-based medical device for the extraction of metals. The medical device is obtained by addition in the dialysate of a functionalized chitosan that can chelate endogenous metals like iron or copper. This water-soluble functionalized chitosan is obtained after controlled reacetylation and grafting of DOTAGA. Due to the high mass of chitosan, the polymer cannot cross through the membrane and the metals are trapped in the dialysate during hemodialysis. Copper extraction has been evaluated in vitro using an hemodialysis protocol. Feasibility study has been performed on healthy sheep showing no acute toxicity througout the entire dialysis procedure and first insights of metallic extraction even on healthy animals.